Fig. 2

RYR1 enhances cell proliferation and metastasis and suppresses cell apoptosis. A, B Growth curves showing the effect of (A) RYR1 silencing using stably expressed shRNAs (shRYR1–1 and shRYR1–2) and control shRNA (shCtrl) and (B) RYR1 overexpression using stably expressed RYR1 construct and empty vector control in USC cells ARK1, ARK2, and HEC50. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (2-way ANOVA). C, F Relative number of (C) USC shRYR1 or shCtrl cells and (F) USC treated with dantrolene (20 or 50 μM) or vehicle migrating through the transwell membrane. D, G Relative frequencies of apoptotic cells in (D) USC shRYR1 or shCtrl cells, and (G) USC treated with dantrolene or vehicle. E Growth curves showing the effect of dantrolene (30 μM) on USC cells ARK1, ARK2 and HEC50. H, I Representative bioluminescence images (H) and bar charts showing relative bioluminescence intensities (I) in nude mice at week 10 after injection with luciferase-labelled ARK1-shRYR1 or shCtrl cells. J Kaplan-Meier analysis for overall survival in mice injected with ARK1-shRYR1 or shCtrl. Log rank test. p = 0.0009. K, L Representative bioluminescence images (K) and bar charts showing relative bioluminescence intensities (L) at week 9 in luciferase-labelled ARK1-bearing mice injected intraperitoneally with dantrolene. N = 10 mice per group. M, N. Representative immunohistochemical staining images (M) and bar charts (N) of numbers of Ki-67-, CD31-, CCP3-positive cells in tumor tissues from mice treated with 50 μM dantrolene or vehicle. Scale bar, 100 μm. In C, D, E, F, G, I, L, N, *p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., no significance (Student t-test). In A-G, N, graphs represent mean ± SEM of 3 independent experiments