Fig. 5: Mechanism of p300/HDAC2-mediated regulation of SmD2.

a Immunoprecipitation coupled with mass spectrometry analysis was used to identify SmD2-interacting proteins in HCCLM3 cells (blue arrow indicates SmD2 signal). b Mass spectrometry spectrum showing the peptide of p300. c Co-immunoprecipitation of exogenous SmD2 and p300 in HEK293T cells after transfection with the indicated plasmids. d Immunofluorescence analysis to detect colocalization of SmD2 and p300 in HCCLM3 cells. The experiment was repeated three times with similar results. e Immunoblot to detect the acetylation of endogenous SmD2 in HCCLM3 cells treated with the SIRT inhibitor (5 mM NAM) or the HDAC inhibitor (1 μM TSA) overnight. Ac-K, acetylated lysine. f Immunoblot to detect the acetylation of exogenous HA-SmD2 in HEK293T cells treated with TSA or NAM. g Immunoblot of input and anti-HA immunoprecipitates (IPs) derived from HEK293T cells transfected with HA-SmD2 and Flag-tagged GCN5, PCAF, TIP60, CBP, or p300. h Immunoblot of input and anti-HA IPs derived from HEK293T cells transduced with HA-SmD2 and shp300. i Immunoblot of SmD2 changes from HCCLM3 cells after EP300 depletion. j Immunoblot to detect the acetylation of exogenous SmD2 from HCCLM3 cells treated with FDA-approved HDAC inhibitors overnight. k Expression levels of SmD2 and γH2A.X from HCCLM3 treated with the HDAC inhibitors. WCL, whole cell lysate. l Immunoblot of input and anti-HA IPs transfected with HA-SmD2 and Flag-tagged HDAC1 or HDAC2. m Immunoblot of input and anti-HA IPs transduced with HA-SmD2 and shHDAC2. n Immunoblot of SmD2 changes from HCCLM3 cells after HDAC2 depletion. o Positive correlations of SNRPD2 (encoding SmD2) mRNA levels with HDAC2 mRNA levels in TCGA-LIHC dataset (n = 424, 374 of which were tumor samples and 50 adjacent normal tissues. r Pearson’s correlation coefficient, two-tailed t-distribution test). p Kaplan–Meier analysis of HCC patients from TCGA, stratified by HDAC2 mRNA expression levels. Patients are categorized into two cohorts: those with HDAC2 expression above the median and those with expression below the median. q Tumor growth of inducible shHDAC2 HCCLM3 cells in nude mice, with doxycycline orally administered, beginning on day 5. 2way ANOVA. r Representative images of tumor from HCCLM3-harboring mice. s Quantitative analysis of tumor (n = 10) volume and weight from (r). Mean values ± SEM. two-sided t-tests. Each Western blot was independently repeated three times. Source data are provided as a Source Data file.