Figure 2.

Transcriptional regulation of HIF‐1α through PI3K mediated RON Kinase pathway: (A) Western blot analysis on the total cell lysates from serum‐starved RON positive BXPC vector control, RON knockdown BXPC3 shRON cl2 cells and AKT inhibitor, MK‐2206 treated BXPC3 cells was performed with various antibodies. RON knockdown or AKT inhibitor, MK‐2206 inhibited PI3K mediated AKT phosphorylation and HIF‐1α expression with no apparent changes in other proteins tested. (B) Quantitative real‐time RT‐PCR using RON, HIF‐1α, and GAPDH internal control primers was performed on the total RNA from BXPC3 vector control, RON knockdown clones 2 and 4. RON knockdown significantly decreased HIF‐1α mRNA expression. *p < 0.05. (C) Sp1 transcription factor effect on HIF‐1α promoter activity was analyzed in breast cancer cells. An Sp dose‐dependent increase in HIF‐1α promoter activity was observed. (D) HIF‐1α promoter activity was analyzed in RON positive BXPC3 vector control in the absence or presence of Sp1 inhibitor, Mithramycin A, and RON knockdown BXPC3 sh RON cl2 cells. HIF‐1α promoter activity was blunted in the Mithramycin A treated and RON knockdown cells