Fig. 2: BBOX1 re-expression suppresses ccRCC tumorigenesis and cell growth in physiological growth conditions.

a Immunoblots of BBOX1 re-expression in multiple ccRCC cell lines. Colony formation assay in DMEM (b), subcutaneous tumor growth (c), and tumor weight (d) of ccRCC cells expressing empty vector (EV) or BBOX1. 786-O, n = 7 mice; A498, n = 7 mice; UMRC2, n = 7 mice; Caki-1, n = 5 mice. Each mouse was inoculated with EV and BBOX1 cells at both flanks. Immunoblots of cell lysates (e), subcutaneous tumor growth (f), and tumor weight (g) of A498 cells with shRNA control (Ctrl) or BBOX1 depletion by two individual sgRNAs. Ctrl, n = 5 mice; sg1, n = 5 mice; sg2, n = 5 mice. Immunoblots of cells with doxycycline (dox) induction (h), representative bioluminescence images (i), corresponding quantification data (j), tumor weight (k), and image of tumors (l) from 786-O luciferase stable cells expressing EV, BBOX1, or catalytic dead (CD) mutant injected orthotopically into the kidney of NSG mice. EV, n = 9 mice; BBOX1, n = 10 mice; CD, n = 9 mice. m Relative cell viability of 786-O cells expressing EV, BBOX1, or CD mutant grown in different growth media. n = 16 independent cell cultures. This experiment has been repeated at least two times with similar results. Representative soft agar growth with colony quantifications of 786-O cells expressing EV, BBOX1, or CD mutant (n = 3 biological cell cultures) (n), or A498 cells expressing Ctrl, BBOX1 sg1 or sg2 (n = 3 biological cell cultures) (o) in indicated growth media. Each experiment has been repeated at least two times with similar results. Scale bar, 1 mm. Statistical analysis was conducted by two-way ANOVA followed by Tukey’s multiple comparison test (c, f, and j) or two-tailed Student’s t test (d, paired; g, k, and m–o, unpaired). Error bars represent SEM, NS denotes no significance. 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.