1. Cancer Biology
  2. Cell Biology
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Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism

  1. William Putzbach
  2. Quan Q Gao
  3. Monal Patel
  4. Stijn van Dongen
  5. Ashley Haluck-Kangas
  6. Aishe A Sarshad
  7. Elizabeth T Bartom
  8. Kwang-Youn A Kim
  9. Denise M Scholtens
  10. Markus Hafner
  11. Jonathan C Zhao
  12. Andrea E Murmann
  13. Marcus E Peter  Is a corresponding author
  1. Northwestern University, United States
  2. European Bioinformatics Institute (EMBL-EBI), United Kingdom
  3. National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, United States
  4. Feinberg School of Medicine, Northwestern University, United States
Research Article
Cite as: eLife 2017;6:e29702 doi: 10.7554/eLife.29702
7 figures, 1 table, 2 data sets and 6 additional files

Figures

Figure 1 with 2 supplements
Exogenous CD95L or CD95 proteins do not protect cells from toxicity of CD95L/CD95-derived shRNAs.

(ALeft: Percent cell confluence over time of NB7 cells after infection with either pLKO-shScr or pLKO-shL1 and concurrent treatment with different concentrations of soluble CD95L protein (S2). Two-way ANOVA was performed for pairwise comparisons of % confluence over time between shScr expressing cells untreated (/) or treated with 100 ng/ml S2. Each data point represents mean ±SE of three replicates. Center: Percent cell confluence over time of NB7 cells after infection with either pLKO-shScr or pLKO-shL1 and concurrent treatment with different concentrations of leucine zipper-tagged (Lz)CD95L protein. Two-way ANOVA was performed for pairwise comparisons of % confluence over time between shScr-expressing cells untreated or treated with 50 ng/ml LzCD95L. Each data point represents mean ±SE of three replicates. Right: Percent nuclear PI staining of MCF-7 cells 24 hrs after adding different amounts of LzCD95L. (B) Schematic of the eight silent mutations introduced to the shL1 and the shL3 target sites of CD95L. (C) Western blot analysis of CD95L and β-actin in NB7 cells over-expressing CD95L-WT, CD95L-L1MUT, or CD95L-L3MUT 3 days after infection with pLKO-shScr, pLKO-shL1, or pLKO-shL3. Shown is one of two repeats of this analysis. (D) Percent nuclear PI staining of NB7 cells expressing empty pLenti vector, CD95L-WT, CD95L-L1MUT, or CD95L-L3MUT 6 days after infection with either pLKO-shScr, pLKO-shL1, or pLKO-shL3. Each bar represents mean ±SD of three replicates. (E) Schematic of the eight silent mutations introduced at the shR6 site of CD95. (F) Western blot analysis of CD95 and β-actin in MCF-7 cells over-expressing CD95-WT or CD95-R6MUT. (G) Percent nuclear PI staining of MCF-7 cells expressing empty pLNCX2 vector, CD95-WT, or CD95-R6MUT 6 days after infection with pLKO-shScr, pLKO-shR6, or pLKO-shR7. Each bar represents mean ±SD of three replicates.

https://doi.org/10.7554/eLife.29702.003
Figure 1—figure supplement 1
The majority of siRNAs and shRNAs targeting CD95L or CD95 are toxic.

(A) Location of target sites, growth inhibitory activities and toxicity of all tested siRNAs, DsiRNAs, and pLKO-shRNAs targeting CD95L and CD95. Experiments were performed in HeyA8 cells at an MOI of 3 for pLKO-shRNA infection, transfected with 25 nM of siRNAs, or 5 nM of DsiRNAs. Color code indicates the level of growth reduction caused by each sh/siRNA. sh/siRNAs labeled with an asterisk induced significant cell death as monitored by nuclear PI staining. Both exon/intron structure and protein domains are shown for both CD95L and CD95. EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain. Data on growth reduction of DsiRNAs were performed in triplicates and in two independent experiments. Data on growth reduction of siRNAs were performed in four replicates and in two independent experiments. Data on growth reduction of shRNAs were performed in triplicate and in two independent experiments. Data on nuclear fragmentation by siRNAs were performed in triplicate in two independent experiments. Data on nuclear fragmentation by shRNAs were performed in triplicate. (B) PI staining was used to quantify percent subG1 of HeyA8 cells 4 days after transfection with 5 nM of CD95L-derived DsiRNAs. Data are representative of three independent experiments. Each bar represents mean ±SD of four replicates (p-value ***p<0.0001, unpaired t-test). (C) Level of underrepresentation (toxicity) of shRNAs targeting either CD95L (left column) or CD95 (right column) across 216 human cancer cell lines as described in (Cowley et al., 2014). The fraction of cell lines for which an shRNA was found to be toxic is given as a percentage, for shL5, data were only available on 197 cell lines. This screen did not include shL3, but another CD95 derived shRNA we did not test (we now call shR3) was found to be toxic to 71.8% of the 216 cell lines.

https://doi.org/10.7554/eLife.29702.004
Figure 1—figure supplement 2
Toxicity of si/shRNAs is dose dependent.

(A) Sequences of the six toxic TRC shRNAs (pLKO vector) that were converted into miR-30-based shRNAs (Tet inducible LT3REPIR vector). (B) Confluence over time of NB7-Venus-CD95L (left) or NB7-Venus CD95 (right) cells infected with the LT3REPIR vector minus/plus Dox to induce expression of the indicated shRNAs. (C) Total green fluorescence over time of the experiment shown in B. (D) Confluence (top) and total green fluorescence (bottom) over time of NB7-Venus-CD95L (left) or NB7-Venus CD95 (right) cells infected with the pTIP vector minus/plus Dox to induce expression of the indicated shRNAs. (E) Confluence over time of HeyA8 cells transfected with the indicated concentration of either siScr or siL3. Each data point represents mean ±SE of six replicates. The experiment was repeated three times.

https://doi.org/10.7554/eLife.29702.005
Figure 2 with 1 supplement
CD95 and CD95L derived si/shRNAs kill cells in the absence of the targeted sites in CD95 or CD95L.

