Fig. 3: Effect of TMEM45A inactivation on the chemosensitivity to cisplatin in SQD9 and RCC4 cells.

SQD9 cells were transfected with siRNA control (siRF = risc free) or siRNA targeting the mRNA of TMEM45A (siTMEM45A). SQD9 cells were incubated with or without 100 mM of cisplatin in normoxic (N) and hypoxic (H) conditions for 24 h. a The apoptosis was studied by western blot analyses of PARP and caspase 3 cleavage. Actin was used as the loading control. Results are expressed as mean ± SD (n = 3). b–d SQD9 cells were transduced with lentiviral particles expressing shRNA control (shCTL) or shRNA targeting the mRNA of TMEM45A (shRNA 22). SQD9 cells were incubated with or without 100 mM of cisplatin in normoxic (N) and hypoxic (H) conditions for 24 h. b After transduction and incubation, the cleavage of PARP and caspase 3 was assessed by western blot. Actin was used as the loading control. Results are expressed as mean ± SD (n = 3). c After the incubation, caspase 3 and 7 activity was assessed by measuring free AFC released from the cleavage of caspase 3 and 7 specific substrate Ac-DEVD-AFC. Results are expressed as mean ± SD (n = 5). d The cytotoxicity of cisplatin was assessed by LDH release. Results are representative of three biological replicates. e–g RCC4 cells were transduced with lentiviral particles expressing shRNA control (shCTL) or shRNA targeting the mRNA of TMEM45A (shRNA 22). RCC4 cells were incubated with or without 20 mM of cisplatin in normoxic (N) or hypoxic (H) conditions for 48 h. e After transduction and incubation, the cleavage of PARP and caspase 3 was assessed by western blot. Actin was used as the loading control. Results are expressed as mean ± SD (n = 3). f The cytotoxicity of cisplatin was assessed by LDH release. Results are expressed as mean ± SD (n = 3). g After the incubation, the number of viable cells was determined using MTT assay. The signal intensity of the control cells was used for normalization a. Results re expressed as mean ± SD (n = 3). N normoxia, NC normoxia + cisplatin, H hypoxia, HC hypoxia + cisplatin. *p < 0.05, **p < 0.01.