Effect of TMEM45A silencing on the protective effect of hypoxia on paclitaxel-induced apoptosis. 8 h post transfection with TMEM45A siRNA (siRNA) or RISC-free control siRNA (RF) (50 nM, 24 h), MDA-MB-231 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without paclitaxel (tax, 50 μM) or epirubicin (epi, 10 μM) for 16 hours. (A) After transfection and incubation, caspase 3 activity was assayed by measuring free AFC released from the cleavage of the caspase 3 specific substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as mean ± 1 SD (n = 3). (B) After the incubation, DNA fragmentation was assayed using an ELISA for soluble nucleosomes (Cell Death Detection Elisa, Roche). Results are expressed as mean ± 1 SD (n = 3). Statistical analyses were determined independently for the 3 subgroups without siRNA, with anti-TMEM45A siRNA (siRNA) and with RISC-free control siRNA (RF) ; N.S. = non significantly different from control (N, N siRNA or N RF), ** = significantly different from control (p < 0.01), *** = significantly different from control (p < 0.001) ; N.S.
(1) = no significant difference between N epi and H epi, #, ### = significant difference between (1) N tax and H tax or (2) N epi and H epi (p < 0.05 ; <0.001), N.S. (2) = no significant difference between H tax and H tax RF, ·· = significant difference between N tax and N tax RF (p < 0.01), ··· = significant difference between (1) N tax and N tax siRNA or (2) H tax and H tax siRNA (p < 0.001).