Extended Data Fig. 6: KD and Na-β-OHB treatment inhibits the mTOR signaling pathway.
From: Ketogenic diet reshapes cancer metabolism through lysine β-hydroxybutyrylation
a, b Immunoblotting shows liver Aldob protein levels in male mice fed Ctrl or KD for 5 weeks (n = 4 per group). The graph showed ratios of Aldob to GAPDH quantified by densitometry using Image J software. c, d Representative immunoblotting of post-translational modifications (PTMs) from SK-Hep1 cells treated with 10 mM Na-β-OHB for 24 h (n = 3 biological replicates). Ctrl Kbhb vs KD Kbhb: p value: 7.2e − 7; e Immunoblotting shows the time course of Kbhb and Kac of Aldob in GFP-Aldob HCC-LM3 cells. HCC-LM3 cells transiently transfected with GFP-Aldob were treated with 10 mM Na-β-OHB for multiple time points, and Aldob was immunoprecipitated by its GFP tag. Total Kbhb or Kac levels were also tested. f, g HCC-LM3 (f) or SK-Hep1 cells (g) were treated with 10 mM Na-β-OHB for 24 h, and whole cell lysates were probed by immunoblotting with indicated antibodies. h HCC-LM3 cells were transiently transfected with GFP-Aldob, treated with 10 mM Na-β-OHB for multiple time points, and Aldob was immunoprecipitated by its GFP tag. Samples were first quantified by immunoblotting. The enzymatic activity of Aldob was measured by monitoring the consumption of NADH. i, j Quantification of p-S6K (i) and p-4EBP1 (j) levels in the livers of male mouse fed Ctrl or KD for 5 weeks (n = 6 per group). k Immunoblotting shows p-AMPKα and total AMPKα levels in the livers of male mice fed Ctrl or KD for 5 weeks (n = 6 per group). l, m Quantification of p-AMPKα levels from liver whole-cell lysates (n = 6 per group). n, o Immunoblotting shows the levels of p-S6K and total S6K in HCC-LM3 cells (n) and SK-Hep1 cells (o) treated with Na-β-OHB at a variety of concentrations (1, 5, 10, 20 mM). Similar results were obtained in three independent experiments (c, e-h, n, o). Each dot represents an independent experiment with three technical replicates (d, h). Statistical data are presented as mean ± SEM (b-m), P values were determined by one-way ANOVA, followed by Dunnett’s test (h), or by unpaired two-tailed t test (b, d, i, j, l, m). ns (not significant).
