Article Figures & Data
Figures
- FIGURE 1.
Inhibition of HIF2α in VHL−/− cells by shRNAi. VHL−/− 786-O cells were stably transfected with pSR or pTU6 plasmids expressing shRNA targeting HIF2α or control empty vector. A. Normalized HRE-driven luciferase activity in shRNAi versus control clones. B. Western blot analysis for HIF2α expression in HIF2α shRNAi and pVHL-reconstituted clones versus vector-only control clones. Bar graph quantitates relative HIF2α levels normalized for actin loading control.
- FIGURE 2.
shRNAi of HIF2α decreases expression of HIF target genes. A. ELISA for secreted VEGF concentration in tissue culture supernatants of HIF2α shRNAi and pVHL-reconstituted clones versus vector-only control clones. Bars, SEM. Differences in VEGF expression between pVHL-reconstituted or HIF2α knockdown clones and their respective control clones were significant (P < 0.01). B. Western blot analysis for Glut-1 expression in the HIF2α shRNAi clones was performed using unboiled samples with one-tenth of the lysate volume loaded in Fig. 1. Bar graph quantitates relative Glut-1 expression normalized for actin loading control (shown in Fig. 1).
- FIGURE 3.
HIF2α inhibition prevents response to hypoxia. A. ELISA for secreted VEGF concentration in tissue culture supernatants of HIF2α shRNAi and pVHL-reconstituted clones versus respective vector-only control clones under normoxic (21% O2, gray bars) and hypoxic (1% O2, white bars) conditions. Bars, SEM. Differences in VEGF expression between pVHL-reconstituted (WT8) or HIF2α knockdown clones (pSR1, pTR1, pTR2, and pTR3) and their respective control clones (pRC3, pSV1, and pTV1) were significant (P < 0.01), whereas differences in the hypoxic induction of VEGF were significant for the pVHL-reconstituted WT8 cells only (P < 0.05). B. Western blot analysis for HIF2α expression in cells under normoxic and hypoxic conditions (denoted as N and H, respectively). Bar graph quantitates relative HIF2α levels normalized for actin loading control. A and B. Gray bars, normoxia; white bars, hypoxia.
- FIGURE 4.
In vitro cell proliferation is not affected by HIF2α-directed shRNAi. Cell proliferation measured via cleavage of the tetrazolium salt WST-1 at the indicated time points. Bars, SEM. Closed squares, PRC3; open squares, WT8; closed triangles, pTV1; open triangles, pTR1. Similar results were obtained for pSV1, pSR1, pTV2, pTR2, and pTR3 cells (data omitted for the sake of clarity).
- FIGURE 5.
HIF2α-directed shRNAi reduces the overall proangiogenic activity of conditioned supernatant. HUVEC proliferation as measured by WST-1 cleavage in the presence of conditioned supernatant from PRC3, WT8, pSV1, pSR1, pTV1, and pTR1 clones. Differences between shRNAi and pVHL-reconstituted clones versus vector-only control clones were significant. Bars, SEM.
- FIGURE 6.
HIF2α-directed shRNAi suppresses tumor formation. A. Average tumor mass resulting from cells injected into the flank of nude mice. n, number of injection sites for each clone. All differences in tumor mass relative to the corresponding vector-only control cell injections were statistically significant (P < 0.01). Bars, SEM. B. Representative mice injected with cells in which pVHL function was reconstituted (WT8) versus HIF activity was inhibited (pSR1) along with corresponding control injections.
Tables
- Table 1.
Description of Cell Lines Used in This Study
Clone Description Reference 786-O Established VHL−/− RCC cell line (39) PRC3 786-O-derived clone stably transfected with empty pcDNA3 vector (11) WT8 786-O-derived clone stably transfected with pcDNA3::HA-VHL30 (11) pSV1 786-O-derived clone stably transfected with empty pSR vector This work pSR1 786-O-derived clone stably transfected with pSR targeting HIF2α This work pTV1 786-O-derived clone stably transfected with empty pTU6IIa vector This work pTV2 786-O-derived clone stably transfected with empty pTU6IIa vector This work pTR1 786-O-derived clone stably transfected with pTU6IIa targeting HIF2α This work pTR2 786-O-derived clone stably transfected with pTU6IIa targeting HIF2α This work pTR3 786-O-derived clone stably transfected with pTU6IIa targeting HIF2α This work
Volume 2, Issue 2
