Fig. 5

BHB suppresses malignant phenotypes of PCa via β-hydroxybutyrylation of INMT. A, Detection of kbhb modifications in 293T cells with IMNT overexpression. The Flag–INMT fusion proteins were purified from 293T cells treated with or without 15 mM BHB and detected via western blot assay using anti-INMT or anti-pan-β-hydroxybutyrylated lysine antibodies (BHB-K). B-D, kbhb modification of endogenous INMT in LNCaP, PC3, and DU145 cells. Cell lysates treated with or without 15 mM BHB were subjected to immunoprecipitation (IP) with BHB-K antibodies and then detected via western blot assay with INMT antibodies. Whole-cell lysates without IP were also detected via western blot assay with INMT, BHB-K, or actin antibodies as the input. E, Identification of enzymes regulating the kbhb of INMT via immunoprecipitation. Cell lysates with indicated treatment were immunoprecipitated (IP) with BHB-K antibodies and then detected via western blot assay with INMT antibodies. Input cell lysates were also subjected to INMT detection via western blot assay. F, Western blot analysis of the effects of BHB-mediated kbhb on INMT expression. Cell lysates treated with 15 mM BHB or 2.5 µM p300 inhibitor A485 were subjected to western blot assay with INMT and actin antibodies. G, Colony formation assays to assess the effects of BHB-mediated kbhb on in vitro tumor growth. H, Transwell assays were performed to evaluate the effects of BHB-mediated kbhb on migration and invasion of indicated cells. Experiments were performed in triplicate, and data were expressed as the mean ± standard deviation (SD). Paired and two-tailed Student’s t-tests were performed, and significant differences were designated as *p < 0.05, **p < 0.01, and ***p < 0.001