Fig. 3
HIF-1α stabilizes C/EBP-β LAP mRNA, increasing ABCB1 and ABCC1, and decreasing ABCA1. NCI-H2228 cells were cultured as indicated in Fig. 1a. a Immunoblot of C/EBP-β LAP in whole cell extracts. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. b Normoxic NCI-H2228 cells, wild-type (wt), overexpressing C/EBP-β LAP (LAP+) or overexpressing C/EBP-β LIP (LIP+), were lysed and immunoprecipitated with an anti-C/EBP-β antibody, recognizing both LAP and LIP isoforms, then immunoblotted for HIF-1α. An aliquot of the lysates before the immunoprecipitation was directly probed with the anti-HIF-1α antibody, to check that the protein was equally present. The image is representative of 1 out of 3 experiments. No Ab: wild-type cells subjected to immunoprecipitation without the anti-C/EBP-β antibody, as negative control. c RNA-IP with an anti-HIF-1α antibody, followed by RT-PCR amplification (in technical triplicates) with primers for C/EBP-β LAP (upper panel) or LIP (lower panel) isoforms. Data are means±SD (n = 4 biological replicates). **p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells. d ChIP of C/EBP-β on ABCB1, ABCC1 and ABCA1 promoter, in technical triplicates. Data are means±SD (n = 4 biological replicates). *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells. e-h Hypoxic (H) NCI-H228 cells were treated with a non-targeting sequence (scr) or with two shRNAs (sh1, sh2) targeting HIF-1α. e HIF-1α mRNA level, measured by RT-PCR in technical triplicates. Data are means±SD (n = 4 biological replicates). ***p < 0.001: H, H/N, H/N/H versus N cells. f Immunoblot of HIF-1α. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. g RNA-IP with an anti-HIF-1α antibody, followed by RT-PCR amplification (in technical triplicates) with primers for C/EBP-β LAP. Data are means±SD (n = 4 biological replicates). ***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001: sh1/sh2-cells versus scr-cells. h ChIP of C/EBP-β on ABCB1, ABCC1 and ABCA1 promoter, in technical triplicates. Data are means±SD (n = 4 biological replicates). *p < 0.05,**p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells, °°°p < 0.001: sh1/sh2-cells versus scr-cells