Fig. 7: The GRAMs are involved in ccRCC survival.

a Samples from The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) study were stratified based on GRAM expression. Overall survival of samples with high GRAM expression (upper quartile, orange line) were compared to low GRAM expression (lower quartile, gray line). The dotted lines represent 95% confidence interval. P values were obtained using Log-rank (Mantel–Cox) test. b For the colony-formation assay, 786-O cells were treated with the indicated siRNAs and incubated for 3 weeks prior to fixation and staining with crystal violet stain. c Colony area was quantified and normalized to control siRNA (siNC). Significance was determined using one-way ANOVA followed by Dunnett’s comparison test from n = 3 independent experiments. Error bar = SEM. P value = 0.001 and 0.0003. d 786-O cells were treated with siRNA against the indicated GRAMs for 72 h prior to fixation and immunostaining for Ki67. e Ki67 positive cells per 100 cells were quantified from 6 independent experiments and represented in a min-max box and whisker plot. The error bars represent max and min points, while the horizontal lines in the box plot represent the upper quartile, median and lower quartile. Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Scale bar = 200 µm. P value = 0.0237, 0.0021 and 0.0073. f A498 cells were treated with siNC or siGRAMD1C for 72 h. The cells were then incubated in DMEM or EBSS ± 100 nM BafA1 for 2 h prior to protein isolation and western blot analysis using the indicated antibodies. g Significance was determined using two-way ANOVA followed by Sidak’s multiple comparisons test from n = 3 experiments. P value = 0.0056 and <0.0001. h ATP-linked respiration, calculated from the difference between the maximal respiratory capacity and the proton leak from Seahorse analysis, in 786-O cells treated with control (siNC) or siRNA against GRAMD1C for 72 h. Significance was determined using one-way ANOVA followed by Dunnett’s comparison test from n = 3 experiments. Error bar = SEM. P value = 0.049 and 0.0129. Source data are provided as a Source Data file.