(A) Schematic of the genomic locations and sequences of the gRNAs used to excise the siL3 (Δ64bp) and shL3 (Δ41bp) target sites from CD95L. PAM site is underlined. Green indicates a gRNA targeting the sense strand. Blue indicates a gRNA targeting the antisense strand. (B) Schematic showing the genomic locations and sequences of the gRNAs used to excise the shR6 (Δ227bp) target site. Mix, pool of three 293T clones with the homozygous shL3 deletion. (C) PCR with flanking (top panels) and internal (bottom panels) primers used to confirm the Δ41 deletion in the shL3 site in one of the three homozygous deletion 293T clones generated. Cells transfected with Cas9 only (Cas9) are wild-type. (D) Quantitative PCR for endogenous CD95L with a primer downstream of the Δ41 shL3 deletion and another primer internal to the deleted region. nd, not detectable. Each bar represents mean ±SD of three replicates. (E) PCR with flanking (top row) and internal (bottom row) primers used to confirm the presence of the shL3 Δ41 (top panel), siL3 Δ64 (middle panel), and shR6 Δ227 (bottom panel) deletions in HeyA8 clones. Mix, HeyA8 cells after transfection with Cas9 and gRNAs but before single cell cloning. (F) Quantitative PCR for CD95 in HeyA8 cells transfected with Cas9 plasmid (Cas9) alone, or the HeyA8 ΔshR6 clone #11. RNA was extracted 5 days after infection with pLKO-shScr, pLKO-shR6, pLKO-shR2, or pLKO-shR6’ (targeting the 3'UTR). Each bar represents mean ±SD of three replicates. (G) Percent cell confluence over time of 293T cells (left) and a pool of three 293T clones with a homozygous deletion of the shL3 target site (right) infected with pTIP-shScr or pTIP-shL3 and treatment with or without Dox. Data are representative of two independent experiments. Each data point represents mean ±SE of six replicates. (H) Left: Percent confluence over time of HeyA8 cells infected with pLKO-shScr, pLKO-shR6, or pLKO-shL3. Center: Percent confluence over time of a HeyA8 clone with a homozygous deletion of the shR6 target site infected with either pLKO-shScr or pLKO-shR6. Right: Percent confluence over time of a pool of three HeyA8 clones with a homozygous deletion of the shL3 site infected with either pLKO-shScr or pLKO-shL3. Data are representative of two independent experiments. Each data point represents mean ±SE of three replicates. (I) Percent confluence over time of a pool of three HeyA8 clones harboring a homozygous deletion of the siRNA siL3 target site after transfection with different concentrations of siScr or siL3. Data are representative of three independent experiments. Each data point represents mean ±SE of three replicates.

https://doi.org/10.7554/eLife.29702.006
Figure 2—figure supplement 1
Knockout of CD95 in HeyA8 cells.

(A) PCR showing a Δ227 shR6 deletion and insertions in HeyA8 clones #1 and #2. (B) Schematic of the Δ227 deletion in allele #1 and partial insertion of a pSC-B plasmid fragment in allele #2 in HeyA8 clone #2 based on Sanger sequencing of isolated bands from PCR shown in A. Note, cl#1 and #2 have the expected Δ227 shR6 deletion in one allele and an insertion in the other. cl#11 has a homozygous Δ227 shR6 deletion. The deleted region is shown in green containing the shR6 target site in red. pSC-B vector sequences are shown in blue letters, and the insertion is shown in orange. (C) Western blot for CD95 and β-actin in Cas9-control transfected HeyA8 cells and HeyA8 shR6 k.o. clones #1, #2, and #11. Shown is one of two repeats of this analysis. (D) Surface staining for CD95 in parental HeyA8 cells and HeyA8 shR6 knockout clones #1, #2, and #11. Shown is one of two repeats of this analysis. (E) Images showing apoptosis induction with LzCD95L treatment (4.5 hr) in parental HeyA8 cells but not in clone #2.

https://doi.org/10.7554/eLife.29702.007
Toxicity of CD95L-derived siRNAs involves canonical RNAi activity.

(A) Percent cell confluence (left) and total green object integrated intensity (right) over time of a HeyA8 CD95 knockout clone (ΔR6 cl#2) expressing the Venus-CD95L sensor either untreated (Ctr) or after transfection with 25 nM of single-stranded sense, single-stranded antisense, or double-stranded (ds) siScr or siL3 siRNAs. The CD95L sensor is schematically shown and comprises the Venus ORF fused to the CD95L ORF lacking the A of the ATG start codon (X). Data are representative of two independent experiments. Each data point represents mean ±SE of three replicates. (B) Percent cell confluence (left) and total green object integrated intensity (right) over time of the HeyA8 CD95L sensor cell used in Figure 3A after transfection with 5 nM siScr or siL3 double-stranded RNA (dsRNA) or double-stranded DNA (dsDNA). Data are representative of two independent experiments. Each data point represents mean ±SE of three replicates. (C) Summary of experiments to test whether siL3 and siL2 siRNAs modified as indicated (left) were active (check mark) or not (X) in reducing green fluorescence or cell growth (both >70% reduction at end point) when transfected at 25 nM (except for blunt end oligonucleotides which were used at 5 nM and compared to 5 nM of siL3) into HeyA8 CD95L sensor cells used in Figure 3A. Endpoints were 164 hrs for blunt end siRNA transfection, 180 hrs for modified siL3 and 144 hrs for modified siL2 siRNA transfections. Every data row is based on cell growth and green fluorescence quantification data executed as shown in A. Each analysis was done in triplicate and based on two independent repeats. (D) Red object count over time of HeyA8 cells (expressing NucRed) after transfection with different ratios of siL3 and mutant siL3 (siL3MUT). Data are representative of two independent experiments. Each data point represents mean ±SE of three replicates. (E) Percent cell confluence over time of HCT116 parental (left) or Dicer-/- (clone #43, another Dicer-/- clone, #45, gave a similar result, data not shown), or Drosha-/- (right) cells after infection with either shScr, shL3 or shR6 pLKO viruses. Inserts show the level of protein expression levels of Drosha/Dicer and AGO2 levels in the tested cells. Data are representative of three independent experiments. Each data point represents mean ±SE of four replicates. Drosha-/- cells were more sensitive to toxic shRNAs than wt cells (p<0.0001, according to a polynomial fitting model). (F) Western blot analysis of HCT116 wt, Dicer-/- or Drosha-/- cells 4 days after infection with either pLKO-shScr or pLKO-shR6. (G) Percent cell confluence over time of HCT116 wt, Dicer-/- (clone #43) and Drosha-/- cells after transfection with 25 nM siScr or siL3. Data are representative of four independent experiments (Dicer-/- clone #45, gave a similar result, data not shown). Each data point represents the mean ±SE of four replicates. Data in insert confirm similar uptake of transfected siRNA (25 nM of siGLO Red) into wild-type, Dicer-/- and Drosha-/- cells. Dicer-/- and Drosha-/- cells were more sensitive to siL3 than wt cells (p<0.0001, according to a polynomial fitting model). (H) Percent reduction in Venus expression (green) and in cell number (red object count [red]) over time of HeyA8 cells expressing the Venus-CD95L sensor and red nuclei after transfection with 5 nM of different chimeric siRNAs generated by substituting nucleotides in the toxic siL3 with the scrambled siRNA sequence beginning at either the seed match end (top) or the opposite end (bottom) of siL3 after 188 hr. The schematic in the middle shows the sequence of siL3 and the siScr siRNA (both sense and antisense strands). The 6mer seed sequence region of siL3 (positions 2 to 7) is highlighted in light blue. Nucleotides shared by siScr and siL3 are shown in grey font. Data are representative of two independent experiments. Each data point represents mean of three replicates. In another independent experiment cells were transfected with 25 nM with a very similar result (data not shown).

https://doi.org/10.7554/eLife.29702.008
Figure 4 with 3 supplements
Toxic shRNAs derived from CD95 and CD95L cause downregulation of critical survival genes.

