FH protein expression id reduced in clear cell renal cancer.

<p>A) Protein was isolated from clear cell (CC) tumor samples (T) in addition to matched normal renal parenchyma (N). Proteins were immunoblotted for FH protein levels. GAPDH immunoblot is included as a loading control. B) Immunohistochemical staining for FH was performed on patient-matched tumor/normal pairs. Images were obtained with a 40× objective lens. C) FH protein levels in RCC lines relative to normal kidney. Actin is included as a loading control.</p

FH mRNA expression is reduced in clear cell kidney cancer.

<p>mRNA levels of <i>FH</i> were determined in a separate set of tumor samples relative to patient matched normal renal parenchyma with real time RT-PCR (p = 0.004). Expression levels were normalized to 18 s rRNA levels prior to comparative analysis.</p

Migration and Invasion assays in 786-O cells transfected with control vector (CV) or FH-FLAG.

<p>A) Wound healing assay images at the indicated time points. B) Wound width distance at the indicated time points. C) Chamber cell migration assay of the indicated clones. Cells were seeded in serum free media with 10% FBS used as the chemotractant. Migrated cell counts of clones at 48 hours from initial seeding. Asterisks (*) indicate statistical significance with p<0.05 relative to control vector transfected cells. D) Matrigel invasion assay with 10% FBS media as the chemotractant. Asterisks (*) indicate statistical significance with p<0.05 relative to control vector transfected cells.</p

FH expression impacts AKT signaling.

<p>A) <i>VHL</i> null 786-O and A498 cells were transfected with siRNA to FH and scramble control (siNC). Forty-eight hours following transfection, protein lysates were analyzed by immunoblotting for total AKT and ser473 phospho-AKT. B) 786-O cells were transiently transfected with control vector (CV) and vector containing FLAG tagged FH. Forty-eight hours following transfection, protein lysates were analyzed by immunoblotting for the indicated proteins. C) 786-O cells were transfected with the indicated siRNA. Twenty-four hours following transfection, media of the cells was replaced with media containing PI3K inhibitor LY-294002 (6.25 µM). Cells were then harvested 24 hours later and protein lysates were subjected to immunoblot analysis for the indicated proteins.</p

HIF-2α promotes migration in RCC cells.

<p>786-O RCC cells were transfected with scramble control siRNA (siNC) or siRNA to HIF-2α. Western blotting results of whole cell extracts are demonstrated in panel A. B) Wound healing assay was performed in transfected cells. Images were serially taken at the indicated time point following “wound” induction. Results are graphically displayed in panel C. Asterisks (*) indicate statistical significance with p<0.05 relative to scramble control transfected cells.</p

FH expression impacts HIF-2α levels.

<p>A) <i>VHL</i> null 786-O and A498 cells were transected with siRNA to FH and scramble control (siNC). Forty-eight hours following transfection, protein lysates were analyzed by immunoblotting for the indicated proteins. B) 786-O cells were transiently transfected with control vector (CV) and vector containing FLAG tagged FH. Forty-eight hours following transfection, protein lysates were analyzed by immunoblotting for the indicated proteins. FLAG immunoblot indicates successful expression of the transgene. C) (Left) 786-O cells were stably transfected with CV and FH-FLAG. After selection in puromycin, single cell clones were harvested and screened for FLAG expression. HIF-2α levels were measured in FH-FLAG expressing clones relative to CV transfected cells. Densitometry of the bands is quantitatively displayed on the right. Mean relative values +/− standard deviation were obtained with ImageJ software from independent experiments.</p