Fig. 5: pRb hyperstabilization promotes ccRCC oncogenesis.

A Representative images showing colony formation of 786-O control and RB1 KO cells in soft agar. Scale bar = 500 µm. B Quantification (relative to control) of the number of colonies formed in A. Colonies were manually counted under a light microscope. Statistical significance was calculated using ordinary one-way ANOVA and Tukey’s post-hoc test (n = 3). C Representative images showing colony formation in soft agar of 786-O control and RB1 KO cells stably transfected with either control vector (Mock) or HA-tagged VHL. Scale bar = 500 µm. D Quantification (relative to control) of the number of colonies formed in C. Colonies were manually counted under a light microscope. Statistical significance was calculated using ordinary two-way ANOVA and Tukey’s post-hoc test (n = 3). E Representative images showing clonogenic outgrowth of 786-O control and RB1 KO cells. F Quantification (relative to control) of the number of colonies in E. Statistical significance was calculated using ordinary one-way ANOVA and Tukey’s post-hoc test (n = 3). G Representative images showing clonogenic outgrowth of 786-O, RCC4 and A-498 cells stably infected with lentivirus containing either control plasmid (Vector) or SKIDA1 cDNA containing plasmid. H Quantification (relative to control) of the number of colonies in (G). Statistical significance was calculated using unpaired t test and Holm-Sidak post-hoc test (n = 3). I Representative images showing colony formation in soft agar of 786-O and A-498 cells stably infected with lentivirus containing either empty (control) plasmid (Vector) or SKIDA1 cDNA-encoding plasmid. Scale bar = 500 µm. J Quantification (relative to control) of the number of colonies formed in (I). Colonies were manually counted under a light microscope. Statistical significance was calculated using unpaired t test and Holm-Sidak post-hoc test (n = 3). K Representative images showing clonogenic outgrowth of 786-O control, RB1 KO and RB1/SKIDA1 double KO cells. L Quantification (relative to control) of the number of colonies in K. Statistical significance was calculated using ordinary one-way ANOVA and Tukey’s post-hoc test (n = 3). M Representative images showing colony formation in soft agar of 786-O control, RB1 KO and RB1/SKIDA1 double KO cells. Scale bar = 500 µm. N Quantification (relative to control) of the number of colonies formed in M. Colonies were manually counted under a light microscope. Statistical significance was calculated using ordinary one-way ANOVA and Tukey’s post-hoc test (n = 6). O Schematic illustration showing the VHL-pRb-SKIDA1 signalling axis and the effect on clonogenicity and soft agar growth. The dotted line suggests alternate pathways. P Scatter dot plot showing quantification of weights of tumors formed by 786-O control and RB1 KO cells injected subcutaneously into the flanks of immunodeficient NOD-scid IL2Rg^null mice, via a tumor xenograft assay. The line indicates the mean. Five injections were performed with control cells and ten injections with RB1 KO cells (five for each clone). One control injection which failed to grow was considered an outlier and omitted during statistical analysis. Statistical significance was calculated using ordinary one-way ANOVA and Tukey’s post-hoc test (n > 3). A–P Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. ‘ns’ denotes not significant.