(A) Strategy used to activate Notch in a tightly regulated fashion. (B) Notch-induced suppression of SC2 cell growth in standard cultures is abrogated by DN-MAML, a specific inhibitor of canonical …
(A, B) Effects of GSI and sham washout on the growth of IC8 and SCCT2 cells transduced with either empty vector (pBABE) or ΔEGF-L1596H. Cell numbers at various times post-GSI washout (DMSO) or sham …
(A) Western blot showing ICN1 levels 4 hr post-GSI washout in IC8-ΔEGF-L1596H clones. (B) Effects of Notch activation on growth of IC8-ΔEGF-L1596H clones. Cell numbers at various times post-GSI …
(A) Volcano plots showing changes in RNA transcript read counts induced by Notch activation in SC2 cells for 4, 24, and 72 hr as compared to control cells treated with sham GSI washout. RNA-seq for …
Duplicate cultures of IC8 cells were treated with GSI or vehicle (DMSO) for 24 hr and transcript abundance was assessed by RNA-seq in two biological replicates. A volcano plot shows no …
The lists of overlapping genes are provided in Supplementary file 6.
(A) Number and overlap of RBPJ and MAML1 binding sites determined by ChIP-Seq of chromatin prepared 4 hr after Notch activation. (B) Genomic distribution of RBPJ/MAML1 co-binding sites 4 hr after …
(A) Chromatin landscapes around IER5 in IC8-ΔEGF-L1596H cells. ChIP-Seq signals for RBPJ, MAML1, and H3K27ac for cells maintained in and 4 hr after GSI washout (WO) are shown. (B, C) Activities of a …
(A, B) TERT-immortalized NOK1 keratinocytes transduced with either empty MigRI retrovirus or MigRI encoding dominant negative MAML1 (DN-MAML) were grown in low Ca2+ medium (Day 0) or were shifted to …
(A) Western blots showing IER5 and ICN1 protein levels in SC2 cells, a single-cell clone derived from SC2-IER5 knockout cells (I5KO), I5KO cells transduced with empty virus (I5/con), and pooled I5KO …
(A) Polypeptides identified by mass spectroscopy in immunoprecipitates prepared from I5 cells expressing tandem-tagged IER5. (B) Cartoon showing the structure of tandem-tagged IER5 polypeptides. FH, …
Gene | Variant | Variant allele frequency |
---|---|---|
IC8 cell | ||
CASP8 | c.971T > C(p.M324T) | 66% of 411 reads |
FBXW7 | c.1633T > C(p.Y545H) | 33% of 195 reads |
KMT2D | c.7412G > A(p.R2471Q) | 42% of 255 reads |
MGA | c.5599G > A(p.V1867I) | 39% of 710 reads |
MTOR | c.4828G > A(p.E1610K) | 56% of 280 reads |
NOTCH1 | c.5059C > T (p.Q1687*) | 100% of 412 reads |
PAXIP1 | c.2023C > T(p.H675Y) | 17% of 384 reads |
PMS1 | c.566_567delTCinsAT(p.V189D) | 36% of 108 reads |
RIF1 | c.658G > A(p.E220K) | 62% of 251 reads |
ROS1 | c.1144T > G(p.Y382D) | 87% of 169 reads |
ROS1 | c.1164+2_1164+8delTTAGTCC () | 19% of 191 reads |
SDHA | c.1627T > C(p.Y543H) | 56% of 668 reads |
SF3B1 | c.2549T > C(p.I850T) | 31% of 246 reads |
TERT | CC242-243TT promoter mutation | 50% of 26 reads |
TP53 | c.451C > T(p.P151S) | 100% of 366 reads |
WHSC1 | c.2185C > T(p.R729C) | 66% of 410 reads |
WWTR1 | c.551T > G (p.V184G) | 64% of 256 reads |
ZNF217 | c.2590C > T(p.L864F) | 39% of 835 reads |
ZNF217 | c.1162delC(p.H388Tfs*77) | 55% of 822 reads |
SCCT2 Cell | ||
ALK | c.2854G > A (p.G952R) | 50% of 441 reads |
ASXL1 | c.3959C > T (p.A1320V) | 31% of 930 reads |