(A) Schematic of RNA-Seq work flow for total RNA sample prepared both before (50 hr) and during (100 hr) DISE after expressing either shR6 or shL3 from different vector systems (i.e. pLKO-shR6 and pTIP-shL3) in different cells (HeyA8 shR6 Δ227 cells and 293T shL3 Δ41 cells). (B) One mRNA was up and 11 mRNAs were downregulated in the cells treated with toxic shL3 and shR6 as shown in Figure 4A. mRNAs shown in red were found to be essential cancer survival genes in two genome-wide lethality screens. The number of essential genes was enriched from 6.6% of the tested genes (Blomen et al., 2015; Wang et al., 2015) to 54.5% in our study (p=3×10−6 according to binomial distribution). (C) The level of growth inhibition observed in HeyA8 cells transfected with siRNA SmartPools (25 nM) individually targeting the listed survival genes. Targeting the seven genes shown significantly reduced cell growth compared to cells transfected with a siScr pool at 140 hrs (samples done in quadruplicate in two independent experiments) with an ANOVA p<0.05. (D) Gene set enrichment analysis for a group of 1846 survival genes (top four panels) and 416 nonsurvival genes (bottom four panels) identified in a genome-wide CRISPR lethality screen (Wang et al., 2015) after introducing Dox-inducible shL3 in 293T ΔshL3 cells (left-most panels), shR6 in HeyA8 ΔshR6 cells (center-left panels), shL1 in parental 293T cells (center-right panels), and siL3 in HeyA8 cells (right-most panels). Scrambled sequences served as controls. p-values indicate the significance of enrichment. (E) Schematics showing all RNAs at least 1.5 fold downregulated (adj p-value<0.05) in cells treated as in Figure 4A. Histones that are underlined contain a 3’UTR. (F) Metascape analysis of the 4 RNA Seq data sets analyzed. The boxed GO term clusters were highly enriched in all data sets.

https://doi.org/10.7554/eLife.29702.009
Figure 4—figure supplement 1
Down-regulation of critical survival genes after treatment with CD95 and CD95L-derived shRNAs and siRNAs.

(A) Arrayed quantitative PCR of genes found to be down-regulated (or upregulated as with ATP13A3) in Figure 4B both in 293T ΔshL3-pTIP-shL3 cells 50 hrs post-Dox treatment and HeyA8 ΔshR6-pLKO-shR6 100 hrs post infection and puromycin selection. Data are representative of two independent experiments. Each bar represents mean ±SD of two biological replicates and two technical replicates (p-value *<0.05, **<0.005, unpaired t-test). (B) Venn diagram showing overlap of genes determined to be down-regulated with both read-based and alignment-based analyses of the RNA-Seq data depicted in Figure 4A with the critical survival genes found in the Sabatini and Brummelkamp studies (Blomen et al., 2015; Wang et al., 2015), all listed in Supplementary file 2. The Venn diagram was generated using http://bioinformatics.psb.ugent.be/webtools/Venn. (C) Kinetic quantitative PCR of the down-regulated genes in the 293T ΔshL3 pTIP-shL3 cells. RNA was collected at 14 hrs, 26 hrs, and 50 hrs after treatment with Dox. NS, not significant. Each bar represents mean ±SD of quadruplicates (p-value *<0.05, **<0.005, unpaired t-test). (D) Table showing which genes were significantly (p<0.05) down-regulated >1.5 fold (indicated by an ‘X’) in parental HeyA8 cells 80 hrs after transfection with siL3 or 100 hrs after infection and puromycin selection with pLKO-shL1, pLKO-shL3, or pLKO-shR7. The following describes the 11 genes that were significantly downregulated after introducing the toxic shRNAs shL3 or shR6 into cancer cells (see Figure 4B) and some of their cancer relevant activities. (1) CAPZA1 (capping actin protein of muscle Z-line alpha subunit 1) is an actin capping protein. CAPZA1 knockdown has been reported to cause disassembly of autophagosomes (Mi et al., 2015). It is overexpressed in malignant melanoma (Sun et al., 2011). (2) CCT3 (chaperonin containing TCP1 subunit 3) is part of a chaperone complex that folds various proteins including actin and tubulin. CCT3 is required for proper mitotic progression (Zhang et al., 2016). (3) FSTL1 (follistatin-like 1) is a putative activin-binding protein. Knockdown of FSTL1 in lung cancer cells resulted in mitotic arrest followed by apoptosis promoted by the activation of caspase-3 and −9 (Bae et al., 2016). FSTL1 is downregulated during cellular senescence of human mesenchymal stem cells (Yoo et al., 2013). (4) FUBP1 (far upstream element binding protein 1). A lack of FUBP1 causes a cell-autonomous defect in the maintenance of fetal and adult hematopoietic stem cells (HSCs). FUBP1-deficient adult HSCs exhibit significant transcriptional changes, including upregulation of the cell-cycle inhibitor p21 and the pro-apoptotic Noxa molecule, suggesting they undergo apoptosis (Rabenhorst et al., 2015). In addition, FUBP1 binds to an upstream element of the c-myc promoter and regulates c-myc mRNA level, thus regulating proliferation (Jang et al., 2009). Finally, FUBP1 is upregulated in many tumors and acts as an oncoprotein by stimulating proliferation and inhibiting apoptosis (Baumgarten et al., 2014). (5) GNB1 (G-protein beta submit 1) is tumor-promoting in breast cancer. Data suggest that GNB1 plays an important role in the mTOR-related anti-apoptosis pathway (Wazir et al., 2013). (6) HIST1H1C. A specific role of this particular histone in cancer cell survival has not yet been described. (Knockdown causes cell cycle arrest in MCF-7 cells; (Sancho et al., 2008). (7) NAA50 (N(alpha)-acetyltransferase 50, NatE catalytic subunit) is required for sister chromatid separation in vivo (Hou et al., 2007). (8) NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a chromatin-associated protein with a role in the DNA damage response. Knocking down NUCKS1 causes chromosomal breaks (Parplys et al., 2015). (9) PRELID3B (PRELI domain containing 3B) is an inner mitochondrial protein. Knocking down PRELID3B decreases cell viability (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRELID3B). (10) SNRPE (small nuclear ribonucleoprotein polypeptide E). siRNA-mediated depletion of SNRPE stimulated autophagy and led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells (Quidville et al., 2013). (11) TFRC (transferrin receptor). Blocking TFRC function with a neutralizing antibody inhibits cell proliferation and survival (Pham et al., 2014). Suppression of TFRC led to apoptosis of renal cells (Gui et al., 2013) and cell cycle arrest in esophageal squamous cell carcinoma cells (Chan et al., 2014).

https://doi.org/10.7554/eLife.29702.010
Figure 4—figure supplement 2
Characterization of the six genes downregulated in shL3 and shR6-treated cells and found to be critical survival genes in lethality screens.
https://doi.org/10.7554/eLife.29702.011
Figure 4—figure supplement 3
Histones are downregulated in all forms of DISE but are not the most highly expressed genes in cells.

