bcl2 flag  (Sino Biological)

Journal: PLOS Biology

Article Title: Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

doi: 10.1371/journal.pbio.3003034

Figure Lengend Snippet: ( A ) Representative western blots of indicated proteins from SKBR3 cells in fed (F) or in amino acid starvation (S) for various time periods, in the absence or presence of 50 nM Bafilomycin A1 (BafA1) in the final 2 h. ( B ) Quantification of LC3B-based autophagy flux in starved cells relative to the fed control, shown in (A). All with BafA1. ( C ) Representative western blots of indicated proteins from SKBR3 cells transfected with scramble, CASP3 and/or CASP7 siRNAs (48 h) and incubated in fed conditions or starved for 8 h with BafA1 (50 nM) for the final 2 h. ( D ) Quantification of LC3BII-based autophagy flux in starved cells relative to the starved scramble-siRNA control, shown in (C). ( E ) Representative western blots of indicated proteins from CASP3, CASP7 single (KO), or double knockout (DKO) SKBR3 cells in fed or starved (8 h) conditions, with BafA1 (50 nM) in the final 2 h. ( F ) Quantification of LC3BII-based autophagy flux in starved cells relative to the parental control, shown in (E). ( G ) Representative western blots of indicated proteins from SKBR3 parental, DKO or CASP3 + 7-WT re-expression in DKO cells in fed or starved (8 h) conditions, with BafA1 (50 nM) in the final 2 h. ( H ) Quantification of LC3BII-based autophagy flux in starved cells relative to starved parental cells, shown in (G). ( I ) Representative immunofluorescence images of SKBR3 parental, DKO or CASP3 + 7-WT re-expression in DKO cells treated with 0.25 µM DALGreen in fed or starved (8 h) conditions. BafA1 (50 nM for 8 h) in both fed and starved conditions serve as controls. Scale bars, 20 µm. ( J ) Quantification of DALGreen immunofluorescence in starved cells shown in (I). Graph shows number of punctae per cell. n = 2, each with 10 random images covering total of 500–700 cells. In graphs, data are shown as mean ± SEM. n = at least 3 independent experiments except in (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS, not significant. In B, D, F, and H, one-way ANOVA with Dunnett’s post-test. In J, one-way ANOVA with Tukey’s post-test. See also . The numerical data presented in this figure can be found in .

Article Snippet: For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection.

Techniques: Western Blot, Control, Transfection, Incubation, Double Knockout, Expressing, Immunofluorescence

Journal: PLOS Biology

Article Title: Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

doi: 10.1371/journal.pbio.3003034

Figure Lengend Snippet: ( A ) Representative western blots of indicated proteins from SKBR3 cells treated with MG132 at increasing dosage for 24 h, with BafA1 (50 nM) in the final 2 h. ( B ) Quantification of LC3B-based autophagy flux in MG132-treated cells relative to untreated (0 μM MG132; DMSO vehicle only; NT) SKBR3 shown in (A). The levels of LC3BII were normalized to loading control and shown relative to the untreated control (NT). ( C ) Graph showing proteasome activity in response to increasing concentrations of MG132 as depicted by chymotrypsin, caspase and trypsin-like activities. ( D ) Representative western blots of indicated proteins from SKBR3 cells transfected with scramble, CASP3 and/or CASP7 siRNAs (48 h) and treated with vehicle DMSO only or MG132 (0.5 μM) for 24 h, with BafA1 (50 nM) in the final 2 h. ( E ) Quantification of LC3B-based autophagy flux in MG132-treated cells relative to the MG132-treated scramble-siRNA control, shown in (D). ( F ) Representative western blots of indicated proteins from CASP3, CASP7 single knockout, or DKO SKBR3 cells treated with vehicle DMSO or MG132 (0.5 μM) for 24 h, with BafA1 (50 nM) in the final 2 h. ( G ) Quantification of LC3B-based autophagy flux in MG132-treated cells relative to the MG132-treated parental control show in (F). ( H ) Representative western blots of indicated proteins from SKBR3 parental, DKO or CASP3 + 7-WT reintroduced into DKO cells treated with vehicle DMSO or MG132 (0.5 or 1.0 μM) for 24 h with BafA1 (50 nM) in the final 2 h. ( I ) Quantification of LC3BII-based autophagy flux in MG132-treated cells relative to DMSO-treated parental control, shown in (H). ( J ) Representative images of crystal violet assay plates of SKBR3 parental and CASP3 + 7 DKO cells treated with indicated concentrations of MG132 for 24 h and continued to grow in drug free media for another 3 days. ( K ) Quantification of cell viability shown in (J). Percentage of stained (viable) cells at each concentration was normalized to respective untreated cells. In graphs, data are shown as mean ± SEM. n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS, not significant. In B, C, E, and G, one-way ANOVA with Dunnett’s post-test and I with Tukey’s post- test. K with two-way ANOVA with Sidak’s post-test. See also . The numerical data presented in this figure can be found in .

