Fig. 4: Endogenous BMAL1 contributes to HIF2α target gene expression in RCC cells.

A Detection of HIF2α, ARNT, BMAL1, and ACTIN by immunoblot in 786O cells expressing the indicated shRNAs. The samples derive from the same experiment but one gel for BMAL1, HIF2α, and ACTIN, and another for ARNT and ACTIN were processed in parallel. Data from one of two independent experiments with similar results. Venn diagrams (B, C) and heatmaps (D, E) depicting all differentially expressed genes (DEGs) (B, significantly downregulated genes (C, D) or downregulated genes in the Hallmark HYPOXIA gene set (E) in 786O cells expressing the indicated shRNAs (n = 3 distinct samples for each condition). DEGs were identified using DESeq2 with a false discovery rate (FDR) cutoff of 0.1. Enrichment plots showing the impact of shARNT (F) or shBMAL1 (G) on genes in the Hallmark HYPOXIA gene set. H Venn diagram depicting overlap of DEGs in 786O cells expressing VHL (WT8 cells) or expressing shBMAL1. I Boxplot depicting changes in gene expression in PDXs treated with PT2399 (data from ref. ²⁶ including sensitive PDXs only, n = 12 biological replicate PDX tumors per condition) for genes grouped by whether their expression in 786 O cells is decreased by shARNT and not by shBMAL1 (ARNTsp, yellow, n = 1069 genes), by shBMAL1 and not by shARNT (BMAL1sp, purple, n = 1707 genes), by either shARNT or shBMAL1 (Overlap, salmon, n = 1273 genes), or neither (NA, gray, n = 15,584 genes). **** P = 3.27e⁻¹⁰ by two-way ANOVA with Tukey’s correction. Boxes depict the median and interquartile range (IQR), whiskers extend either to the minimum or maximum data point or 1.5 × IQR beyond the box, whichever is shorter. Outliers (values beyond the whisker) are shown as dots. J–L Volcano plots depicting expression changes for individual genes in groups depicted in (I). Genes with padj <0.05 are colored in red (fold change >1.5) or blue (fold change <0.67). padj is calculated in DESeq2 using p-values attained by the Wald test and corrected for multiple testing using the Benjamini and Hochberg method. Top non-redundant GOBP (M) or KEGG (N) pathways with ≥15 genes, FDR < 0.05, fold enrichment ≥2 enriched among ARNT-specific or BMAL1-specific target genes in 786O cells. Source data are provided as a Source Data file.