MA plots comparing the expression level (counts per million, CPM) and fold change in the four RNA Seq data sets in this study. Shown are all RNAs that were >1.5 fold deregulated with an adjusted p-value of <0.01. Significantly downregulated RNAs are shown in green, upregulated RNAs in cyan. All 73 histones are shown as dark blue dots and the 12 histones downregulated in all four data sets are shown as red dots.

https://doi.org/10.7554/eLife.29702.012
Figure 5 with 3 supplements
DISE inducing si/shRNAs target critical survival genes through RNAi.

(A) Sylamer plots for the list of genes in the shL3 experiment (left) and the shR6 experiment (right) ordered from down-regulated to up-regulated. The most highly enriched sequence is shown which in each case is the 8mer seed match of the introduced shRNA. The red line corresponds to a p-value threshold of 0.05 after Bonferroni correction for the number of words tested (65536). Bonferroni-adjusted p-values are shown. The unadjusted p-values are 1.58E-24 and 1.35E-26, respectively. The black line represents the sequences carrying the let-7 8mer seed match. (B) Location of the 6mer seed matches of either shL3 or shR6 in the 3'UTRs of the 11 genes (shown at scale) identified in the RNA-Seq experiment described in Figure 4A. Red font indicates a critical survival gene. (C) A series of six 2 × 2 contingency tables comparing whether or not a critical survival gene is downregulated after treatment with the indicated siRNA or shRNA to whether or not its 3’UTR contains at least one seed match for the introduced sh/siRNA. p-values were calculated using Fisher’s Exact Test to determine any significant relationship between gene downregulation and presence of seed matches in 3’UTR.

https://doi.org/10.7554/eLife.29702.013
Figure 5—figure supplement 1
Quantification of the mature shRNA forms.

(A) Graphical representation of the percentage of the different Dicer cut sites to produce the mature passenger (top) and guide (bottom) strands of 3 shRNAs expressed from two vectors. All analyses were performed with cells 50 hrs after either Dox addition (in pTIP expressing cells) or 50 hrs post puromycin selection after infection with the pLKO virus. Letters in green: vector sequences; black: passenger and guide strands of shRNAs; Arrow heads label the most highly cleaved residues; the darker the arrow head the more highly cleaved. Numbers in yellow box represent total number of reads detected for passenger and guide strands. (B) Percent cell confluence in HeyA8 cells after transfection with shL3 => siL3 (shL3 converted to an siRNA) or shR6 => siR6 (shR6 converted to an siRNA). Conversion was based on the most common mature double-stranded RNA form produced as indicated by the results in A. Data are representative of two independent experiments. Each bar represents mean ±SE of four replicates. Insert: percent DNA fragmentation in the same samples. Data are representative of two independent experiments. Each bar represents mean ±SD of four replicates, ***p<0.0001, unpaired t-test.

https://doi.org/10.7554/eLife.29702.014
Figure 5—figure supplement 2
Identification of seed matches targeted by shL1 and siL3.

Sylamer plots for the list of genes in the shL1 experiment (293T cells 100 hrs post puromycin selection after infection with pLKO-shL1) (left) and the siL3 experiment (48 hrs after transfection of HeyA8 cells with siL3) (right) ordered from down-regulated to up-regulated. The most highly enriched sequences are shown which in each case is the 7mer seed match of the introduced shRNA. The red line corresponds to a p-value threshold of 0.05 after Bonferroni correction for the number of words tested. Bonferroni-adjusted p-values are shown.

https://doi.org/10.7554/eLife.29702.015
Figure 5—figure supplement 3
Activity to knockdown CD95 does not determine shRNA toxicity.

HeyA8 cells infected with the indicated shRNAs in the pLKO vector were analyzed for toxicity (top, % percent reduction at half maximal confluency), CD95 expression by Western blot analysis (center, 2 days after puromycin addition) and qPCR analysis (bottom, 3 days after puromycin addition). Shown data are representative of two independent experiments. +++,>75%; ++,>50%; +,>10%; -,<10% growth reduction.

https://doi.org/10.7554/eLife.29702.016
Figure 6 with 2 supplements
Identifying all toxic shRNAs derived from CD95L and CD95.

(A) Schematic showing the cloned shRNAs covering the ORF of Venus and the ORFs and 3'UTRs of CD95L and CD95. The 3’UTR is displayed as a dashed line because it was not included in the full-length Venus-CD95L/CD95 sensors. (B) Work-flow of pTIP-shRNA library synthesis, shRNA screen and data analysis. (C) Ranked fold reduction of shRNAs spanning Venus and CD95L (ORF and 3'UTR) (left three panels) and Venus and CD95 (ORF and 3'UTR) (right three panels). The ranked lists were separated into the shRNAs derived from Venus (top), the ORFs (center) and the 3'UTRs (bottom). The p-value of enrichment for each ranked set of shRNAs is given. Only the parts of the ranked lists are shown with the downregulated shRNAs. For all six panels, the top section of each panel (boxed in blue) contains the data on shRNAs downregulated after infection of cells and cultured for 9 days without Dox when compared to the composition of the shRNA plasmid library and the bottom half (boxed in orange) contains the data on shRNAs downregulated after culture with Dox for 9 days when compared to the culture without Dox. P-values were calculated using Mann Whitney U tests with a one-sided alternative that the rank was lower. (D) The location of all shRNAs significantly downregulated at least five fold along the sequences of Venus, CD95L ORF, CD95L 3'UTR (left panel) and Venus, CD95 ORF, and CD95 3'UTR (right panel). The top half of each sub panel (blue ticks) shows the shRNAs downregulated after infection and the bottom half (orange ticks) contains the data on shRNAs downregulated after culture with Dox for 9 days. Significance of enrichment in the different subpanels is shown. p-values were calculated according to statistical tests of two proportions. Each data set was compared to the corresponding Venus distribution. Green line: sequence that corresponds to the intracellular domain of CD95L.

https://doi.org/10.7554/eLife.29702.017
Figure 6—figure supplement 1
Toxicity and RNAi of individual shRNA pools.

(A) Top panels: Green object intensity over time of NB7 Venus-CD95L sensor cells infected with the pTIP-Venus shRNA pool (left panel), pTIP-CD95L ORF shRNA pool (center panel), or pTIP-CD95L 3’UTR shRNA pool (right panel) with or without Dox treatment. Bottom panels: Green object intensity over time of NB7 Venus-CD95 sensor cells infected with the pTIP-Venus shRNA pool (left panel), pTIP-CD95 ORF shRNA pool (center panel), or pTIP-CD95 3’UTR shRNA pool (right panel) with or without Dox treatment. Values were calculated from samples done in quadruplicates shown as mean ±SE. (B) Percent confluence over time of parental NB7 cells infected with the pTIP-Venus shRNA pool (top left panel), pTIP-CD95L ORF shRNA pool (top center panel), pTIP-CD95L 3’UTR shRNA pool (top right panel), pTIP-CD95 ORF-shRNA pool (bottom center panel), and pTIP-CD95 3’UTR shRNA pool (bottom right panel) with or without Dox treatment. Values were calculated from samples done in triplicate shown as mean ±SE. P-values were calculated using two-way ANOVA with a factor for Dox treatment and a factor for time. Similar data were obtained when either HCT116 or 293T cells were treated with each of the five shRNA pools (data not shown).

https://doi.org/10.7554/eLife.29702.018
Figure 6—figure supplement 2
Fold change in shRNA representation after infection of NB7 cells and after treatment with Dox.