Article Snippet: For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection.

Techniques: Western Blot, Control, Activity Assay, Transfection, Knock-Out, Crystal Violet Assay, Staining, Concentration Assay

Journal: PLOS Biology

Article Title: Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

doi: 10.1371/journal.pbio.3003034

Figure Lengend Snippet: ( A – D ) Representative western blots showing changes in CASP7 and CASP3 in SKBR3 or MDA-MB-231 cells in amino acid starvation (A, B) or treated with MG132 (C, D). Non-canonical CASP7 bands (p29/p30) at 30 kDa are indicated by blue arrows. A non-canonical CASP3 band (p27) at 27 kDa is also detected in some conditions. ( E ) Representative western blots comparing CASP7, CASP3, and PARP1 processing patterns in SKBR3 cells following 4-h incubation in fed conditions (F), non-lethal stress (starvation) or lethal stress (apoptosis induction by staurosporine; STS, 1 or 2 µM). ( F ) Representative western blots comparing CASP7 and PARP1 processing in SKBR3 parental cells stably transfected with vector control or BCL2 expression construct, following 4-h incubation in fed conditions (F), non-lethal stress (starvation) or lethal stress (apoptosis induction by staurosporine; STS, 1 or 2 µM). ( G ) Representative western blots showing the effect of rapamycin on the formation of CASP7-p29/p30 bands in SKBR3 cells. Cells in fed, starved (4 h), and/or treated with 10 nM rapamycin (Rap; 24 h). Blot was immunolabeled with mTOR activity reporters P70S60K and p70S6K-PO4. ( H ) Representative western blot showing CASP7 immunolabeling in tissues from a female CD-1 mouse. Ponceau S labeling serves as the loading control. n = 3 mice. ( I ) Representative western blot showing CASP7 immunolabeling in human breast cancer PDX specimens from indicated subtypes. Starved SKBR3 serves as the positive control for p29/p30 fragments. ( J ) Representative western blot showing CASP7 immunolabeling in parental MDA-MB-231 cells and derivative epirubicin-resistant (R8a, R8b, R3-1, R3-2) and 5Fu-resistant (R6-1, R6-2) cells in fed conditions. In each, at least n = 2 independent experiments. See also .

Article Snippet: For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection.

Techniques: Western Blot, Incubation, Stable Transfection, Transfection, Plasmid Preparation, Control, Expressing, Construct, Immunolabeling, Activity Assay, Labeling, Positive Control

Journal: PLOS Biology

Article Title: Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

doi: 10.1371/journal.pbio.3003034

Figure Lengend Snippet: ( A ) Representative western blot showing CASP7 immunolabeling in CASP3 + 7 DKO SKBR3 cells stably transfected with CASP7-WT or CASP7-delta-Pro (CASP7-Δpro) constructs in fed or starvation conditions. Arrowhead indicates CASP7-Δpro band and arrow indicates CASP7-p29/p30 bands. n = 2 independent experiments. ( B ) Schematic of subunits, canonical cleavage sites and non-canonical cleavage sites in CASP7 wild-type or in various CASP7 fragments used or observed in this study. Black scissors denote the canonical cleavage sites and the order of processing in apoptosis. Purple scissors, bars, and black arrows denote the putative calpain cleavage sites (non-canonical). ( C ) The sequences on the left show the putative calpain cleavage sites in CASP7-WT and the amino acid replacements made in the CASP7 construct with putative calpain-cleavage sites mutated (CASP7-CCM). Representative western blot of CASP7 from CASP3 + 7 DKO SKBR3 cells transfected with CASP7-WT or CASP7-CCM constructs grown in fed (F) or starved (S) conditions. n = 2 independent experiments. ( D , E ) Representative western blots of indicated proteins from SKBR3 cells transfected with scramble, calpain 1 or calpain 2 siRNAs (48 h) and then continued to be cultured in fed (F) conditions or starved (S) for 8 h (D) or treated with MG132 for 24 h (E), with BafA1(50 nM) in the final 2 h. ( F – I ) Quantification of LC3B-based autophagy flux and CASP7 bands shown in (D) and (E) relative to scramble-siRNA control. In graphs, data are shown as mean ± SEM. n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS, not significant. See also . In F–I, one-way ANOVA with Dunnett’s post-test. The numerical data presented in this figure can be found in .

Article Snippet: For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection.

