Fig 2: Measures of MPS and anabolic signaling in response to treatment with CON, NAFLD and ESLD serum. Myotubes were treated for 4 h with ex vivo serum from age-matched control (CON), non-alcoholic fatty liver disease (NAFLD) and end stage liver disease (ESLD) patients. (a) Representative western blot for puromycin and total protein, (b) representative western blots for anabolic signaling targets and loading control, (c) puromycin incorporation, (d) phospho-mTOR (Ser2448)/total-mTOR, (e) phospho-eEF2 (Thr56)/total-eEF2, (f) phospho-p70S6K (Thr389)/total-p70S6K, (g) phospho-AktSer473/total-Akt, (h) phospho-RPS6 (Ser240/244)/total-RPS6, (i) phospho-4EBP-1 (Thr37/46)/total-4EBP-1. Data are expressed as the mean (cross), median (central horizontal line), 25th and 75th percentiles (box) and the minimum and maximum values (vertical lines), with n = 4 per group and each data point corresponding to the average of 3 technical repeats.

Fig 3: Evaluation of metabolic contributions to the enhanced nephrogenic phenotype in Six2TGC;Tsc1.A Immunofluorescence staining for the phosphorylated ribosomal protein S6 (p-rpS6, red) at P0 to assess mTorc1 activity in Tsc1 hemi- and homozygous NPC. Genotype noted on the top, key to antibody staining on the left. Scale bar denotes 50 µm. Magnified view of NPC (green) in the dashed area below each panel. B, C Seahorse analysis of cultured NPCs reveals no change in glycolytic function (n = 3 control Six2TGC and n = 3 Six2TGC;Tsc1 animals) or in mitochondrial respiration (n = 3 control Six2TGC and n = 4 Six2TGC;Tsc1 animals); error bars represent standard deviation from sample group mean. D Representative image of staining by the hypoxia indicator pimonidazole reveals strong staining in the proximal tubules relative to the GFP (Six2) expressing NPCs. Scale bar denotes 50 µm. E Quantification of pimonidazole staining intensity in NPC normalized to adjacent tubular regions to control for differences in dose efficacy (n = 4 littermate animals in sample group; n = 15 control Six2TGC and n = 12 Six2TGC;Tsc1 niches measured). Two-tailed unpaired t tests were performed in GraphPad Prism version 8 to evaluate statistical significance of pimonidazole signal; n.s. denotes not significant (p = 0.9613). Error bars represent standard deviation from the mean. Source data are provided as a Source Data file.

Fig 4: p110a is the principal PI3K isoform engaged in insulin signalling in brown adipocytes. Brown pre-adipocytes or differentiated adipocytes were treated with A66 (1 µM, blue graph bars) or TGX221 (0.5 µM, red graph bars) or a combination of both inhibitors (purple graph bars) followed by stimulation with 100 nM of insulin for 15 min at 37 °C. (A) Levels of Akt (T308) phosphorylation were determined by immunoblot analysis. Phosphorylation levels were normalised to total Akt levels detected in a second blot performed in parallel using the same lysates. (B) Ribosomal protein S6 (rpS6) (S240/244) phosphorylation was also detected on the same immunoblots. (C) The levels of expression of p110a and p110ß in pre-adipocytes and differentiated adipocytes used in the signalling experiments were also determined by immunoblot analysis. p110a and p110ß signal intensities were normalised to vinculin (used as a loading control). (D) Akt (T308) phosphorylation in p110a-deficient (p110aDEL) brown adipocytes treated and stimulated as above. Representative immunoblots and bar graphs with data from three or four (n = 3–4) independent experiments are shown. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparisons test between insulin-stimulated vehicle-treated and insulin-stimulated inhibitor-treated samples (A,B,D) or by unpaired two-tailed t-test (C). Comparisons between pre-adipocytes and differentiated adipocytes in (B) were performed by two-way ANOVA with Sidak’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 5: p110a is the principal PI3K isoform engaged in insulin signalling in 3T3-L1 adipocytes and Hepa 1.6 cells. 3T3-L1 pre-adipocytes or differentiated adipocytes or murine Hepa 1.6 hepatocytes were treated with A66 (1 µM, blue graph bars) or TGX221 (0.5 µM, red graph bars) or a combination of both inhibitors (purple graph bars) followed by stimulation with 100 nM of insulin for 15 min at 37 °C. (A) Levels of Akt (T308) phosphorylation in 3T3-L1 pre-adipocytes and adipocytes were determined by immunoblot analysis. Phosphorylation levels were normalised to total Akt levels detected in a second blot performed in parallel using the same lysates. (B) Ribosomal protein S6 (rpS6) phosphorylation (S240/244) was also detected in the same immunoblots. (C) The levels of expression of p110a and p110ß in pre-adipocytes and differentiated 3T3-L1 adipocytes used for the signalling experiments were also determined by immunoblot analysis. (D) Akt (T308) and rpS6 (S240/244) phosphorylation in Hepa 1-6 murine hepatoma cells treated and stimulated as above. Representative immunoblots and bar graphs with pooled data from three or four (n = 3–4) independent experiments are shown. Data are presented as mean ± SEM. (E) Insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with inhibitors as above. Data from three (n = 3) independent experiments are shown. Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparisons test between insulin-stimulated vehicle-treated and insulin-stimulated inhibitor-treated samples (A,B,D,E) or by unpaired two-tailed t-test (C). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.