(A) Change in green fluorescence (top panels) and percent cell confluence (bottom panels) over time of NB7 cells expressing either Venus-CD95 (left panels) or Venus-CD95L (right panels). Cells were infected with the Tet-inducible pTIP-shR6 virus, selected for two days with puromycin and then subjected to an analysis in the IncuCyte Zoom. No Dox was added. Two other inducible constructs (pTIP-shL1 and pTIP-shL3) were tested in the same way and no evidence of leakiness was observed (data not shown), supporting the finding in the shRNA screen that certain shRNA constructs display leakiness while others do not. Values were calculated from samples done in triplicate shown as mean ±SE. (B) Scatterplot showing the fold down of shRNAs after infection of cells and culture for 9 days without Dox when compared to the composition of the shRNA plasmid library (X axis) and the fold down of shRNAs after culture with Dox for 9 days when compared to the culture without Dox (Y axis). The red dots are the shRNAs that were significantly downregulated at least five fold. The number of shRNAs labeled in red in each quartile is given in blue. Two of the shRNAs tested before are labeled in green.

https://doi.org/10.7554/eLife.29702.019
Figure 7 with 1 supplement
In silico prediction of DISE activity tracks with experimental determined toxicity of shRNAs.

(A) Left: Schematic showing the preferential targeting of seed matches present in the 3'UTRs (red marks) of survival genes by toxic si/shRNAs. Right: The toxicity index (TI) is the normalized ratio of the number of 6mer or 8mer seed matches present in a list of survival genes versus a list of nonsurvival genes. (B) Fold downregulation versus ranked (8mer seed matched based) Toxicity Index for shRNAs of the Venus/CD95L pool (left three panels) and the Venus/CD95 pool (right three panels). Orange and blue tick marks indicate the same as in Figure 6D. To test if higher TI is enriched in shRNAs that were highly downregulated, p-values were calculated based on permutated datasets using Mann-Whitney U tests. The ranking of TI was randomly shuffled 10,000 times and the W statistic from our dataset was compared to the distribution of the W statistic of the permutated datasets. (C, D) Plot of fold downregulation of toxic shRNAs derived from CD95L ORF of the toxicity screens -Dox (left) or +Dox (center) versus GC content the 6mer seed in each shRNA. (E) Plot of the log(TI) of all 4092 possible 6mers versus GC content of the seeds. Pearson correlation coefficient and significance (p values) are given.

https://doi.org/10.7554/eLife.29702.020
Figure 7—figure supplement 1
DISE does not just target all highly expressed genes.

(A) Correlation between 850 survival genes (genes identified as critical survival genes in two genome-wide lethality screens (Blomen et al., 2015; Wang et al., 2015) and expressed at least at 100 reads in all of the 16 control RNA Seq samples in this study) and 850 expression matched nonsurvival genes (genes not identified as critical survival genes in two genome-wide lethality screens (Blomen et al., 2015; Wang et al., 2015) and expressed at least at 100 reads in all of the 16 control RNA Seq samples in this study). (B) Reanalysis of the CD95L ORF data in Figure 7B using two alternative ways to calculate the toxicity index (TI). Left: the analysis shown in Figure 7B with the data ranked using the original TI (using all known 3'UTRs for each gene group). Center: analysis with the data ranked using the original TI but based on only the longest 3'UTR for each gene. Right: analysis with the data ranked using the new TI based on expression matched SGs and nonSGs identified in A and using the longest 3'UTR for each gene. To test if higher TI is enriched in shRNAs that were highly downregulated, p-values were calculated based on permutated datasets using Mann-Whitney U tests.

