Figure 3

Analysis of HIF-1α protein expression, HIF-1 DNA binding activity, HIF-1 transcriptional activity and gene expression level of HIF-1 inducible gene LDHA. 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) HIF-1α was detected in total cell extracts by western blotting, using specific antibody. β-actin was used to assess the total amount of proteins loaded on the gel. (B) After the incubation, nuclear extracts were performed and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-HIF-1α antibody. Results are expressed in absorbance, as mean ± 1 SD (n = 3). (C) Cells were co-transfected with the pUAS-tk-Luc reporter plasmid encoding the firefly luciferase and the pCMVβ normalisation plasmid before incubated. Results are expressed as mean of the ratio between firefly luciferase activity and the β-galactosidase activity ± 1 SD (n = 3). (D) After incubation, total RNA has been extracted and retro-transcribed in cDNA. A real time PCR has been performed with specific primers for LDHA and for RPL13A, a house-keeping gene. Results are expressed in induction level by comparison with the reference condition, normoxia. N.S. = non significantly different from control, *** = significantly different from control (p < 0.001); N.S. = 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 < 0.001).