BRD3 | c.533C > T (p.S178F) | 49% of 281 reads |
BRD4 | c.3915_3917dupTGC (p.A1306dup) | 45% of 170 reads |
CDH4 | c.1801C > T (p.L601F) | 30% of 447 reads |
CDKN2A | c.*151–1G > A () | 100% of 172 reads |
CDKN2A | c.212A > T (p.N71I) | 100% of 184 reads |
CREBBP | c.5842C > T (p.P1948S) | 74% of 77 reads |
CREBBP | c.2116G > A (p.G706R) | 45% of 172 reads |
DDB1 | c.327+6G > A () | 47% of 451 reads |
DICER1 | c.775C > T (p.P259S) | 42% of 301 reads |
DOCK8 | c.185T > A (p.V62E) | 100% of 597 reads |
EGFR | c.1955G > A (p.G652E) | 48% of 518 reads |
EGFR | c.298C > T (p.P100S) | 49% of 595 reads |
ERCC2 | c.886A > T (p.S296C) | 48% of 165 reads |
ERCC5 | c.264+1G > A () | 50% of 442 reads |
ETV4 | c.1298C > G (p.P433R) | 45% of 302 reads |
FANCF | c.494C > T (p.T165I) | 50% of 644 reads |
FANCL | c.155+1G > A () | 51% of 220 reads |
FAT1 | c.9076–1G > A () | 49% of 367 reads |
FH | c.681G > T (p.Q227H) | 4% of 756 reads |
FLT4 | c.2224G > A (p.D742N) | 49% of 346 reads |
GALNT12 | c.1035+5G > A () | 52% of 523 reads |
GLI2 | c.1859C > A (p.T620K) | 45% of 351 reads |
HNF1A | c.1640C > T (p.T547I) | 54% of 392 reads |
JAZF1 | c.477C > T (p.I159I) | 47% of 606 reads |
JAZF1 | c.328C > T (p.P110S) | 44% of 211 reads |
KMT2D | c.10355+1G > A () | 49% of 622 reads |
LIG4 | c.1271_1275delAAAGA (p.K424Rfs*20) | 40% of 659 reads |
MAP2K1 | c.568+1G > A () | 53% of 239 reads |
MED12 | c.2080G > A (p.E694K) | 100% of 269 reads |
MYB | c.1461+5G > A () | 41% of 430 reads |
NF1 | c.2608G > A (p.V870I) | 50% of 615 reads |
NF2 | c.813T > G (p.F271L) | 48% of 168 reads |
NOTCH1 | c.1226G > T (p.C409F) | 44% of 519 reads |
NOTCH1 | c.1406A > G (p.D469G) | 50% of 912 reads |
NOTCH1 | c.1245G > T (p.E415D) | 42% of 495 reads |
NOTCH2 | c.5252G > A (p.G1751D) | 44% of 459 reads |
NOTCH2 | c.1298G > A (p.C433Y) | 50% of 484 reads |
NOTCH2 | c.1108+1G > A () | 53% of 305 reads |
NSD1 | c.7669G > A (p.G2557R) | 49% of 743 reads |
PDGFRB | c.2586+2T > A () | 43% of 380 reads |
PHOX2B | c.181A > T (p.T61S) | 52% of 222 reads |
POLQ | c.6565G > A (p.A2189T) | 27% of 462 reads |
POLQ | c.1634G > A (p.S545N) | 33% of 667 reads |
PPARG | c.819+6T > C () | 100% of 134 reads |
PRKDC | c.6436G > A (p.A2146T) | 42% of 471 reads |
RAD51C | c.996G > A (p.Q332Q) | 45% of 302 reads |
RHEB | c.443C > T (p.S148F) | 46% of 120 reads |
ROS1 | c.6871C > T (p.P2291S) | 45% of 605 reads |
ROS1 | c.3342A > T (p.Q1114H) | 48% of 274 reads |
ROS1 | c.137A > T (p.D46V) | 42% of 215 reads |
RPTOR | c.2992G > A (p.V998I) | 48% of 352 reads |
RUNX1T1 | c.1039G > A (p.D347N) | 45% of 715 reads |
SDHA | c.1151C > T (p.S384L) | 53% of 446 reads |
SLC34A2 | c.1700T > A (p.I567N) | 50% of 460 reads |
SMARCA4 | c.3947T > G (p.F1316C) | 55% of 431 reads |
SMARCE1 | c.395C > T (p.A132V) | 51% of 587 reads |
STAT3 | c.1852G > A (p.G618S) | 47% of 527 reads |
TDG | c.166+4G > A () | 48% of 329 reads |
TP53 | c.375+1G > T | 47% of 173 reads |
TP53 | c.832_833delCCinsTT (p.P278F) | 46% of 418 reads |
UIMC1 | c.971T > C (p.V324A) | 48% of 745 reads |
XPC | c.571C > T (p.R191W) | 100% of 219 reads |
*Based on analysis of 16,131,317 unique, high-quality sequencing reads (mean, 406 reads per targeted exon, with 98% of exons having more than 30 reads).
