(A) Representative immunofluorescence staining of MCF10A cells with cleaved caspase 3 (green) and DAPI (blue). Scale bar represents 10 μm. (B) The proportion of nonapoptotic MCF10A cells presenting …
Data for Figure 1B.
Data for Figure 1E.
Data for Figure 1G.
Data for Figure 1—figure supplement 3A.
Data for Figure 1—figure supplement 3B.
Data for Figure 1—figure supplement 5.
Data for Figure 1—figure supplement 6B.
Data for Figure 1—figure supplement 6C.
Data for Figure 1—figure supplement 7B.
Data for Figure 1—figure supplement 7C.
There was not a significant increase in annexin V-high cells in Casp3-GFP high cells.
(A) Average number of γH2AX foci per cell in Myc-transduced MCF10A cells with or without CASP3 gene ablation. (B) Average number of chromosomal aberrations per cell in Myc-transduced MCF10A cells …
Notice the heterogeneous nature of Myc expression.
Cells were synchronized and irradiated with 3Gys of X-rays. At different time points after radiation exposure, they were fixed and stained with fluorescence-labeled γH2AX antibody. The fraction of …
(A) Western blot confirmation of Myc over-expression and re-expression Casp3 in Casp3KO cells. (B) The effect of Casp3 re-expression on Myc-induced γH2AX foci in MCF10A-CASP3KO cells. (C) The effect …
(A) Western blot showing CASP3 gene knockout in immortalized human BJ1 fibroblasts. (B) Quantitative estimate of Myc induced γH2AX foci in control and CASP3 knockout BJ1 fibroblasts. (C) …
(A) Depicts colonies which grew in soft agar. (B) Average number of colonies in soft agar in control and Casp3-deficient cells with or without Myc expression. (C) Average number of colonies in soft …
Data for Figure 2B.
Data for Figure 2C.
Data for Figure 2D&E.
(A) Immunofluorescence staining of of MCF10A with antibodies to EndoG (green), mitochondria (Orange) and DAPI (blue). Scale bar represents 20 μm. (B) Fraction of MCF10A cells with activated EndoG …
Data for Figure 3B.
Data for Figure 3D.
Data for Figure 3E.
Data for Figure 3—figure supplement 1.
Data for Figure 3—figure supplement 4.
Error bar indicates SEM. * indicates p<0.01, Student’s t-test, n = 3. Each data point was derived from the average of three triplicate groups of 150 cells each.
Top panels. ROS levels in MCF10A vs CASP3KO cells (left) and MCF10A vs ENDOGKO cells (right). Lower panels, ROS levels in wild type (left), CASP3KO (middle), and ENDOGKO (right) MCF10A cells with or …
Cytoplasmic mtND5 of indicated cells were analyzed by use of quantitative RT-PCR. The levels of 18S rDNA were also determined to serve as genomic DNA control. Error bars represent standard error of …
(A) A diagram showing a re-engineered endoG with a nucleus localization signal (NLS) at its tagged N-terminal. (B) Western blot demonstrating exogenous myc and NLS-EndoG expression by use of an …
Data for Figure 4D.
Data for Figure 4E.
Data for Figure 4F.
Primary antibodies used in this study.
Target protein | Antibody source | Clone information |
---|---|---|
γH2AX (Ser139) | Upstate Biotechnology | JBW301, Mouse mAb |
Caspase-3 (full length) | Cell Signaling Technology | 8G10, Rabbit mAb |
Caspase-3 (cleaved,Asp175) | Cell Signaling Technology | 5A1E, Rabbit mAb |
EndoG | Chemicon | Rabbit polyclonal |
HA epitope | Novus Biologicals | Goat polyclonal |
β-Actin | Novus Biologicals | Mouse mAb |
c-Myc | Cell Signaling Technology | Rabbit mAb |
Mito Marker | Thermo Fisher Scientific | N/A |
Single guided RNA (sgRNA) sequences used in this study.
Gene | Accession | sgRNA oligo(5`−3`)* | Targeted exon |
---|---|---|---|
CASPASE3 | NC_000004 | CACCGcatacatggaagcgaatcaa AAACttgattcgcttccatgtatgC | Exon4 |
CASPASE3 | NC_000004 | CACCGggaagcgaatcaatggactc AAACgagtccattgattcgcttccC | Exon4 |
EndoG | NC_000009 | CACCGgggctgggtgcggtcgtcga AAACtcgacgaccgcacccagcccC | Exon1 |
EndoG | NC_000009 | CACCGcgacttccgcgaggacgact AAACagtcgtcctcgcggaagtcgC | Exon1 |
*Capital letters: enzyme overhangs; non-capital letters: sgRNA target guide sequence.
Mutations at target sequences in various CRISPR knockout MCF10A and BJ-1 hTERT cells.
5`……3` | Mutation | ||
---|---|---|---|
Casp3 KO | MCF10A | Clone1: AAAGATCATACATGGAAGCGAATCAATGGA - - - - - - - ATAT Casp3: AAAGAT ACTCTGGAATAT | 7 bp deletion |
Casp3 KO | BJ-1hTERT | Clone28: AAAGATCATACATGGAAGCGAATCAATG - - - deletion-------- Casp3: AAAGAT ACTCTGGAATAT | 193 bp deletion |
EndoG KO | MCF10A | Clone13: -------- deletion-------- EndoG: TGCCA GGCTTCGACCGCG | 169 bp deletion |
Note: Red: sgRNA sequence; Yellow: PAM sequence; Bold: insertion sequence; -: deletion sequence. In all cases, knockout clones that showed both clear absence of target protein expression and gene mutations were chosen. In addition, in most cases, only those clones with homozygous mutations (where both copies of the gene showed the same mutation) were chosen for convenience.