https://doi.org/10.7554/eLife.29702.021

Tables

Key resource table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)CD95LNANM_000639
Gene (H. sapiens)CD95NANM_000043
Cell line (H. sapiens)NB7PMID: 10802708BRENDA Tissue and
Enzyme Source
Ontology: BTO_0003439;
RRID:CVCL_8824
Human neuroblastoma derived from
autonomic ganglia; carries a deletion
in both alleles of CASP8
Cell line (H. sapiens)HeyA8PMID: 4016745;
PMID: 25984343
RRID: CVCL_8878;
RRID:CVCL_8878
Human high grade ovarian
serous adenocarcinoma; derived
from parent Hey cells (RRID: CVCL_0297)
Cell line (H. sapiens)HeyA8 ΔshL3this paperNAPool of three HeyA8 cell clones with
homozygous 41 nucleotide deletion
of the shL3 target site
(chr1:172,665,726–172,655,766; Human
Dec. 2013 GRCh38/hg38 assembly)
produced using CRISPR/Cas9 technology.
Cell line (H. sapiens)HeyA8 ΔsiL3this paperNAPool of three HeyA8 cell clones with
homozygous 64 nucleotide deletion of
the siL3 target site
(chr1:172,669,178–172,659,241; Human
Dec. 2013 GRCh38/hg38 assembly)
produced using CRISPR/Cas9 technology.
Cell line (H. sapiens)HeyA8 ΔshR6; shR6
k.o. clone #11
this paperNAHeyA8 cell clone #11 with homozygous
227 nucleotide deletion of the shR6
target site (chr10:89,008,920–89,009,146;
Human Dec. 2013 GRCh38/hg38
assembly) produced using
CRISPR/Cas9 technology; verified
homozygous CD95 protein knockout
Cell line (H. sapiens)HeyA8 shR6 k.o.
clone #1
this paperNAHeyA8 cell clone #1 with a small deletion
and the 227 nucleotide deletion of the
shR6 target site and an insertion of the
pMJ920 plasmid fragment in CD95
produced using CRISPR/Cas9
technology; verified homozygous
CD95 protein knockout
Cell line (H. sapiens)HeyA8 shR6 k.o.
clone #2
this paperNAHeyA8 cell clone #2 with a 227
nucleotide deletion of the shR6
target site (chr10:89,008,920–89,009,146;
Human Dec. 2013 GRCh38/hg38 assembly)
in one allele and an insertion of the
pSCB plasmid fragment in the other
in CD95 produced using
CRISPR/Cas9 technology; verified
homozygous CD695 protein knockout
Cell line (H. sapiens)MCF-7ATCCATCC: HTB-22;
RRID:CVCL_0031
Human adenocarcinoma of the mammary
gland, breast; derived from metastatic
site: pleural effusion
Cell line (H. sapiens)HCT116Korean Collection for
Type Cultures (KCTC)
KCTC: cat#HC19023;
ATCC: CCL_247;
RRID:CVCL_0291
Human colorectal carcinoma
Cell line (H. sapiens)Drosha-/-; Drosha-/-
clone #40
Korean Collection for
Type Cultures (KCTC);
PMID: 26976605
KCTC: cat#HC19020HCT116 clone #40 with homozygous
protein knockout of Drosha; knockout
achieved using CRISPR/Cas9 which
resulted in a single nucleotide insertion
in one allele and a 26 nuceotide
deletion in the other
Cell line (H. sapiens)Dicer-/-; Dicer-/-
clone #43
Korean Collection for
Type Cultures (KCTC);
PMID: 26976606
KCTC: cat#HC19023HCT116 clone #43 with homozygous
protein knockout of Dicer; knockout
achieved using CRISPR/Cas9 which
resulted in a three nucleotide
insertion and 14 nucleotide deltion
in one allele and a 35 nuceotide
deletion in the other
Cell line (H. sapiens)Dicer-/-; Dicer-/-
clone #45
Korean Collection for
Type Cultures (KCTC);
PMID: 26976607
KCTC: cat#HC19024HCT116 clone #45 with homozygous
protein knockout of Dicer; knockout
achieved using CRISPR/Cas9 which
resulted in a 53 nucleotide deltion
in one allele and a 28 nuceotide
deletion in the other
Cell line (H. sapiens)293TATCCATCC: CRL-3216;
RRID:CVCL_0063
Derived from HEK293 cells
(ATCC: CRL-1573); express large T
antigen; used for packaging viruses
Cell line (H. sapiens)293T ΔshL3this paperNAPool of three 293T cell clones with
homozygous 41 nucleotide deletion
of the shL3 target site
(chr1:172,665,726–172,655,766;
Human Dec. 2013 GRCh38/hg38
assembly) produced using
CRISPR/Cas9 technology.
Cell line (H. sapiens)Phoenix-AMPHOATCCATCC: CRL-3213;
RRID:CVCL_H716
Second generation retrovirus
producer cell line
Antibodyanti-β-actin antibody
(mouse monoclonal)
Santa CruzSanta Cruz:
cat#sc-47778;
RRID:AB_626632
1:2000; for western blot;
primary Ab
Antibodyanti-human CD95L
(Mouse IgG1
monoclonal)
BD BiosciencesBD Biosciences:
cat#556387;
RRID:AB_396402
1:500; for western blot;
primary Ab
Antibodyanti-human CD95
(rabbit polyclonal)
Santa CruzSanta Cruz:
cat#sc-715;
RRID:AB_2100386
1:1000; for western blot;
primary Ab
Antibodyanti-human AGO2
(rabbit monoclonal)
AbcamAbcam: cat#AB186733;
RRID:AB_2713978
1:2000; for western blot;
primary Ab
Antibodyanti-human Drosha
(rabbit monoclonal)
Cell SignalingCell Signaling:
cat#3364;
RRID:AB_2238644
1:1000; for western blot;
primary Ab
Antibodyanti-human Dicer
(rabbit polyclonal)
Cell SignalingCell Signaling:
cat#3363;
RRID:AB_2093073
1:1000; for western blot;
primary Ab
AntibodyGoat anti-rabbit,
IgG-HRP
Southern BiotechSouthern Biotech:
cat#SB-4030–05;
RRID:AB_2687483
1:5000; for western blot;
secondary Ab
AntibodyGoat anti-rabbit,
IgG-HRP
Cell SignalingCell Signaling:
cat#7074; RRID:AB_2099233
1:2000; for western blot;
secondary Ab
AntibodyGoat anti-mouse;
IgG1-HRP
Southern BiotechSouthern BioTech:
cat#1070–05;
RRID:AB_2650509
1:5000; for western blot;
secondary Ab
Isotype controlFITC-mouse IgG1,
κ isotype control
BD BiosciencesBD Biosciences:
cat#551954;
RRID:AB_394297
4 uL used for 1 × 10^6 cells;
for flow cytometry
AntibodyFITC-mouse
anti-Human CD95
BD BiosciencesBD Biosciences:
cat#556640;
RRID:AB_396506
4 uL used for 1 × 10^6 cells;
for flow cytometry
Recombinant protein
reagent
sCD95L (S2)PMID: 14504390NASoluble form of human CD95L
(amino acids 137–281);
recombinant protein
Recombinant protein
reagent
LzCD95LPMID: 14504390NALeucine zipper tagged CD95L;
recombinant protein
Chemical compoundpropidium iodideSigma-AldrichSigma-Aldrich:
cat#P4864
Used for subG1 flow cytometry analysis
Chemical compoundpuromycinSigma-AldrichSigma-Aldrich:
cat#P9620
Used for selection of cells expressing
puromycin resistance cassettes
Chemical compoundG418AffymetrixAffymetrix:
cat#11379
Used for selection of cells
expressing G418 resistance cassette
Recombinant DNA
reagent
venus-CD95L
sensor (plasmid)