†Based on analysis of 20,972,158 unique, high-quality sequencing reads (mean, 413 reads per targeted exon, with 99% of exons having more than 30 reads).
Chromosome | Type | Genes affected |
---|---|---|
IC8 cell line | ||
1q | Gain | MCL1, GBA, RIT1, NTRK1, DDR2, PVRL4, SDHC, CDC73, MDM4, PIK3C2B, UBE2T, PTPN14, H3F3A, EGLN1, AKT3, EXO1, FH |
2 | Loss | XPO1, FANCL, REL, MSH6, EPCAM, MSH2, SOS1, ALK, BRE, DNMT3A, GEN1, MYCN, TMEM127, GLI2, ERCC3, CXCR4, RIF1, ACVR1, ABCB11, NFE2L2, PMS1, CASP8, SF3B1, CTLA4, ERBB4, IDH1, BARD1, XRCC5, DIS3L2 |
3p | Loss | MITF, BAP1, PBRM1, COL7A1, RHOA, SETD2, CTNNB1, MLH1, MYD88, XPC, PPARG, RAF1, FANCD2, OGG1, VHL |
3q | Gain | NFKBIZ, CBLB, POLQ, GATA2, MBD4, TOPBP1, FOXL2, ATR, MECOM, PRKCI, TERC, PIK3CA, SOX2, ETV5, BCL6 |
4 | Loss | PHOX2B, RHOH, SLC34A2, FGFR3, WHSC1, KDR, KIT, PDGFRA, FAM175A, HELQ, TET2, FBXW7, NEIL3, FAT1 |
5 | Gain | RICTOR, IL7R, SDHA, TERT, MAP3K1, PIK3R1, XRCC4, RASA1, APC, RAD50, CTNNA1, PDGFRB, ITK, NPM1, TLX3, FGFR4, NSD1, UIMC1, FLT4 |
6 | Gain | CCND3, NFKBIE, POLH, VEGFA, CDKN1A, PIM1, RNF8, FANCE, DAXX, HFE, HIST1H3B, HIST1H3C, ID4, PRDM1, ROS1, RSPO3, MYB, TNFAIP3, ESR1, ARID1B, PARK2, QKI |
7 | Gain | EGFR, IKZF1, JAZF1, ETV1, PMS2, RAC1, CARD11, SBDS, CDK6, SLC25A13, CUX1, RINT1, MET, POT1, SMO, BRAF, PRSS1, EZH2, RHEB, XRCC2, PAXIP1 |
8p | Loss | KAT6A, POLB, FGFR1, WHSC1L1, NRG1, WRN, NKX3-1, PTK2B, GATA4, NEIL2 |
8q11.21-q21.11 | Loss | PRKDC, MYBL1, TCEB1 |
8q21.3-q24.3 | Gain | NBN, RUNX1T1, RAD54B, RSPO2, EXT1, RAD21, MYC, RECQL4 |
9p13.2-p21.3 | Loss | PAX5, FANCG, RMRP, CDKN2A, CDKN2B, MTAP |
9p24.1-p24.3 | Gain | CD274, JAK2, PDCD1LG2, DOCK8 |
11p11.2-p13 | Gain | EXT2, LMO2 |
13q33.1 | Loss | ERCC5 |
15q | Gain | FAN1, GREM1, BUB1B, MGA, RAD51, TP53BP1, B2M, USP8, MAP2K1, PML, NEIL1, FAH, NTRK3, BLM, FANCI, IDH2, IGF1R |
16p13.3 | Loss | CREBBP, SLX4 |
19 | Loss | BABAM1, CRTC1, JAK3, KLF2, MEF2B, BRD4, NOTCH3, CALR, KEAP1, SMARCA4, ELANE, GNA11, MAP2K2, STK11, TCF3, CCNE1, C19orf40, CEBPA, AKT2, AXL, CIC, XRCC1, ARHGAP35, ERCC1, ERCC2, BCL2L12, PNKP, POLD1, PPP2R1A |
20 | Gain | MCM8, ASXL1, BCL2L1, MAFB, AURKA, ZNF217, GNAS, CDH4 |
SCCT2 Cell Line | ||
1q32.1 | Loss | UBE2T |
1q42.12-q42.2 | Gain | H3F3A, EGLN1 |
1q43 | Loss | AKT3, EXO1 |
1q43 | Gain | FH |
3 p Arm level | Loss | MITF, BAP1, PBRM1, COL7A1, RHOA, SETD2, CTNNB1, MLH1, MYD88, XPC, PPARG, RAF1, FANCD2, OGG1, VHL |
3q Arm level | Gain | NFKBIZ, CBLB, POLQ, GATA2, MBD4, TOPBP1, FOXL2, ATR, MECOM, PRKCI, TERC, PIK3CA, SOX2, ETV5, BCL6 |
8q Arm level | Gain | PRKDC, MYBL1, TCEB1, NBN, RUNX1T1, RAD54B, RSPO2, EXT1, RAD21, MYC, RECQL4 |
