Fig. 5: KMT5C competes against RNF34 to interact with PGC-1α.

a HEK293T cells were co-transfected with plasmids as indicated for 48 h, followed by treatment with 10 µM MG132 for 6 h. Immunoprecipitation was then performed. b, c The truncated HA-Pgc-1α plasmids and Flag-Kmt5c (b) or Flag-Rnf34 (c) were transfected into HEK293T cells. Immunoprecipitation was conducted by HA antibody after transfection for 36 h. d The indicated plasmids were transfected into HEK293T cells. Immunoprecipitation was conducted to show the interaction among PGC-1α, RNF34, and KMT5C (upper). The level of PGC-1α associated with RNF34 was quantified (lower panel, n = 3). e The indicated plasmids were transfected into HEK293T cells. At 36 h after transfection, cells were treated with CHX (50 μM) for the indicated time, followed by western blot analysis. f Western blot (left) and densitometry analyses (right) of indicated proteins in primary hepatocytes (n = 3 per group). Primary hepatocytes isolated from 2-month-old WT and KO mice were infected with shRnf34 or control lentivirus for 48 h following the western blot analysis. g Glucose production using pyruvate and lactate as substrates was measured in the primary hepatocytes. The cells were treated as in (f) (n = 3 per group). h TBG promoter-driven AAV-shRnf34 and AAV-Scr were injected into 2-month-old WT and KO mice. After three weeks, blood glucose levels were measured under both fed (n = 7 each group, male) and 16-hour fasting conditions (n = 7 each group, male). i, j PTT (i) (n = 6 each group, male) and GcTT (j) (n = 6 each group, male) assay in the mice of (h). k Representative western blot analysis (top) and densitometry analyses (bottom) in the livers of the indicated mice after 16 h fasting (WT + Scr, n = 3; KO + Scr, n = 3; WT + shRnf34, n = 4; KO + shRnf34, n = 4, male). Mice were treated as in (h). l Serum insulin levels in mice of (h) underfeeding and 16-hour fasting condition (WT + Scr, n = 7; KO + Scr, n = 7; WT + shRnf34, n = 6; KO + shRnf34, n = 7, male). m Immunoprecipitation assay to detect interaction among PGC-1α, RNF34, and KMT5C in the mouse liver. Liver nuclear extracts were prepared from fed and 24h-fasting mice. The PGC-1α protein complex was immunoprecipitated using PGC-1α antibody, followed by western blot analysis with a similar amount of PGC-1α in input and IP samples. n Immunoprecipitation assay to detect interaction among PGC-1α, RNF34, and KMT5C proteins in primary hepatocytes. Primary hepatocytes isolated from C57BL/6 mice were treated with 100 nM glucagon for 16 h, followed by immunoprecipitation using PGC-1α antibody. KMT5C and RNF34 levels in the precipitates were analyzed by western blot. d, f–l, data present mean ± s.e.m. d, f, g n represents biologically independent cell samples; (h–l) n presents biologically independent animals. Significance analysis was performed using one-way ANOVA. All experiments here were repeated independently three times with similar results. Source data are provided as a Source Data file.