this paperNAModified CD510B-1 lentiviral vector
(PMID: 25366259) was used as backbone;
vector expresses a venus-human
CD95L conjugate mRNA that can be
used to monitor RNAi activity of
si/shRNAs targeting CD95L using
venus fluorescence.
Recombinant DNA
reagent
venus-CD95
sensor (plasmid)
this paperNAModified CD510B-1 lentiviral vector
(PMID: 25366259) was used as backbone;
vector expresses a venus-human CD95
conjugate mRNA that can be used
to monitor RNAi activity of
si/shRNAs targeting CD95
using venus fluorescence.
Recombinant DNA
reagent
pLenti-GIII-CMV-
RFP-2A-Puro
vector; pLenti
ABM IncNApLenti control empty lentiviral vector;
carries an RFP-2a-puromycin
resistance cassette
Recombinant DNA
reagent
pLNCX2ClontechClontech:
cat#631503
pLNCX2 control empty
retroviral vector;
carries a neomycin
resistance cassette
Recombinant DNA
reagent
pTIPPMID: 24656822NALentivirus used for doxycycline-
induced expression of shRNAs;
contains puromycin resistance
cassette; modified from the
original backbone which
contained a GFP cassette instead
of a puromycin cassette
(PMID: 17311008); original
backbone from the Rossi lab.
Recombinant DNA
reagent
pLenti-CD95L-WTthis paperNApLenti-GIII-CMV-RFP-2A-Puro vector
that expresses human wild type
CD95L cDNA (NM_000639.2);
used to express wt human
CD95L upon infection
with lentiviral particles
Recombinant DNA
reagent
pLenti-CD95L-
L1MUT
this paperNApLenti-GIII-CMV-RFP-2A-Puro vector
that expresses human CD95L
cDNA (NM_000639.2) with 8
silentmutations overlapping
the shL1 target site
(GCATCATCTTTGGAGAAGCAA
- > GCCTCGTCCCTAGAAAAACAG); used
to express shL1-resistant human
CD95L upon infection
with lentiviral particles
Recombinant DNA
reagent
pLenti-CD95L-
L3MUT
this paperNApLenti-GIII-CMV-RFP-2A-Puro vector
that expresses human CD95L
cDNA (NM_000639.2) with 8 silent
mutations overlapping the shL3
target site
(ACTGGGCTGTACTTTGTATAT
- > ACCGGATTATATTTCGTGTAC);
used to express shL3-resistant
human CD95L upon infection
with lentiviral particles
Recombinant DNA
reagent
pLNCX2-CD95-WTthis paperNApLNCX2 vector that expresses
human CD95 cDNA (BC012479.1);
used to express wild type CD95
upon infection with lentiviral particles
Recombinant DNA
reagent
pLNCX2-CD95-R6MUTthis paperNApLNCX2 vector that expresses mutant
human CD95 cDNA (BC012479.1) which
contains 8 silent mutations
overlapping the shR6 site
(GTGTCGCTGTAAACCAAACTT
- > ATGTCGCTGCAAGCCCAATTT);
used to express shR6-resistant
CD95 upon infection with
lentiviral particles
Transfected constructgRNA scaffoldPMID: 23287722IDT: synthesized
as gene block
455 nucleotide CRISPR/Cas9
gRNA scaffold synthesized as a
gene block; contains promoter,
gRNA scaffold, target sequence,
and termination sequence; scaffold
transcribes gRNAs that target Cas9
endonuclease to cut at target sites;
target sequences consist of 19
nucleotides that are complementary to
the target site of choice; co-transfected
with Cas9 to catalyze cleavage.
Transfected constructpMJ920 Cas9 plasmidAddgene;
PMID: 23386978
Addgene: cat#42234Plasmid that expresses a human
codon-optimized Cas9 tagged with
GFP and HA; used to express Cas9 for
CRISPR-mediated deletions.
Chemical compoundLipofectamine 2000ThermoFisher ScientificThermoFisher Scientific:
cat#11668019
Transfection reagent
Chemical compoundLipofectamine RNAiMAXThermoFisher ScientificThermoFisher Scientific:
cat#13778150
Transfection reagent; used for
transfection of small RNAs such as siRNAs
Commercial assay or kitStrataClone Blunt
PCR Cloning Kit
Agilent TechnologiesAgilent Technologies:
cat#240207
Used to blunt-end clone the gRNA
scaffolds into the pSC-B plasmid
Commercial assay or kitHigh-Capacity cDNA
reverse transcription kit
Applied Biosystems4368814
Array cards preloaded
with primers
384-well TLDA cardsApplied Biosystems43422489
Commercial assay kit Taqman Gene
expression master mix
ThermoFisher Scientific4369016
Sequence-based reagentshL3 flanking Fr primerIDTIDT: custom DNA oligoFr primer that flanks shL3 site;
used to detect 41 nt shL3 deletion;
5’-TCTGGAATGGGAAGACACCT-3’
Sequence-based reagentshL3 flanking Rev primerIDTIDT: custom DNA oligoRev primer that flanks shL3 site;
used to detect 41 nt shL3 deletion;
5’-CCTCCATCATCACCAGATCC-3’
Sequence-based reagentshL3 internal Rev primerIDTIDT: custom DNA oligoRev primer that overlaps with the shL3 site;
used to detect 41 nt shL3 deletion;
5’-ATATACAAAGTACAGCCCAGT-3’
Sequence-based reagentshR6 flanking Fr primerIDTIDT: custom DNA oligoFr primer that flanks shR6 site;
used to detect 227 nt shR6 deletion;
5’-GGTGTCATGCTGTGACTGTTG-3’
Sequence-based reagentshR6 flanking Rev primerIDTIDT: custom DNA oligoRev primer that flanks shR6 site;
used to detect 227 nt shR6 deletion;
5’-TTTAGCTTAAGTGGCCAGCAA-3’
Sequence-based reagentshR6 internal Rev primerIDTIDT: custom DNA oligoRev primer that overlaps with the shR6 site;
used to detect 227 nt shR6 deletion;
5’-AAGTTGGTTTACATCTGCAC-3’
Sequence-based reagentsiL3 flanking Fr primerIDTIDT: custom DNA oligoFr primer that flanks siL3 site;
used to detect 64 nt siL3 deletion;
5’-CTTGAGCAGTCAGCAACAGG-3’
Sequence-based reagentsiL3 flanking Rev primerIDTIDT: custom DNA oligoRev primer that flanks siL3 site;
used to detect 64 nt siL3 deletion;
5’-CAGAGGTTGGACAGGGAAGA-3’
Sequence-based reagentsiL3 internal Rev primerIDTIDT: custom DNA oligoRev primer that is internal to the siL3 site;
used to detect 64 nt siL3 deletion;
5’-ATATGGGTAATTGAAGGGCTG-3’.
Sequence-based reagentsiScrIDT; DharmaconDharmacon
#D-001810-02-05
sense: UGGUUUACAUGUUGUGUGA
Sequence-based reagentsiL1DharmaconL-011130-00-0005sense: UACCAGUGCUGAUCAUUUA
Sequence-based reagentsiL1IDTcustomer synthesissense: UACCAGUGCUGAUCAUUUA
Sequence-based reagentsiL2IDTcustomer synthesissense: CAACGUAUCUGAGCUCUCU
Sequence-based reagentsiL3IDTcustomer synthesissense: GCCCUUCAAUUACCCAUAU
Sequence-based reagentsiL3MUTIDTIDT #51-01-14-03sense: GGACUUCAACUAGACAUCU
Sequence-based reagentsiL4IDTcustomer synthesissense: GGAAAGUGGCCCAUUUAAC
Sequence-based reagentshL3 => siL3IDTcustomer synthesissense: GACUGGGCUGU
