Fig. 3: HIF1α regulates RNA Pol I activity.
A–C Immunoblot analysis of HIF1α protein levels in SKBR3 (A), T47D (B), and SUM1315 (C) cells exposed to hypoxia (1% O2) for 0, 4, 8, and 24 h. D Immunoblot analysis of HIF1α stabilization in EMT6 cells exposed to hypoxia for 0, 4, 8, 16, and 24 h. E Schematic representation of HIF1α knockdown experimental design under short-term hypoxia. F, H Immunoblot analysis of HIF1α knockdown efficiency following siRNA transfection in SUM1315 (F) and SKBR3 (H) cells after 8-h hypoxia exposure. G, I Pol I activity and pre-rRNA synthesis measured by 5′ ETS qRT-PCR following HIF1α knockdown in SUM1315 (G) and SKBR3 (I) cells under hypoxia. Relative expression levels were normalized to β-Actin. Data are presented as mean ± SEM of three biological replicates. Statistical significance was assessed using a two-sided Student’s t-test. J Immunoblot analysis of HIF1α protein levels in SUM1315 and SKBR3 cells transfected with HIF1α overexpression (OE) or empty vector (CTRL EV). K Pol I activity quantified by 5′ ETS qRT-PCR in HIF1α OE and CTRL EV cells. Data are presented as mean ± SEM of three biological replicates. Two-sided Student’s t-test was used. For all immunoblots, densitometric quantifications were first normalized to β-Actin loading control and then further normalized to corresponding control conditions (either normoxia, empty vector, or non-target siRNA control), as appropriate. Immunoblotting was repeated in at least two independent biological experiments with comparable results; a representative blot is shown.