9q Arm level | Gain | GNAQ, NTRK2, FANCC, PTCH1, GALNT12, XPA, KLF4, TAL2, ENG, ABL1, TSC1, BRD3, NOTCH1 |
18q11.2 | Gain | GATA6, RBBP8 |
18q11.2-q21.33 | Gain | SS18, SETBP1, SMAD2, SMAD4, BCL2 |
20 | Gain | MCM8, ASXL1, BCL2L1, MAFB, AURKA, ZNF217, GNAS, CDH4 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Rabbit monoclonal anti-MAML1 | Cell Signaling Technology | Cat. #: 12166 | ChIP, 1 ml per1 × 106 cells |
Antibody | Rabbit monoclonal anti-RBPJ | Cell Signaling Technology | Cat. #: 5313 | ChIP, 2.5 ml per1 × 106 cells |
Antibody | Rabbit polyclonal anti-histone H3 acetyl K27 | Abcam | Cat. #: ab4729 | ChIP, 9 ml per1 × 106 cells |
Antibody | Mouse monoclonal anti-involucrin | Sigma | Cat. #: I9018 | IF, 1:500; IHC, 1:10,000 |
Antibody | Rabbit polyclonal anti-plakophilin-1 | Sigma | Cat. #: HPA027221 | IF, 1:300; IHC, 1:500 |
Antibody | Rabbit monoclonal anti-activated NOTCH1 (ICN1) | Cell Signaling Technology | Cat. #: 4147 | IHC, 1:50; WB, 1:1000 |
Antibody | Rabbit monoclonal anti-keratin-1 | Abcam | Cat. #: ab185628 | IHC, 1:1000 |
Antibody | Rabbit monoclonal anti-Ki-67 | Biocare | Cat. #: CRM325 | IHC, 1:100 |
Antibody | Mouse monoclonal anti-B55a | Cell Signaling Technology | Cat. #: 5689 | WB, 1:1000 |
Antibody | Rabbit monoclonal anti-NOTCH2 | Cell Signaling Technology | Cat. #: 5732 | WB, 1:1000 |
Antibody | Rabbit monoclonal anti-NOTCH3 | Cell Signaling Technology | Cat. #: 5276 | WB, 1:1000 |
Antibody | Rabbit monoclonal anti-GADD45A | Cell Signaling Technology | Cat. #: 4632 | WB, 1:1000 |
Antibody | Rabbit monoclonal anti-ID3 | Cell Signaling Technology | Cat. #: 9837 | WB, 1:1000 |
Antibody | Horse polyclonal anti-mouse Ig linked to HRP | Cell Signaling Technology | Cat. #: 7076 | WB, 1:1,000-1:20,000 |
Antibody | Goat polyclonal anti-rabbit Ig linked to HRP | Cell Signaling Technology | Cat. #: 7074 | WB, 1:1000 |
Antibody | Mouse monoclonal anti-actin | Sigma | Cat. #: A1978 | WB, 1:10,000 |
Antibody | Mouse monoclonal anti-FLAG | Sigma | Cat. #: F3165 | WB, 1:1000 |
Antibody | Rabbit polyclonal anti-IER5 | Sigma | Cat. #: HPA029894 | WB, 1:1000 |
Antibody | Mouse monoclonal anti-filaggrin | Santa Cruz Biotechnology | Cat. #: sc-66192 | IHC, 1:100 |
Antibody | Mouse monoclonal anti-p63 | Biocare Medical | Cat. #: CM163A | IHC, 1:250 |
Antibody | Rabbit polyclonal anti-loricrin | BioLegend | Cat. #: 905103 | IHC, 1:800 |
Antibody | Mouse monoclonal anti-BCL6 | Cell Marque Tissue Diagnostics | Cat. #: 227 M-95 | IHC, 1:500 |
Antibody | Rabbit monoclonal anti-keratin5 | Cell Signaling Technology | Cat. #: 71536 | IHC, 1:2000 |
