Fig. 4

Depletion of UNC5B or SIRT4 suppresses CDDP-mediated apoptotic cell death. a Knockdown of UNC5B or SIRT4. T24 (left panels) and 5637 (right panels) cells were transfected with the non-targeting scramble siRNA or with siRNA against UNC5B or SIRT4. Forty-eight hours after transfection, cell lysates were extracted and analyzed for UNC5B (upper panels) and SIRT4 (lower panels) by Western blotting. β-actin was used as a loading control. b Silencing of UNC5B or SIRT4 decreases CDDP sensitivity of bladder cancer cells. T24 (left panel) and 5637 (right panel) cells were transfected as in (a). Twenty-four hours after transfection, cells were treated with 10 μM of CDDP or left untreated. Twenty-four hours after treatment, cells were subjected to FACS analysis. (c) Knockdown of UNC5B or SIRT4 attenuates CDDP-induced proteolytic cleavage of PARP. T24 cells were transfected and treated as in (b). Twenty-four hours after CDDP treatment, cell lysates were prepared and subjected to Western blotting with anti-PARP antibody. β-actin was used as a loading control. d miR-424-caused suppression of CDDP-mediated apoptotic cell death is partially restored by ectopic expression of UNC5B or SIRT4. T24 (left panel) and 5637 (right panel) cells were transfected with the indicated combinations of the expression vectors. Twenty-four hours after transfection, cells were exposed to 10 μM of CDDP. Twenty-four hours after treatment, cells were processed for FACS analysis. The data are presented as the mean ± S.D. of triplicate experiments. *P < 0.05, **P < 0.01