PIN1 silencing leads to decreases in the protein levels of HIF-1α.

<p>A) Silencing of PIN1 expression via RNAi reduces the protein levels of HIF-1α in HCT116 cells. Western blot analysis was performed with 50 μg total cell lysate from HCT116 cells transfected with control or PIN1 si-RNA. B) mRNA levels of HIF-1α and PIN1 were determined by RT-PCR analysis at the indicated time points under hypoxic conditions. GAPDH was used as internal controls. C) The PIN1 inhibitor, PiB, reduces the protein levels of HIF-1α in HCT116 cells. HCT116 cells were mock-treated or treated with PiB and harvested 8 h after treatment for the analysis of the HIF-1α and PIN1 levels. D) HCT116 cells were mock-treated or treated with PiB and harvested 8 h later. mRNA levels of HIF-1α and PIN1 were determined by RT-PCR analysis at the indicated time points under hypoxic conditions. GAPDH was used as internal controls. E) ATRA decreases the expression of HIF-1α and PIN1 in HCT116/5xHRE-ODD-luc cells in dose-dependent manner. F) The luciferase activity of HRE in HCT116/5xHRE-ODD-luc cells treated ATRA was reduced in dose-dependent manner. G) Nuclear co-localization of HIF-1α and PIN1 was determined by immunocytochemistry. HCT116 cells plated on the chamber slide were transfected PIN1 si-RNA or treated PiB and grown at 1% O₂ in a hypoxia chamber for additional 4 h. The samples were co-incubated with anti HIF-1α and PIN1 antibodies and then co-incubated with FITC-conjugated secondary antibodies. Lastly, samples were stained with DAPI. The signals were detected using a confocal microscope or a fluorescent microscope. Scale bar, 10 μm.