Extended Data Fig. 8: Aldob K108bhb attenuates the mTOR signaling.
From: Ketogenic diet reshapes cancer metabolism through lysine β-hydroxybutyrylation
a, b HCC-LM3 cells were transfected with Aldob WT, K108Q, or K147Q, respectively. After 24 h, cell lysates were immunoprecipitated with GFP beads and probed with antibodies against Aldob and LKB1 (a). The ratios of LKB1 to GFP-Aldob derived from a densitometric analysis of the blot were shown (b). c Cells were stably expressed with Aldob WT, K108Q, K230A, D34S, or K147Q, respectively, and starved for glucose (GS) for 2 h or not starved. The indicated protein levels were detected by immunoblotting. d Representative confocal images showing the colocalization between LAMP1 (purple) and mTORC1 (red) in cells stably expressed with Aldob WT, K108Q, K230A, D34S, or K147Q, respectively. Cells were starved for glucose (GS) for 2 h or not starved, GFP indicates Aldob (green). e Colocalization analysis of LAMP1 and mTORC1 in cells in (d). Each dot represents Mander’s overlap coefficients from one cell. At least 30 cells from each group were used for analysis. MOCK (-GS) vs MOCK ( + GS): p value: 3.2e − 6; WT (-GS) vs WT ( + GS): p value: 2.8e − 5; f Cells stably expressing RNAi-resistant Aldob WT or K108Q were transfected with control or a mix of siRNA targeting Aldoa, Aldob, and Aldoc. The p-S6K and total S6K protein levels were detected by immunoblotting. g Cells stably expressed Aldob WT, K108R, or K147R were treated with 10 mM Na-β-OHB for 24 h. The p-S6K, total S6K and total Aldob protein levels were detected by immunoblotting. h Representative confocal images showing the colocalization between LAMP1 (purple) and mTORC1 (red) in cells stably expressed with Aldob WT, K108R, or K147R. The cells were treated with 10 mM Na-β-OHB for 24 h. GFP indicates Aldob (green). Scale bar: 2 μm. i Colocalization analysis of LAMP1 and mTORC1 in cells in (h). Each dot represents Mander’s overlap coefficients from one cell. At least 30 cells from each group were used for analysis. Similar results were obtained in three independent experiments (a-i). Statistical data are presented as mean ± SEM (a-i), P values were determined by one-way ANOVA, followed by Dunnett’s test (b, i), or by two-way ANOVA, followed by Tukey’s test (e). ns (not significant).
