Fig 2: S70pBcl2 protects cancer cells from oxidative damage-induced cell death. (A) Fold change in cell viability of Jurkat cells treated with increasing doses of DDC (μM) 3 separate times at intervals of 24 h for 72 h. Cell viability measured by MTT assay. N = 3. (B, C) Fold change in cell viability of Jurkat cells pre-treated with NAC (10 mM) for 2 h and treated with DDC (400 μM) three separate times at intervals of 24 h for 72 h. Cell viability measured by MTT or CellTiterGlo assay. N = 3. (D) Fold change in cell viability of Jurkat cells pre-treated with NAC (10 mM) for 2 h and treated with either etoposide (2.5 μM) or doxorubicin (1 μM) for 24 h. Cell viability measured by MTT assay. N = 3. One-way ANOVA and Sidak's multiple comparison were performed for A–D. (E, F) Fold change in cell viability of pcDNA3.1, WT BCL2, S70A or S70E transiently-transfected Jurkat cells treated with either DDC (400 μM) three separate times at intervals of 24 h for 72 h or etoposide (2.5 μM) for 24 h. Cell viability measured by MTT assay. N = 4. Bar graphs showing mean and SD. Two-way ANOVA and Sidak's multiple comparison were performed for E and F. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Fig 3: S70pBcl2 prevents chemotherapeutic-induced ROS and subsequent DNA Damage. (A, B) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, β-actin of Jurkat cells following pre-treatment with NAC (10 mM) for 2 h and treatment with etoposide (2.5 μM) or doxorubicin (1 μM) for 18 h. (C, D) Fold change in DCFDA or Mitosox staining levels in Jurkat cells following pre-treatment with NAC (10 mM) for 2 h and treatment with etoposide (2.5μM) or doxorubicin (1 μM) for 18 h. N = 3. (E, F) Fold change in DCFDA or Mitosox staining levels in pcDNA3.1, WT BCL2, S70A or S70E transiently-transfected Jurkat cells, treated with either DMSO or etoposide (2.5 μM) for 18 h. N = 4. (G) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, β-actin of pcDNA3.1, WT BCL2, S70A or S70E transiently-transfected Jurkat cells, treated with either DMSO or etoposide (2.5 μM) for 18 h. (H) Confocal microscopy images showing γH2AX, 8-OHdG and DAPI (Nuclear) staining of pcDNA3.1, WT BCL2, S70A or S70E transiently-transfected Jurkat cells, DMSO (merged) or treated (individual and merged) with etoposide (2.5 μM) for 18 h. (I) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, β-actin of Jurkat cells following treatment with increasing doses of doxorubicin, cisplatin, etoposide or 5-fluorouracil (5-FU) for 4 h. (J) Diagram showing the inverse relationship between redox-sensitive S70pBcl2 and ROS-induced DNA damage. Bar graphs showing mean and SD. One-way ANOVA and Sidak's multiple comparison were performed unless otherwise specified. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Fig 4: S70pBcl2 modulates mitochondrial COX activity, respiration rate and ROS production. (A) Fold change in COX activity of Jurkat cells following treatment with DDC (400 μM) for 4 h. N = 3. (B) Fold change in COX activity of Jurkat cells following treatment with two independent siSOD1 (150 nM) for 48 h. N = 3. (C) Fold change in COX activity of Jurkat cells transiently transfected with 2 μg pcDNA3.1, WT BCL2, S70A or S70E for 30 h. N = 3. (D, E) One representative line graph showing the kinetics of OCR in Jurkat cells following treatment with DDC (400 μM) or increasing doses of etoposide for 4 h. Fold change of OCR taken at point 3 ∼20 min. * indicates oligomycin, FCCP or rotenone and antimycin-A injections in a timely order. N = 4. (F) One representative line graph showing the kinetic of OCR in Jurkat cells transiently transfected with 2 μg pcDNA3.1, WT BCL2, S70A or S70E for 30 h. Fold change of OCR taken at point 3 ∼20 min. * indicates oligomycin, FCCP or rotenone and antimycin-A injections in a timely order. N = 6. (G, H) Fold change of Mitosox staining levels in Jurkat cells following treatment of siSOD1 for 48 h or transiently transfected with 2 μg pcDNA3.1, WT BCL2, S70A or S70E for 30 h. N = 3 and 4 respectively. Bar graphs showing mean and SD. Two-tailed t-test was performed for A, D and G. One-way ANOVA and Sidak's multiple comparison were performed unless otherwise specified. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Fig 5: S70pBcl2 suppresses the increase in ROS and ROS-induced DNA damage. (A) Fold change in intracellular O2.− levels in Jurkat cells following treatment with increasing doses of DDC (μM) for 4 h. N = 4. (B) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, β-actin of Jurkat cells following treatment with increasing doses of DDC after 4 h. Fold changes (FC) showing densitometry values of S345pChk1/Chk1 and γH2AX/β-actin below their respective targets (similarly shown in subsequent figures in the article). (C) Fold change in intracellular O2.− levels in Jurkat cells following 2 independent siRNA treatments (150 nM) against SOD1 for 48 h. N = 4. (D) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, SOD1, β-actin of Jurkat cells following two independent siRNA treatments (150 nM) against SOD1 for 48 h. (E) Fold change in intracellular O2.− levels in Jurkat cells following pre-treatment with NAC (10 mM) for 2 h and treatment of DDC (400 μM) for 4 h. N = 4. (F) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, β-actin of Jurkat cells following pre-treatment with NAC (10mM) for 2 h and treatment of DDC (400 μM) for 4 h. (G) Western blot showing S345pChk1, γH2AX, Chk1, S70pBcl2, Bcl-2, β-actin of Jurkat cells following pre-treatment with FeTPPS (50 μM) for 2 h and treatment with DDC (400 μM) for 4 h. (H) Fold change in intracellular O2.− levels in Jurkat cells following a 4- and 8-h time-kinetic treatments with 400 μM DDC. N = 5. (I) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, β-actin of Jurkat cells following a 4- and 8-h time kinetic treatment with 400 μM DDC. (J) Fold change in intracellular O2.− levels in pcDNA3.1, wild-type (WT) BCL2, S70A or S70E transiently-transfected Jurkat cells, untreated or treated with DDC (400 μM) for 4 h. N = 5. (K) Western blot showing S345pChk1, γH2AX, Chk1, β-actin of pcDNA3.1, WT BCL2, S70A or S70E transiently-transfected Jurkat cells, untreated or treated with DDC (400 μM) for 4 h. Bar graphs showing mean and standard deviation (SD). One-way ANOVA and Sidak's multiple comparison were performed unless otherwise specified. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.