Figure 2.

ACF differentially modulates PDK1 and VEGF transcription in melanoma cells. (A) Western blot experiments for the effect of ACF on HIF-1α expression in the absence or the presence of MG132. SK-MEL-28 cells were treated for 12 h with ACF alone or simultaneously co-treated with ACF and MG132. High and low exposure make reference to the exposure time during development of the chemiluminescent signal. HIF-1α expression in the absence of MG132 (w/o MG132) was quantified on high exposure membranes, while that of HIF-1α expression in the presence of MG132 was evaluated on low exposure membranes to avoid overexposure (histograms). In both cases, relative HIF-1α content in the ACF-treated samples was compared with untreated controls in membranes developed under the respective exposure conditions (* p < 0.05). (B) Dose-dependent effect of ACF (24 h) on HIF-1α and IOD quantification (histogram; * p < 0.05). (C) Semiquantitative determination of PDK1 and VEGF mRNAs in SK-MEL-28 cells. Relative levels of mRNA (with respect to β-actin) in ACF-treated samples (24 h) were compared to the expression levels in untreated controls (* p < 0.05). (D) Effective silencing of HIF-1α was determined by both western blot experiments and mRNA determinations in SK-MEL-28 melanoma cells. To visualize the silencing of HIF-1α in western blots, the protein was stabilized with CoCl₂ (200 µM). Images were obtained under high and low exposure as indicated in Figure 2A and quantified in indicated membranes (histograms) in the absence (w/o CoCl₂) or the presence of CoCl₂ (* p < 0.05). HIF-1α-mRNA in silenced samples were compared with their respective ACF treatments (24 h) in siControls (siCN) samples and differences were found statistically significant (* p < 0.05). (E) Semiquantitative determination of PDK1 and VEGF mRNAs in HIF-1α-silenced SK-MEL-28 cells. HIF-1α-mRNA in silenced samples were compared with their respective treatments in siCN samples (* p < 0.05). Although VEGF-mRNA decrease in siHIF-1α cells was found to be statistically significant with respect to siCNs, silencing of HIF-1α did not completely abolish ACF-dependent induction of VEGF. Cells were treated with ACF for 24 h. (F) Effective silencing of ATF4 with two different siRNAs (left panel) did not influence VEGF-mRNA expression in SK-MEL-28 cells. * p < 0.05 and ** not significant when compared with siATF4 samples with their respective treatments in siCN samples. When indicated, cells were treated with ACF for 24 h.