Figure 7. RSK4 activates the Wnt/β-catenin pathway.

(A) Control or RSK4-overexpressing ECA109 cells were incubated with CHX (10 μg/mL) for the indicated durations. High RSK4 expression prolonged the half-life of β-catenin degradation (n = 3 independent experiments). (B) HEK293T cells were transfected with the indicated plasmids, and cell extracts were immunoprecipitated with an anti-His antibody. Ubiquitinated β-catenin was detected by immunoblotting. (C) Control or RSK4-silenced TE10 cells were incubated with CHX (10 μg/mL), CHX plus BI-D1870 (10 μM), or MG132 (10 μM) for the indicated durations. The indicated proteins were detected by immunoblotting (n = 3 independent experiments). (D) HEK293T cells were transfected with the indicated plasmids and incubated with CHX (10 μg/mL) for the indicated durations. The indicated proteins were detected by immunoblotting (n = 3 independent experiments). (E) HEK293T cells were transfected with the indicated plasmids, and cell extracts were immunoprecipitated with an anti-His antibody. Ubiquitinated β-catenin was detected by immunoblotting. (F and G) ECA109 cells were transfected with the indicated plasmids, and then CSC markers (F) and caspase-3 activity after IR (10 Gy) (G) of the indicated groups were detected (n = 3 independent experiments). (H) Representative images of the nuclear localization of β-catenin in ESCC cells from the indicated groups detected using immunofluorescence microscopy. Scale bars: 100 μm. (I) Effects of knockdown or overexpression of RSK4 on the indicated proteins in ΔNp63α-overexpressing or ΔNp63-suppressing ESCC cells. (J and K) Treatment of RSK4-overexpressing cells with 50 μM iCRT3 (an inhibitor of β-catenin signaling) for 24 hours greatly reduced their sphere-forming ability (J) and increased caspase-3 activity after IR (K) (n = 3 independent experiments). BI, BI-D1870; AR, AR-A014418. Data represent the mean ± SD. *P < 0.05 and **P < 0.01. Differences were tested using 1-way ANOVA with Tukey’s post hoc test (G, J, and K).