Antibody | Chicken polyclonal anti-keratin14 | BioLegend | Cat. #: 906004 | IHC, 1:800 |
Antibody | Chicken polyclonal anti-SUMO | Lifesensors | Cat. #: AB7002 | WB, 1:2000 |
Antibody | Sheep polyclonal anti-rabbit Ig linked to Dynabeads | ThermoFisher Scientific | Cat. #: 11203D | ChIP, 100 μl beads per 20 × 106 cells |
Antibody | Mouse monoclonal anti-FLAG epitope linked to magnetic beads | Sigma | Cat #: M8823 | Tandem purification,40 μl to 1 ml beads |
Cell line (Homo sapiens) | IC8 | 10.1038/s41467-018-06027-1 | Dr. Andrew South (Thomas Jefferson University) | |
Cell line (H. sapiens) | SCCT2 | 10.1038/s41467-018-06027-1 | Dr. Andrew South (Thomas Jefferson University) | |
Cell line (H. sapiens) | NOK1 | Piboonniyom et al., 2003; 63:476–83 | Dr. Karl Munger (Tufts University) | |
Commercial assay or kit | CellTiter Blue | Promega | Cat. #: G8080 | |
Commercial assay or kit | ChIP Assay Kit | Millipore | Cat. #: 17–295 | |
Commercial assay or kit | Next Ultra II DNA Library Prep Kit | New England BioLabs | Cat. #: E7645 | |
Commercial assay or kit | Next Ultra II RNA Library Prep Kit | New England BioLabs | Cat. #: E7775 | |
Commercial assay or kit | QuickChange II Kit | Agilent Technologies | Cat. #: 200523 | |
Commercial assay or kit | Dual Luciferase Kit | Promega | Cat. #: E1910 | |
Chemical compound, drug | Compound E | Tocris | Cat. #: CAS 209986-17-4 | |
Recombinant DNA reagent | pL-CRISPR. SFFV.GFP | Addgene | Cat. #: #57827 | |
Recombinant DNA reagent | pL-CRISPR.SFFV.tRFP | Addgene | Cat. #: #57826 | |
Recombinant DNA reagent | lentiCRISPRv2 neo | Addgene | Cat. #: 98292 | |
Recombinant DNA reagent | lentiCRISPRv2 hygro | Addgene | Cat. #: 98291 | |
Recombinant DNA reagent | pVL1392 | Expression Systems | Cat. #: 91–012 |
Differentially expressed genes after 4 hr of Notch activation, SC2 cells.
Differentially expressed genes after 24 hr of Notch activation, SC2 cells.
Differentially expressed genes after 72 hr of Notch activation, SC2 cells.
Unclustered GO annotations of Notch-sensitive genes, SC2 cells, at 72 hr of Notch activation.
Clustered GO annotations of Notch-sensitive genes, SC2 cells, at 72 hr of Notch activation.
Comparison of Notch-responsive genes in SC2 cells, MB157 triple-negative breast cancer cells, REC1 mantle cell lymphoma cells, and DND41 T-cell acute lymphoblastic leukemia cells.
Unclustered GO annotations of IER5-dependent Notch-sensitive genes, SC2 cells.
Clustered GO annotations of IER5-dependent Notch-sensitive genes, SC2 cells.
IER5-interacting proteins identified by tandem affinity purification.