Figure 3 from SET1B Drives Sustained HIF Activity and Disease Progression in Clear-Cell Renal Cell Carcinoma

<jats:p>&lt;p&gt;SET1B is required for sustained HIF2-dependent activity in ccRCC. &lt;b&gt;A,&lt;/b&gt; High expression of SETD1B (&lt;i&gt;SET1B&lt;/i&gt; gene name) in kidney cancer is associated with poor outcomes. Kaplan–Meier survival analysis of TCGA data for ccRCC, comparing tumors in the highest and lowest quartiles of SETD1B mRNA expression. &lt;i&gt;n&lt;/i&gt; = 130 patients for each group. Log-rank test. &lt;b&gt;B,&lt;/b&gt; VEGFA ELISA in control, SET1B depletion, SET1B KO, and HIF2α KO 786-0 cells. Cells were incubated for 48 hours and before supernatants were collected. The graph is representative of 3 biological replicates. Two-way ANOVA. &lt;b&gt;C,&lt;/b&gt; Graphs showing the percentage engraftment of control, HIF1β, and SET1B KO cells in the CAM assay. &lt;b&gt;D,&lt;/b&gt; Representative images of 786-O parental (&lt;i&gt;n&lt;/i&gt; = 17), SET1B KO (&lt;i&gt;n&lt;/i&gt; = 16), and HIF1β KO (&lt;i&gt;n&lt;/i&gt; = 15) CAM assay. The xenografts and surrounding CAM was fixed &lt;i&gt;in ovo&lt;/i&gt; to preserve blood in the vessels and then imaged from the underneath after dissection. Scale bars, 1 mm. Vessel density was calculated as total vessel length (pixels)/total area analyzed (pixels&lt;sup&gt;2&lt;/sup&gt;) per ROI. Brown–Forsythe ANOVA test, F(2,33.38) = 7.156, &lt;i&gt;P&lt;/i&gt; = 0.0026. Dunnett T3 multiple comparison test; parental (P) vs. SET1B KO (S), &lt;i&gt;P&lt;/i&gt; = 0.0076; parental vs. HIF1β KO (H), &lt;i&gt;P&lt;/i&gt; = 0.4873; SET1B KO vs. HIF1β KO, &lt;i&gt;P&lt;/i&gt; = 0.0136. &lt;b&gt;D,&lt;/b&gt; Branching points relative to area analyzed (mm&lt;sup&gt;2&lt;/sup&gt;). Ordinary one-way ANOVA, F(2, 45) 4.019, &lt;i&gt;P&lt;/i&gt; = 0.0248. Tukey multiple comparison test; parental vs. SET1B &lt;i&gt;P&lt;/i&gt; = 0.0076; parental vs. HIF1β &lt;i&gt;P&lt;/i&gt; = 0.4873; SET1B vs. HIF1β &lt;i&gt;P&lt;/i&gt; = 0.0136. &lt;b&gt;E,&lt;/b&gt; IHC of SET1B was performed on a ccRCC tissue microarray (TMA). Samples were assessed and graded 1–4 with 1 being the least aggressive and grade 4 being the most aggressive. SET1B staining intensity across the tumor was calculated as an H score which accounts for the staining intensity and the % of positive cells detected. Samples were subdivided based on grade, and SET1B intensity was plotted. Significance was assessed using a two-way ANOVA. &lt;b&gt;F,&lt;/b&gt; Control, HIF2α KO, and SET1B KO 786-0 cells were embedded in Matrigel, and cellular invasion was measured over indicated time using Incucyte. Graphs are representative for 3 biological replicates and depict the mean ± SD. Two-way ANOVA. &lt;b&gt;G,&lt;/b&gt; qPCR of HIF targets associated with metastasis (&lt;i&gt;MAFF&lt;/i&gt;, &lt;i&gt;AKAP12&lt;/i&gt;, and &lt;i&gt;ANGPTL4&lt;/i&gt;) in 786-0 cells depleted of HIF1β and SET1B using CRISPR (&lt;i&gt;n&lt;/i&gt; = 3 biologically independent samples, mean ± SD). &lt;b&gt;H,&lt;/b&gt; ccRCC mouse xenograft model. WT, HIF1β, and SET1B-depleted 786-0 cells expressing luciferase were injected into the tail vein of nude mice. Bioluminescence was measured and quantified from the lungs on day 40 and day 54 (control = 5; HIF1β KO = 6; and SET1B KO = 6). Mean ± SD. Two-way ANOVA. ROI, region of interest; siSET1B, SET1B siRNA.&lt;/p&gt;</jats:p>