Extended Data Fig. 8: ENO2-derived PEP induces β-catenin acetylation by inhibiting HDAC1 activity in CRC.
a, Co-IP assay was performed to evaluate the effect of ENO2 on the binding of β-catenin to CBP, PCAF, and P300 in HCT116 cells. b, c, ENO2-silenced HCT116 cells were treated with different HDAC inhibitors (ENT, Entinostat, 5 μM; Vorinostat, 10 μM; BEL, Belinostat, 1 μM; TSA, Trichostatin A, 1 μM; SAN, Santacruzamate A, 1 μM; TUB, Tubacin, 5 μM; TMP, TMP269, 10 μM) for 24 h, and (b) Ac-K49-β-catenin expression and (c) β-catenin transcriptional activity were determined. d, e, Western blot analysis of the expression of HDACs in (d) ENO2-transfected HCT116 cells and (e) in HCT116 cells transfected as indicated. f, g, ENO2-silenced HCT116 cells were transfected with the indicated siRNAs, and (f) Ac-K49-β-catenin expression and (g) β-catenin transcriptional activity were evaluated. h, The effects of PEP (1 mM) and HDAC1 on CRC cell viability were assessed by MTT assay. i, The levels of β-catenin target genes in CRC cells transfected and treated as indicated were evaluated by qPCR assay. j, The interaction between β-catenin and HDAC1 in ENO2-silenced HCT116 cells was assessed by Co-IP assay. k, The inhibitory effect of ENO2 on HDAC1 activity was evaluated in HCT116 cells. l, The effect of PEP on the interaction of β-catenin with HDAC1 in HCT116 cells was determined by Co-IP assay. m, The interaction of PEP with wild-type or the mutant HDAC1 (K58R, H141D, and Y303D) in SW620 cells was determined by DARTS approach. n, The effects of PEP (1 mM) and HDAC1 on CRC cell viability were assessed by MTT assay. o, qPCR assay was conducted to assess the effect of HDAC1 on the expression of β-catenin target genes in PEP-treated CRC cells. (a, b, d–f, j, l, m, representative of n = 3 independent replicates. c, g–i, k, n, o, n = 3 biologically independent experiments). Data are presented as the mean ± s.e.m. ns, not significant. P values were determined by unpaired two-tailed Student’s t-test (c, g, i, k and o) or two-way ANOVA with Tukey’s multiple comparison test (h and n).
