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. 2020 Jul 13;130(8):4301–4319. doi: 10.1172/JCI134930

Figure 5. RSK4 is essential for ΔNp63α-mediated CSC properties and radioresistance of ESCC cells.

Figure 5

(A) Knockdown of RSK4 abolished ΔNp63α-enhanced sphere formation ability, whereas RSK4 overexpression partially restored the effects of ΔNp63-suppressed sphere formation (n = 3 independent experiments). Scale bars: 100 μm. (B) Flow cytometric analysis of ALDH activity and the proportion of CD90+ cells in ESCC cells from the indicated groups (n = 3 independent experiments). (C) Western blot analysis of ESCC CSC markers in the indicated groups. (D) Clonogenic survival assays of ESCC cells in the indicated groups at IR doses of 0, 3, and 6 Gy (n = 3 independent experiments). (E) Relative caspase-3 activity 24 hours after IR (10 Gy) of ESCC cells in the indicated groups (n = 3 independent experiments). (F) Western blot analysis of phosphorylated and total amounts of the checkpoint proteins ATM and CHK2 from the indicated groups 1 hour after 10 Gy IR. (G) ESCC cells from the indicated groups were treated with 10 Gy IR and recultured under normal conditions for 1 and 6 hours, and then subjected to Western blot analysis with γ-H2AX antibody. 0 h, cells with IR treatment but with no time for DNA repair. Data represent the mean ± SD. **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired, 2-sided Student’s t test (A, B, D, and E).