Hypoxia induces IL-6 and IL-8 production solely in RCC cells.

<p>A) Interleukin-6 (IL-6) and B) Interleukin-8 (IL-8) secretion by normal proximal tubular epithelial cells (HK2) exposed to normoxic (norm) or hypoxic conditions for short (T15-180min) or long (T24-T72hr) time points was determined by ELISA. C, D) IL-6 and E, F) IL-8 secretion by renal carcinoma cells (RCC 786-O and RCC4) exposed to normoxic (norm) or hypoxic conditions for short (T15-180min) or long (T24-T72hr) time points was determined by ELISA as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#s2" target="_blank">materials and methods</a>.</p

Effects of IL-6 and IL-8 on RCC cell invasion.

<p>A) RCC 786-O cells were treated with recombinant IL-6 and/or IL-8 for 18 hours and cell invasion was analyzed by Boyden chamber as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#s2" target="_blank">materials and methods</a>. <i>Left panel,</i> the invaded cells were photographed and <i>right panel,</i> invaded cells were counted and quantified. The graph is representative of ten independent experiments and the results are expressed as the means <u>+</u> S.E. ***, p<0.001 versus buffer control (-). B) RCC 786-O cells were incubated with condition media from RCC 786-O cells subjected to 48hr norm or hypoxic conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#pone-0030712-g004" target="_blank">Fig. 1C,E</a>) for 18 hours and cell invasion was analyzed by Boyden chamber. C) RCC 786-O cells were incubated with condition media from RCC 786-O cells in normoxic media (norm) or hypoxic media from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#pone-0030712-g004" target="_blank">Fig. 2 D</a>, E transfected with scrambled control (scr) or siRNA for Nox4 (siNox4). D) RCC 786-O cells were incubated with condition media from RCC 786-O cells in normoxic media (norm) or hypoxic media from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#pone-0030712-g004" target="_blank">Fig. 3A</a>,B incubated with buffer control (-) or AICAR. B, C, D, Invaded cells were quantified from three independent experiments and expressed as the means <u>+</u> S.E. **, p<0.01 versus norm, <i>##</i>, p<0.01 versus hypoxic control (-).</p

Effects of AMPK activation on Hypoxia induced IL-6 and IL-8 production in RCC cells.

<p>A) Interleukin-6 (IL-6) and B) Interleukin-8 (IL-8) secretion was measured using ELISA analysis in RCC 786-O cells pretreated for 30 min in buffer alone (-), or the AMPK activator, aminoimidazole-4-carboxamide-1-riboside 5-aminoimidazole-4-carboxamide-1-riboside (AICAR) and exposed to normoxic (norm) or hypoxic conditions for 48 hrs. The data were quantitated from three independent experiments and the results are expressed as the means <u>+</u> S.E. **, p<0.01 versus norm, <i>##</i>, p<0.01 versus hypoxic control (-). C) Cell lysates were prepared from cells treated with AICAR, (Fig. 3 <i>A, B)</i> and analyzed for Nox4 expression or GAPDH control by Western blot analysis.</p

Effects of antioxidants on Hypoxia induced IL-6 and IL-8 production in RCC cells.

<p>A) Interleukin-6 (IL-6) and B) Interleukin-8 (IL-8) secretion was measured using ELISA analysis in RCC 786-O cells pretreated for 30 min in buffer alone (-), N-Acetyl-L-cysteine (NAC) or a flavoprotein inhibitor (DPI) and exposed to normoxic (norm) or hypoxic conditions for 48 hrs. The data were quantitated from three independent experiments and the results are expressed as the means <u>+</u> S.E. **, p<0.01 versus norm and #<i>#</i>, p<0.01 versus hypoxic control (-). C) IL-6 and D) IL-8 secretion was measured using ELISA analysis in RCC 786-O cells transfected with scrambled control (Scr) or small interfering RNA to Nox4 (siNox4) exposed to normoxic (Norm) or hypoxia for 48 hrs. The data were quantitated from three independent experiments and the results are expressed as the means <u>+</u> S.E. **, p<0.01 versus norm, <i>#</i>, p<0.05, and <i>##</i>, p<0.01 versus hypoxic scr control. E) RCC 786-O cells transfected with scrambled control (Scr), small interfering RNA to Nox4 from C, D were analyzed by Western blot with Nox4 antibody or GAPDH control.</p

<i>Ex-vivo</i> human RCC tumor cells demonstrate reduced cell invasion with AICAR treatment.

<p>Human RCC cells were cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030712#s2" target="_blank">materials and methods.</a> Freshly prepared <i>ex-vivo</i> cells were incubated with AICAR for 48 hours and A) Interleukin-6 (IL-6) and B) Interleukin-8 (IL-8) was measured by ELISA analysis. C) Freshly prepared <i>ex-vivo</i> human RCC cells were plated in a Boyden chamber in the presence (+) or absence (-) of AICAR for 18 hours. Invaded cells were quantified from three independent experiments and expressed as the means <u>+</u> S.E. ***, p<0.001 versus buffer control (-).</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

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

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

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

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