Figure 2
Identification of Rab5GEFs induced by HIF-1α. (A) A549 cells were incubated in normoxia (N) and hypoxia (H) for 24 h and total RNA was extracted, for subsequent RT-qPCR analysis of RIN1, RIN2, RIN3, RABEX5 and ALS2. VEGFA was measured as positive control of hypoxia-induced transcription. Relative abundance of all screened Rab5GEFs and VEGFA was normalized to actin, by using the ΔΔCT method, as described in the materials and methods. Graphs show data obtained by averaging 4 independent experiments (mean ± SEM; *p < 0.05). (B) RCC and RCC+VHL cells were grown for 24 h, total RNA was extracted and relative levels of RIN2, RIN3, ALS2 and VEGFA were quantified by RT-qPCR, as described in (A). Data are shown as the average of 4 independent experiments (mean ± SEM; *p < 0.05). (C) Upper panel, putative hypoxia response elements (HRE, gray boxes) are shown within the proximal gene promoter of ALS2 (the transcription start site (+ 1) is shown for reference). Lower panel, bioinformatics analysis showing histone acetylation (H3K27ac) marks and DNAse I hypersensitivity within the promoter region of the human genetic sequence of ALS2 (hg38, chromosome 2). H3K27Ac enrichment was obtained from the Chip-Seq database (UCSC Genome Browser Output, hg18) and is shown in mountain/valley color code. Color superposition represents the analysis of at least 7 different cell lines. DNAse I hypersensitivity data was obtained from 95 cell models and shown with respect to intensity and extension. Black rectangle, 95/95 cases; gray rectangle, 52/95 cases. (D) Binding of HIF-1α to the proximal promoter of ALS2 was assessed by Chromatin immunoprecipitation (ChIP) assay. HIF-1α was immunoprecipitated with a specific, ChIP-grade antibody in A549 cells exposed to 24 h of normoxia or hypoxia. HSP90 was immunoprecipitated as control (IgG). The enrichment of ALS2 (putative HRE − 792/-788), RhoA (positive control, HRE -1263/-1259, see text for details) and actin was evaluated by qPCR, using specific primers, as described in the materials and methods. The graph shows the quantification of 3 independent experiments (mean ± SEM; *p < 0.05). (E) A549 cells were incubated in normoxia (N) or hypoxia (H) for 24 h and whole cell lysates were prepared for Western blot analysis of HIF-1α, ALS2 and actin. Representative Western blot images are shown, and relative levels of HIF-1α and ALS2 were quantified by scanning densitometry and normalized to actin. Graphs represent the average from 3 independent experiments (mean ± SEM; **p < 0.01). (F) RCC4 and RCC4+VHL cells were grown for 24 h and whole cell lysates were prepared for Western blot analysis of HIF-1α, ALS2 and actin. HIF-1α and ALS2 relative levels were quantified as described in (E). Graphs represent the average from 3 independent experiments (mean ± SEM; *p < 0.05, ***p < 0.001). (G) A549 cells were transfected with either siRNA control or mix of 3 different siRNA sequences targeting endogenous HIF-1α for 16 h. Subsequently, cells were incubated in normoxia (N) or hypoxia (H) for an additional 24 h, and whole cell lysates were prepared for Western blot analysis of HIF-1α, ALS2 and actin. Representative Western blot images are shown, and relative levels of HIF-1α and ALS2 were quantified by scanning densitometry and normalized to actin. Graphs represent the average from 3 independent experiments (mean ± SEM; *p < 0.05, ***p < 0.001).
