Extended Data Fig. 10: The ENO2/PEP/HDAC1 signaling contributes to ENO2-mediated β-catenin acetylation in CRC cells.
a, Western blot analysis of ENO1, ENO3, and PKM2 in drug-sensitive, Bev-resistant, and Rego-resistant CRC xenograft tumors and the corresponding drug-sensitive cells and tumors. Bev, bevacizumab. Rego, regorafenib. b, IHC staining and quantification of ENO1, ENO3, and PKM2 in antiangiogenic drug-sensitive and -resistant CRC xenograft tumors. Scale bar, 50 µm. n = 5 mice per group. Representative IHC images are shown. c, Western blot analysis of ENO1, ENO2, ENO3, and PKM2 in antiangiogenic drug-sensitive and -resistant HCT116 cells. d, Western blot analysis of ENO1 and ENO2 in parental and drug-resistant HCT116 cells. e, Western blot analysis of ENO1, ENO2, and ENO3 in HCT116 cells transfected or treated as indicated. f, Western blot analysis of Ac-K49-β-catenin, β-catenin, CHGA, SYP, and TUBB3 in CRC cells transfected or treated as indicated. g, Identification of direct binding of PEP to rhHDAC1 and rhHDAC2 was determined by DARTS approach. rhHDAC1, recombinant human HDAC1. rhHDAC2, recombinant human HDAC2. h, i, The binding sites and hydrogen-binding numbers of the interaction of PEP with HDAC1 (PDB: 4BKX) and HDAC2 (PDB: 6XEB) were predicted using AutoDock Vina software. j. The binding modes of the interaction of PEP with HDAC1 and HDAC2 were predicted by molecular dynamics (MD) simulation using GROMACS 2020.3 software. k, An in vitro enzyme activity assay was conducted to evaluate the effect of PEP on the activity of HDAC1 and HDAC2. l, Western blot analysis of Ac-K9-Histone H3 and Ac-K27-Histone H3 in PEP-treated CRC cells transfected as indicated. (a, c, d-f, g, l, representative of n = 3 independent replicates. b, n = 5 mice per group. k, 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 (b and k).
