Fig. 3

ADGRG6 enhancer mutations are associated with poor survival and tumor angiogenesis in UBC. a Sanger sequencing detected ADGRG6 enhancer mutations in the additional 196 UBCs. b Immunohistochemical staining of UBC tumors displaying consequences of ADGRG6 enhancer mutations. ADGRG6 enhancer mutations lead to ADGRG6 protein overexpression in tumor tissues. The three different groups of UBC tumors: G > A Mut (n = 25), ADGRG6 enhancer only with 142,706,209 G > A mutation; C > T Mut (n = 25), ADGRG6 enhancer only with 142,706,206C > T mutation; No Mut (n = 25), ADGRG6 enhancer with no mutation. c CD31 immunostained microvessel in the additional 196 UBCs. The three different groups of UBC tumors: G > A Mut (n = 70), ADGRG6 enhancer with 142,706,209G > A mutation; C > T/G Mut (n = 51), ADGRG6 enhancer with 142,706,206 C > T/G mutation; No Mut (n = 94), ADGRG6 enhancer with no mutation. The three horizontal lines represent mean ± SD for the subjects. *P < 0.05, **P < 0.01, ***P < 0.001 vs. No Mut group, two-sided unpaired t test with Welch’s correction (b) or Mann–Whitney test (c). d Kaplan–Meier survival curves show that the patients with the ADGRG6 enhancer mutations were significantly associated with a shorter overall survival than those with no mutation in both nonmuscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) subcohorts. Statistical significance was determined by log-rank test. e, f Knockdown of ADGRG6 in 5637 and SW780 cells compromised their abilities to recruit human umbilical vein endothelial cells (e) and induce tube formation (f). Scale bars, 100 µm. Error bars represent the SEM. The data shown represent averages from three independent experiments and were statistically analyzed by two-sided t test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. siCon