Fig. 3: BBOX1 attenuates mTORC1 signaling in ccRCC.

Venn diagram for upregulated (a) or downregulated (b) differentially expressed genes (DEGs, BBOX1 vs EV log2FC > 0.5 or < −0.5, P < 0.05) in EV/BBOX1 786-O xenograft tumors or in vitro cells grown in DMEM. The number of unique or overlapped DEGs in each condition was indicated. c–f Gene set enrichment analysis (GSEA) using normalized gene expression values of EV or BBOX1 samples from 786-O xenograft tumors (c) or in vitro cells grown in DMEM (e), n = 3 biological replicate samples per group. Hallmark gene sets were used. Downregulated or upregulated signaling pathways with FDR  <  0.05, P < 0.05 are shown. GSEA enrichment plot of the mTORC1 signaling from 786-O xenograft tumors (d) or in vitro cells grown in DMEM (f) were shown. NES, Normalized Enrichment Score. g Heatmap reveals gene expression of mTORC1 signaling related genes in EV/BBOX1 786-O xenograft tumors or in vitro cells grown in DMEM. n = 3 biological tumor or cell samples per group. Immunoblots and gel quantifications of tumor lysates from 786-O (h) or A498 (i) xenograft tumors expressing EV or BBOX1. n = 6 paired tumors, each mouse was inoculated with EV and BBOX1 cells at both flanks. j Immunoblots and gel quantifications of tumor lysates from A498 xenograft tumors expressing control sgRNA (Ctrl), or BBOX1 sgRNA 1 and 2. n = 4 mice. k Immunoblots of 786-O or UMRC2 cells expressing empty vector (EV), BBOX1, or catalytic dead (CD) mutant. l Immunoblots of A498 cells expressing Ctrl, or BBOX1 sgRNA 1 and 2. m Representative immunohistochemistry (IHC) staining images of BBOX1, phospho-S6 (at S240/244), and phospho-4EBP1 (at T37/46) in 39 ccRCC tumor samples. Scale bar, 100 μm. Spearman correlation between BBOX1 and phospho-S6 (n) or between BBOX1 and phospho-4EBP1 (o) in 39 ccRCC tumor tissues. p H-score of phosphor-S6 in ccRCC tumor thrombus samples stratified by low or high levels of BBOX1 mRNA expression from a previous study⁴⁶. Two-tailed Student’s t test (h, i paired and j, n–p unpaired), two-sided permutation test (c–f). Error bars represent SEM. Representative immunoblots and gels shown in figures were repeated at least two times independently with similar results. Source data are provided as a Source Data file.