Techniques: Western Blot, Immunolabeling, Stable Transfection, Transfection, Construct, Cell Culture, Control

Journal: PLOS Biology

Article Title: Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

doi: 10.1371/journal.pbio.3003034

Figure Lengend Snippet: ( A , B ) Representative western blots of indicated proteins from parental and CASP3 + 7 DKO SKBR3 cells cultured in fed conditions (F) or starved (S) for 8 h (A) or treated with MG132 (0.5 μM) for 24 h (B). ( C ) Quantification of cleaved-PARP1 shown in (A). Values were normalized to parental fed conditions. (D) Quantification of cleaved-PARP1 shown in (B). Values were normalized to parental NT. NT = No Treatment (vehicle DMSO). ( E , F ) Representative western blots of PAR immunolabeling from parental and CASP3 + 7 DKO SKBR3 (E) or MDA-MB-231(F) cells treated with vehicle (DMSO) or MG132 (0.5 μM) for 24 h. ( G , - H ) Quantification of PAR levels shown in (E) and (F). Values were normalized to the parental NT condition. (I) RT-qPCR analyses of LC3B in SKBR3 parental, CASP3 + 7 DKO or CASP3 + 7-WT re-expression in CASP3 + 7 DKO SKBR3 cells. Cells were treated with vehicle (DMSO) or 0.5 μM of MG132 for 24 h. ( J ) Representative western blots of indicated proteins from SKBR3 parental and CASP3 + 7 DKO cells transfected with scramble or CASP2 siRNAs (72 h). ( K ) Quantification of cleaved-PARP1 levels (shown in J) relative to scramble-siRNA control. ( L ) Quantification of CASP2 levels relative to scramble-siRNA-treated parental control. ( M ) Quantification of PAR levels shown in (J) relative to scramble-siRNA-treated parental control. ( N ) Representative western blots of indicated proteins from SKBR3 parental and CASP3 + 7 DKO cells transfected with scramble, CASP2 or CASP8 siRNAs (42 h) and treated with vehicle DMSO or MG132 (0.5 μM) for 24 h, with BafA1(50 nM) in the final 2 h. ( O ) Quantification of LC3B-based autophagy flux in MG132-treated cells, shown in (N). In graphs, data are shown as mean ± SEM. n = 3–6 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS, not significant. In C, D, G, and H, one-way ANOVA with Tukey’s post-test. In I, K–M, and O, one-way ANOVA with Dunnett’s post-test. See also . The numerical data presented in this figure can be found in .

Article Snippet: For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection.

Techniques: Western Blot, Cell Culture, Immunolabeling, Quantitative RT-PCR, Expressing, Transfection, Control

Journal: PLOS Biology

Article Title: Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

doi: 10.1371/journal.pbio.3003034

Figure Lengend Snippet: ( A ) Representative western blots of indicated proteins from SKBR3 parental and CASP3 + 7 DKO cells treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Ponceau S staining was used as the loading control. ( B ) Quantification of γ-H2AX/total H2AX shown in (A). ( C ) Representative images from alkaline comet assay from SKBR3 parental and CASP3 + 7 DKO cells treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Cells treated with H 2 O 2 (100 μM for 4 h) serve as the positive control. ( D ) Quantification of tail moments of comets shown in (C). Tukey’s box-and whisker plots are based on tail moments determined by CometScore from 200 cells in each of two independent assays. ( E ) Representative western blots of indicated proteins from SKBR3 parental, CASP3 + 7 DKO or single KO cells treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Ponceau S staining was used as the loading control. ( F ) Quantification of γ-H2AX/total H2AX shown in (E). ( G ) Representative western blots of indicated proteins from CASP3 + 7 DKO SKBR3 cells stably transfected with indicated CASP7 constructs treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Ponceau S staining was used as the loading control. ( H ) Quantification of γ-H2AX/total H2AX shown in (G). ( I ) Representative images from alkaline comet assay from CASP3 + 7 DKO SKBR3 cells stably transfected with indicated CASP7 constructs and treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. Cells treated with H 2 O 2 (100 μM for 4 h) serve as the positive control. ( J ) Quantification of tail moments of comets shown in (I). Tukey’s box-and-whisker plots are based on tail moments obtained from 200 cells in each of two independent assays. ( K ) Representative images of crystal violet assay plate. CASP7-CCM, CASP7-p30, and CASP7-p29 SKBR3 cells treated with indicated concentrations of proteasome inhibitor MG132 for 24 h and continued to grow in drug free media for another 3 days. ( L ) Quantification of cell viability data presented in (K). The percentage of stained (viable) cells at each concentration was normalized to respective untreated cells. ( M ) Representative western blots of indicated proteins from CASP3 + 7 DKO SKBR3 cells re-expressing vector, CASP7-WT (catalytically active) or CASP7-inactive (catalytically inactive) constructs, treated with vehicle DMSO or MG132 (0.5 µM) for 24 h. H3 serves as the loading control. ( N ) Quantification of γ-H2AX/total H2AX shown in (M). ( O ) Representative western blots of LC3B-based autophagy flux in CASP3 + 7 DKO SKBR3 cells re-expressing the indicated CASP7 constructs, treated with vehicle DMSO or MG132 (0.5 µM) for 24 h, with BafA1(50 nM) in the final 2 h. ( P ) Quantification of autophagy flux in MG132 treated cells relative to MG132 treated CCM expressing cells, shown in (O). In graphs, data are shown as mean ± SEM. n = 3 independent experiments except for comet assays n = 2. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS, not significant. In B, F, L, N, and P, one-way ANOVA with Dunnett’s post-test. In D and H, one-way ANOVA with Tukey’s post- test. In J, two-way ANOVA with Sidak’s post-test. See also . The numerical data presented in this figure can be found in .

