Figure 5. CSAG2 Promotes Cellular Resistance to Genotoxic Stress through Regulation of SIRT1.

• A, B
  Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) or H₂O₂ (B) for 24 h before cell viability was determined by alamarBlue (n = 6 biological replicates).
• C, D
  shCTRL, shCSAG2‐1, or shCSAG2‐2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) or H₂O₂ (D) for 24 h before cell viability was determined by alamarBlue (n = 6 biological replicates).
• E, F
  HCT116 cells were transiently transfected with the indicated shRNAs for 48 h before treatment with 1 μM doxorubicin/0.4 μM TSA for 6 h. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n = 3 biological replicates) of normalized cleaved PARP levels is shown (F).
• G
  H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 h. Cells were then treated with the indicated concentrations of H₂O₂ for 24 h before cell viability was determined by alamarBlue (n = 6 biological replicates).
• H
  Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 overexpressing H460 cells were treated with the indicated concentrations of H₂O₂ with or without 1 μM EX‐527 for 24 h before cell viability was determined by alamarBlue (n = 6 biological replicates).
• I, J
  Control or Myc‐CSAG2 expressing HCT116 p53^−/− cells were treated with the indicated concentrations of H₂O₂ (I) or doxorubicin (J) for 24 h before cell viability was determined by alamarBlue (n = 6 biological replicates).
• K, L
  shCTRL, shCSAG2‐1, or shCSAG2‐2 stable HCT116 p53^−/− cells were treated with the indicated concentrations of H₂O₂ (K) or doxorubicin (L) for 24 h before cell viability was determined by alamarBlue (n = 6 biological replicates).
• M, N
  Control or Myc‐CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H₂O₂ (M) or doxorubicin (N) for 24 h before cell viability was determined by alamarBlue (n = 6 biological replicates).

Data information: Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired, two‐tailed Student t‐test. Source data are available online for this figure.