FIGURE 6.

TRIB2‐mediated activation of the Akt/mTOR pathway induces histone deacetylase 2 (HDAC2) phosphorylation and subsequent transcriptional activation of p21 in ESCC cells (A) Cell viability assays showed that HDAC2 inhibition by santacruzamate (SCA; 2 μM) completely blocked TRIB2‐mediated growth of Eca109 cells. (B) The indicated proteins were detected by western blotting of Eca109 and TE‐1 cells after TRIB2 overexpression or silencing. (C) The indicated proteins were detected by western blotting of Eca109 and TE‐1 cells after TRIB2 overexpression or silencing. (D) Phosphorylation of Akt (S473) in ESCC cells after the indicated treatments. (E) The phosphorylation of Akt (S473) and HDAC2 (S394) in ESCC cells transfected with the indicated constructs and/or with siRNA was detected by western blotting. (F) The phosphorylation of HDAC2 (S394) and S6K1 (S411) in Eca109 cells transfected or not with TRIB2 constructs was detected by western blotting after treatment of the cells with rapamycin (100 nM). (G) The phosphorylation of S6K1, p38, and HDAC2 in Eca109 cells transfected with the indicated constructs were detected by western blotting after treatment of the cells with rapamycin (100 nM), SB203580 (10 μM), or anisomycin (1 g/ml). (H) The phosphorylation of S6K1 (S411) and HDAC2 (S394) in Eca109 cells transfected with TRIB2 constructs and/or with S6K1 siRNA was detected by western blotting after treatment of the cells with or without rapamycin (100 nM). (I) The protein expression of p21 in Eca109 cells transfected or not with TRIB2 constructs was detected by western blotting after treatment of the cells with an HDAC2 inhibitor. (J) ChIP‐PCR was performed using the indicated ESCC cells to detect H3K9Ac and H3K14Ac enrichment at p21 promoter regions after treatment of the cells with or without an HDAC2 inhibitor (SCA, 2 μM). (K) The protein levels of p21 and phosphorylation of Akt (S473), S6K1 (S411), and HDAC2 (S394) in Eca109 cells transfected with or without TRIB2 constructs were detected by western blotting after the treatment of the cells with rapamycin (100 nM). (L) The mRNA levels of p21 in Eca109 cells transfected with or without TRIB2 constructs were detected by RT‐PCR after treatment of the cells with rapamycin (100 nM). (M) The protein levels of p21 and phosphorylation of S6K1 (S411) and HDAC2 (S394) in Eca109 cells transfected or not with S6K1 siRNA were detected by western blotting after treatment with rapamycin (100 nM). (N) The mRNA levels of p21 in Eca109 cells transfected or not with S6K1 siRNA were detected by RT‐PCR after treatment of the cells with rapamycin (100 nM). (O) The expression of METTL14, TRIB2, p21, HDAC2, and its phosphorylated form in CSC and non‐CSC subpopulations of ESCC cells were analyzed by western blotting. (P) The abundance of total‐HDAC2, p‐HDAC2, and p21 in the HDAC2‐KO or WT 293T cells transfected with the indicated constructs were determined by western blotting. (Q) ChIP‐PCR was performed in the HDAC2‐KO or WT 293T cells with or without TRIB2 overexpression to detect H3K9Ac and H3K14Ac enrichment at p21 promoter regions. (R) The abundance of total‐HDAC2, p‐HDAC2, and p21 in the HDAC2‐KO or WT 293T cells with or without SCA treatment was determined by western blotting. The data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. p‐values were determined by one‐way ANOVA with Tukey's post hoc test