Article Snippet: For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection.

Techniques: Western Blot, Staining, Control, Alkaline Single Cell Gel Electrophoresis, Positive Control, Whisker Assay, Stable Transfection, Transfection, Construct, Crystal Violet Assay, Concentration Assay, Expressing, Plasmid Preparation

Journal: PLOS Biology

Article Title: Caspase 3 and caspase 7 promote cytoprotective autophagy and the DNA damage response during non-lethal stress conditions in human breast cancer cells

doi: 10.1371/journal.pbio.3003034

Figure Lengend Snippet: ( A ) Representative images of crystal violet assays. SUM149PT, MDA-MB-231, SKBR3, or JIMT-1 cells transfected with scramble-siRNA or CASP3 and CASP7 siRNAs (10 nM) for 2 days and then cultured in siRNA-free media for another 3 days. ( B ) Quantification of cell viability from data represented in (A). Percentage of stained (viable) cells at each concentration was normalized to respective untreated cells. ( C ) Representative western blots showing CASP3 and CASP7 levels following siRNA treatment conditions used in crystal violet assays shown in ( A ). Immunolabelling of cleaved-PARP1 is also shown. n = 2 independent experiments. (D) Representative western blots of indicated proteins from SUM149PT cells transfected with scramble, CASP3 and/or CASP7 siRNAs (48 h) and treated with vehicle DMSO or MG132 (0.5 μM) for 24 h, with BafA1 (50 nM) in the final 2 h. ( E ) Quantification of LC3B-based autophagy flux in proteasome inhibitor (MG132) treated cells shown in (D). The levels of LC3BII in MG132 treated cells were normalized to loading control and shown relative to the MG132 treated scramble-siRNA control. ( F , G ) Representative images of crystal violet assay plates (F) and quantification of percentage of cell viability (G). Indicated siRNA transfected cells treated with indicated concentrations of Olaparib for 24 h and continued to be cultured in drug free media for another 3 days. Graph (G) show the percentage of stained (viable) cells at each concentration normalized to scramble-siRNA-treated and Olaparib untreated cells. ( H ) Dot plot showing DepMap cancer cell lines with bottom 10% of CASP3 + 7 protein expression ( n = 16) and top 10% CASP3 + 7 protein expression ( n = 10). Normalized CASP3/7 protein expression (scaled and mean centered) were extracted from Nusinow and colleagues using GRETTA. ( I ) Ranked CASP3 + 7 genetic interaction scores generated using GRETTA. The cell lines at the bottom 10% of CASP3 and CASP7 protein expression are compared against cells lines at the top 10%. Rank is based on lethal GI scores. The red points indicate true candidate CASP3 + 7 lethal genetic interactions (GIs). The top most lethal genetic interactors are labeled. In graphs, unless otherwise noted, data are shown as mean ± SEM. n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS, not significant. In B and E, one-way ANOVA with Sidak’s and Dunnett’s post-test, respectively. In G, two-way ANOVA with Sidak’s post-test. In I, Mann–Whitney U -test p -value < 0.05. See also . The numerical data presented in this figure can be found in . The code related to and is publicly available in a GitHub repository ( https://github.com/MarraLab/Caspase_GRETTA_analysis ) and archived on Zenodo ( https://doi.org/10.5281/zenodo.14722298 ).

Article Snippet: For CASP3 and BCL2, a clone from Sino Biological (HG10050-CH) or Addgene (N-FLAG-BCL2, 18003) was obtained, respectively, and used directly for transfection.

Techniques: Transfection, Cell Culture, Staining, Concentration Assay, Western Blot, Control, Crystal Violet Assay, Expressing, Generated, Labeling, MANN-WHITNEY