Figure 4.

CLIC5A protects cells from hydrogen peroxide-induced apoptosis. (A) pLENTI control and CLIC5A stably infected Reh cells were challenged for 20h with 35μM hydrogen peroxide (PRXD), 250nM camptothecin (CPT), or 150nM doxorubicin (DOXO) and analyzed by flow cytometry with Alexa Fluor 488-coupled Annexin V and propidium iodide (PI) staining. (B) A representative example of staining following PRXD treatment is presented with PI on the x axis and Alexa Fluor 488 on the y axis. (C) Total apoptosis is calculated for each sample. CLIC5A overexpressing cells displayed reduced apoptosis compared to control only when treated with PRXD. Each experiment was performed 3 times in triplicates (n=9). (D) IM9 cells transduced with CLIC5A or pLENTI empty vector control were challenged with 50μM PRXD and processed similarly to assess apoptosis. (E) A representative example of staining and (F) total apoptosis is shown for the IM9 cell line. Again, CLIC5A overexpression leads to reduced apoptosis. Two independent experiments carried out in triplicate and an additional single test were performed (n=7). Error bars in (C) and (F) represent the standard deviation. Statistical significance is calculated by two-tailed Mann-Whitney U test. For (A) and (D) adjustments of brightness and contrast were applied to the whole image. DMSO: dimethyl sulfoxide.