Figure 2

Analysis of TMEM45A gene expression level obtained with real time PCR and Affymetrix microarrays and effect of TMEM45A silencing on TMEM45A expression. (A) For Affymetrix microarray results: 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 before total RNA extraction. Results are expressed as mean ratios indicating a fold-increase or decrease in gene expression by comparison with the reference condition (normoxia) ± 1 SD (n = 3). For Real Time PCR results: after incubation, total RNA has been extracted and retro-transcribed in cDNA. A real time PCR has been performed with specific primers for TMEM45A and for RPL13A, a house-keeping gene. Results are expressed as mean of induction level by comparison with the reference condition (normoxia) ± 1 SD (n = 3). (B) Effect of TMEM45A silencing on TMEM45A expression. MDA-MB-231 cells were transfected 24 h with TMEM45A siRNA (siRNA) or RISC-free control siRNA (RF) (50 nM). Cells were then incubated in normoxia or hypoxia with paclitaxel (tax, 50 μM) for 16 hours. TMEM45A mRNA expression was quantified by real-time RT-PCR using RPL13A as the house-keeping gene. Results are expressed in induction level by comparison with the reference condition, normoxia. (C) Effect of HIF-1α silencing on TMEM45A expression. MDA-MB-231 cells were transfected 24 h with HIF1A siRNA (HIF) or RISC-free control siRNA (RF) (50 nM). Cells were then incubated in normoxia or hypoxia for 16 hours. TMEM45A mRNA expression was quantified by real-time RT-PCR using RPL13A as the house-keeping gene. Results are expressed in induction level by comparison with the reference condition, normoxia, as means ± 1 SD (n = 3).