Fig. 4 | Nature Communications

Fig. 4

From: A RASSF1A-HIF1α loop drives Warburg effect in cancer and pulmonary hypertension

Fig. 4

RASSF1A regulates HIF1α protein stability and transcriptional activity. a, b Human PASMCs were transfected with a RASSF1 siRNA (si-RASSF1) and control siRNA (si-Control) or b RASSF1A-FLAG plasmid or EV. 24 h after transfection, cells were exposed to hypoxia or normoxia for further 24 h. Cell lysates were subjected to a, b, upper western blotting for indicated proteins, followed by a, b, lower densitometric quantification of relative RASSF1A expression. c A luciferase reporter under control of multiple HIF1α binding sites was transfected into HeLa cells with c, upper si-RASSF1 or c, lower RASSF1A-FLAG. 6 h after transfection, cells were exposed to hypoxia for 24 h. Cells were lysed and luciferase activity was measured and normalized to co-transfected Renilla luciferase internal control. RLU relative luciferase units. d From hypoxic HEK293 cells transfected with RASSF1A-FLAG or EV, chromatin was precipitated with anti-HIF1α (HIF1A) antibody or rabbit IgG (IgG) and was analyzed by real time PCR with primers spanning the hypoxia-response element (HRE) regions of mentioned genes (PDK1, LDHA, HK2). e Human PASMCs were transfected with EV, RASSF1A-FLAG, RASSF1A-FLAG (S203A) or RASSF1A-FLAG (S203D). 24 h later, cells were exposed to hypoxia or normoxia for further 24 h. Cell lysates were subjected to e, upper western blotting for indicated proteins, followed by e, lower densitometric quantification of relative HIF1A expression. f A luciferase reporter under control of multiple HIF1α binding sites (HRE) was transfected into HeLa cells with EV, RASSF1A-FLAG, RASSF1A-FLAG (S203A) or RASSF1A-FLAG (S203D). 6 h after transfection, cells were exposed to hypoxia for 24 h. Cells were lysed and luciferase activity was measured and normalized to co-transfected Renilla luciferase internal control. *P < 0.05, **P < 0.01, ***P < 0.001 compared to a, c si-Control (hypoxia) or b, c EV (normoxia) or e, f EV (hypoxia), §P< 0.05, §§P < 0.01, §§§P < 0.001 compared to bd EV (Hypoxia) or e, f RASSF1A-FLAG (S203A), one-way ANOVA followed by SNK multiple comparison test. Data represent mean ± s.e.m. n = 3 independent experiments from 3 biological replicates

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