Fig. 4: KMT5C suppresses PGC-1α degradation.

a Western blot analysis (top) (n = 5 each group) and protein quantification (bottom) of indicated proteins in the livers from 12-week-old female WT and KO mice after 24 h fasting. b Western blot (top) and densitometry analyses (bottom) were conducted in mouse livers expressing AAV-KMT5C or AAV-Mut (AAV-Ctrl, n = 5; AAV-KMT5C, n = 5; AAV-Mut, n = 4, male). Mice were fasted for 16 h before the assay. c AAV8-TBG-Ctrl or AAV8-TBG-Pgc-1α viruses were infused into the WT and KO mice. Four weeks later, the mice were starved for 24 h followed by western blot (top) analysis. The quantification of proteins was shown at bottom (WT + AAV-Ctrl, n = 5; KO + AAV-Ctrl, n = 4; KO + AAV-Pgc-1α, n = 5, 12-week-old male mice). d, e PTT (d) (n = 7 each group, male) and GcTT (e) (n = 7 each group, male) were performed in mice of (c) in the 3rd weeks after AAV infusion. f Representative western blot (left) and densitometry analysis (right) in primary hepatocytes expressing lentiviral Pgc-1α, Kmt5c, or Kmt5c Mut (n = 3 per group). Western blot analysis was performed 48 hours after lentivirus-mediated gene expression. g Lentiviruses carrying the shRNA targeting Pgc-1α infected Kmt5c-overexpressing primary hepatocytes for 48 h. Indicated proteins were detected by western blot analysis (n = 2 per group). h Co-immunoprecipitation analysis was conducted to assess the interaction between PGC-1α and KMT5C in liver tissues. Mice were starved for 16 h following the immunoprecipitation with PGC-1α antibody in the hepatic nuclear extracts. i The binding affinity between PGC-1α and KMT5C was determined by microscale thermophoresis (MST) assay (n = 4 per group). Inset: thermophoretic movement of fluorescently labeled proteins. Kd represents the dissociation constant. j Immunoblot analysis was conducted to assess the ubiquitination level of PGC-1α in Kmt5c-overexpressed primary hepatocytes. Primary hepatocytes were infected with lentivirus expressing vector, KMT5C, or Mut for 48 h, followed by treatment with 10 µM MG132 for 6 h. Then the ubiquitination of PGC-1α was analyzed. k Immunoblot to detect the ubiquitination level of PGC-1α in primary hepatocytes. Primary hepatocytes isolated from WT and KO mice were treated with 10 µM MG132 for 6 hours, followed by immunoprecipitation and western blot analysis. a–f, i data present mean ± s.e.m. a–e, n presents biologically independent animals; f, g, i, n represents biologically independent cell samples. a two-tailed unpaired Student’s t test; b–f one-way ANOVA. All experiments here were repeated independently three times with similar results. Source data are provided as a Source Data file.