ACUUUGUAdTdA
antisense: UACAAAGUACA
GCCCAGUUdTdT
Sequence-based reagentshR6 => siR6IDTcustomer synthesissense: GGGUGCAGAU
GUAAACCAAAdCdT;
antisense: UUUGGUUUACA
UCUGCACUUdTdT
Sequence-based reagentDsi-13.2IDTcustomer synthesissense: AUCUU ACCAGUGC
UGAUCAUUUAdTdA
Sequence-based reagentDsi-13.3IDTcustomer synthesissense: AAAGUAUACUU
CCGGGGUCAAUCdTdT
Sequence-based reagentDsi-13.9IDTcustomer synthesissense: CUUCCGGGG
UCAAUCUUGCAACAdAdC
Sequence-based reagentDsi-13.xIDTcustomer synthesissense: CAGGACUGAGAAG
AAGUAAAACCdGdT
Sequence-based reagentDsiL3IDTcustomer synthesissense: CAGCCCUUCAAU
UACCCAUAUCCdCdC
Sequence-based reagentsiScr poolDharmaconD-001810–10
Sequence-based reagentsmartpool siRNA
targeting NUCKS1
DharmaconL-014208–02
Sequence-based reagentsmartpool siRNA
targeting CAPZA1
DharmaconL-012212–00
Sequence-based reagentsmartpool siRNA
targeting CCT3
DharmaconL-018339–00
Sequence-based reagentsmartpool siRNA
targeting FSTL1
DharmaconL-013615–00
Sequence-based reagentsmartpool siRNA
targeting FUBP1
DharmaconL-011548–00
Sequence-based reagentsmartpool siRNA
targeting GNB1
DharmaconL-017242–00
Sequence-based reagentsmartpool siRNA
targeting NAA50
DharmaconL-014597–01
Sequence-based reagentsmartpool siRNA
targeting PRELID3B
DharmaconL-020893–01
Sequence-based reagentsmartpool siRNA
targeting SNRPE
DharmaconL-019719–02
Sequence-based reagentsmartpool siRNA
targeting TFRC
DharmaconL-003941–00
Sequence-based reagentsmartpool siRNA
targeting HIST1H1C
DharmaconL-006630–00
Sequence based reagent
(human)
GAPDH primerThermofisher ScientificHs00266705_g1
Sequence based reagent
(human)
CD95 primerThermofisher Scientificcustom probeFr primer: GGCTAACCCC
ACTCTATGAATCAAT
Rev primer: GGCCTGCCT
GTTCAGTAACT
Probe: CCTT
TTGCTGAAATATC
Sequence based reagent
(human)
CD95 primer
(Figure 5—figure supplement 3)
Thermofisher ScientificHs00163653_m1
Sequence based reagent
(human)
CD95L primersThermofisher ScientificHs00181226_g1;
Hs00181225_m1
Sequence based reagent
(human)
shL3 target
site in CD95L
Thermofisher Scientificcustom probeFr primer: GGTGGCC
TTGTGATCAATGAAA
Rev primer: GCAAGA
TTGACCCCGGAAGTATA
Probe: CTG
GGCTGTACTTTGTATATT
Sequence based reagent
(human)
downstream of shL3 siteThermofisher Scientificcustom probeFr primer: CCCC
AGGATCTGGTGATGATG
Rev primer: ACTG
CCCCCAGGTAGCT
Probe: CCCAC
ATCTGCCCAGTAGT
Sequence based reagent
(human)
GAPDH primer
(TLDA card)
Thermofisher ScientificHs99999905_m1
Sequence based reagent
(human)
ATP13A3 primer
(TLDA card)
Thermofisher ScientificHs00225950_m1
Sequence based reagent
(human)
CAPZA1 primer
(TLDA card)
Thermofisher ScientificHs00855355_g1
Sequence based reagent
(human)
CCT3 primer
(TLDA card)
Thermofisher ScientificHs00195623_m1
Sequence based reagent
(human)
FSTL1 primer
(TLDA card)
Thermofisher ScientificHs00907496_m1
Sequence based reagent
(human)
FUPB1 primer (
TLDA card)
Thermofisher ScientificHs00900762_m1
Sequence based reagent
(human)
GNB1 primer
(TLDA card)
Thermofisher ScientificHs00929799_m1
Sequence based reagent
(human)
HIST1H1C
primer (TLDA card)
Thermofisher ScientificHs00271185_s1
Sequence based reagent
(human)
NAA50 primer
(TLDA card)
Thermofisher ScientificHs00363889_m1
Sequence based reagent
(human)
NUCKS1 primer
(TLDA card)
Thermofisher ScientificHs01068059_g1
Sequence based reagent
(human)
PRELID3B primer
(TLDA card)
Thermofisher ScientificHs00429845_m1
Sequence based reagent
(human)
SNRPE primer
(TLDA card)
Thermofisher ScientificHs01635040_s1
Sequence based reagent
(human)
TFRC primer
(TLDA card)
Thermofisher ScientificHs00951083_m1
SoftwareStata 14StataRRID:SCR_012763
SoftwareRstudio (R3.3.1)RstudioRRID:SCR_000432
sequence based reagentshScrSigmaSHC002VNon-targeting shRNA
control transduction particles
sequence based reagent
(human)
shL1SigmaTRCN0000058998GCATCATCTTTGGAGAAGCAA
sequence based reagent
(human)
shL2SigmaTRCN0000058999CCCATTTAACAGGCAAGTCCA
sequence based reagent
(human)
shL3SigmaTRCN0000059000ACTGGGCTGTACTTTGTATAT
sequence based reagent
(human)
shL4SigmaTRCN0000059001GCAGTGTTCAATCTTACCAGT
sequence based reagent
(human)
shL5SigmaTRCN0000059002CTGTGTCTCCTTGTGATGTTT
sequence based reagent
(human)
shL6SigmaTRCN0000372231TGAGCTCTCTCTGGTCAATTT
sequence based reagent
(human)
shL2'SigmaTRCN0000372232TAGCTCCTCAACTCACCTAAT
sequence based reagent
(human)
shL5'SigmaTRCN0000372175GACTAGAGGCTTGCATAATAA
sequence based reagent
(human)
shR2SigmaTRCN0000218492CTATCATCCTCAAGGACATTA
sequence based reagent
(human)
shR5SigmaTRCN0000038695GTTGCTAGATTATCGTCCAAA
sequence based reagent
(human)
shR6SigmaTRCN0000038696GTGCAGATGTAAACCAAACTT
sequence based reagent
(human)
shR7SigmaTRCN0000038697CCTGAAACAGTGGCAATAAAT
sequence based reagent
(human)
shR8SigmaTRCN0000038698GCAAAGAGGAAGGATCCAGAT
sequence based reagent
(human)
shR27'SigmaTRCN0000265627TTTTACTGGGTACATTTTATC
sequence based reagent
(human)
shR7'SigmaTRCN0000255407TTAAATTATAATGTTTGACTA
sequence based reagent
(human)
shR8'SigmaTRCN0000255408ATATCTTTGAAAGTTTGTATT
sequence based reagent
(human)
shR6'SigmaTRCN0000255406CCCTTGTGTTTGGAATTATAA

Data availability

The following data sets were generated
  1. 1
    Cell Death-Inducing Sequences with RNAi Activity Embedded in the Genes of CD95 and CD95L
    1. Peter M
    (2017)
    Publicly available at NCBI Gene Expression Omnibus (accession no: GSE87817).
The following previously published data sets were used
  1. 1

Additional files

Supplementary file 1

Results of the RNA-Seq analysis used to generate Figure 4B.

https://doi.org/10.7554/eLife.29702.022
Supplementary file 2

Gene lists used in this work.

https://doi.org/10.7554/eLife.29702.023
Supplementary file 3

shRNA screen data.

https://doi.org/10.7554/eLife.29702.024
Supplementary file 4

The 6mer and 8mer toxicity index.

https://doi.org/10.7554/eLife.29702.025
Supplementary file 5

Correlation between experimental shRNA toxicity and TI.

https://doi.org/10.7554/eLife.29702.026
Transparent reporting form
https://doi.org/10.7554/eLife.29702.027

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