Fig 2: Upregulation of IGF1R mediates resistance to p110α + p110β dual blockade.a, b Growth assay of PDO lines MSK-PCa11 and MSK-PCa12 treated with BYL719 (1 µM), AZD8186 (250 nM), BYL719 + AZD8186 or Veh. Cell number was read using CellTiter-Glo assay on Day 1, Day 4, and Day 7, respectively. c Western blot showing levels of IGF1R/INSR and AKT signaling in PDO lines MSK-PCa11 and MSK-PCa12 treated with BYL719 (1 µM) + AZD8186 (250 nM) or Veh for 4 h. d Western blot showing levels of IGF1R and AKT signaling in MSK-PCa12-shRenilla or MSK-PCa12-shIGF1R lines treated with BYL719 (1 µM) + AZD8186 (250 nM) or Veh for 4 h. e CellTiter-Glo assay showing cell viability of MSK-PCa12-shRenilla or MSK-PCa12-shIGF1R lines treated with BYL719 (1 µM) + AZD8186 (250 nM) or Veh, Day 7. f Growth of MSK-PCa12-shRenilla or MSK-PCa12-shIGF1R tumors in SCID mice treated with BYL719 (25 mg/Kg) + AZD8186 (75 mg/Kg) or Veh (n = 5 mice per group). g Western blow showing levels of INSR, IGF1R, pAKT, pPRAS40, and pS6 in MSK-PCa12-shRenilla or MSK-PCa12-shIGF1R tumors from (f), 14 days post treatment. h Volume of MSK-PCa12 tumors in SCID mice treated with linsitinib (40 mg/kg), BKM120 (50 mg/kg), linsitinib + BKM120, or Veh, 14 days post treatment, box and whiskers Min to Max.all points (n = 10 mice per group). i Body weight analysis of mice in (h) (n = 10 mice per group). Source data are provided as Source data file. All assays were performed with three biological replicates. ****p < 0.0001, ***p = 0.0003, **p = 0.0041, n.s: not significant, a, b: multiple t-test two-sided, e, h: one-way ANOVA compared to Veh group, f: one-way ANOVA compared to shIGF1R group treated with BYL719 + AZD8186, a, b, e: error bar represents mean values ± SD, f, h: error bar represents mean values ± SEM, i: error bar represents ± SD.

Fig 3: Tacrolimus (TAC) treatment increased the amount of triglycerides in vivo and in vitro.a, b Triglyceride and cholesterol levels measured by the glycerol lipase oxidase (GPO-PAP) method in Huh7 and Hep-G2 cells treated with different concentrations of TAC. c FASN, SREBP2, and AKT protein expression in Huh7 and Hep-G2 cells analyzed by western blotting. d Body weight of mice assessed every week in the control (black), low-dose TAC (red), and high-dose TAC (blue) mouse groups (n = 8 per group). e, f Serum total cholesterol (TC) and triglycerides (TG) assessed 8 weeks after the initial TAC injection. g Hepatic triglyceride levels were measured by Oil red O staining. h FASN, SREBP2, and AKT protein expression in mouse liver was analyzed by western blotting. Data represent the mean ± SD. *P < 0.05, **P < 0.01, NS, not significant, TAC groups vs. control. The experiments were independently repeated at least three times.

Fig 4: Inhibition of PHDs increases insulin and decreases glucagon sensitivity in primary mouse hepatocytes. (A–C) Primary mouse hepatocytes were isolated from NCD/lean WT mice, treated with 10 μg/ml (or indicated doses of) PHDi for 24 h, and subjected to Western blot and qRT-PCR analyses for determining protein expression of HIF-1α (A) and HIF-2α (B) and mRNA expression of HIF target genes (C; n = 3 mice for vehicle and 4 mice for PHDi-treated groups). (D) Insulin-stimulated Akt phosphorylation. Primary WT mouse hepatocytes were treated with or without 500 μM PA, +/− 10 μg/ml PHDi for 24 h, and then stimulated with or without 100 nM insulin for 15 min. Total (tAkt) and phosphorylated (pAkt) Akt levels were determined by Western blots. Samples from 3 independent experiments were ran in a single gel, and the pAkt:tAkt ratio in each sample was calculated and graphed. (E) Western blot analysis of insulin-stimulated FOXO1 phosphorylation in primary hepatocytes treated with or without PHDi. Mouse primary hepatocytes were treated with 10 μg/ml PHDi for 24 h and then stimulated with insulin for 10 min. (F) HIF-1α, HIF-2α, and IRS-2 protein levels were determined by Western blots in primary WT mouse hepatocytes treated with or without 500 μM PA, +/− 10 μg/ml PHDi for 24 h. (G) Western blot analysis of insulin-stimulated Akt phosphorylation. Primary mouse hepatocytes transfected with mock or Irs2-specific siRNAs. Twenty-four hours after siRNA transfection, cells were treated with 500 μM palmitic acid (PA) + vehicle or PA + PHDi for an additional 24 h, followed by 10 min insulin simulation. (H) Intracellular cAMP levels. Primary WT mouse hepatocytes were pretreated with or without PHDi for 24 h. After washing, cells were stimulated with or without 50 ng/ml glucagon (Gcg) for 7 min and subjected to cAMP assays (n = 3 wells per group). AU, arbitrary unit. (I) Primary WT mouse hepatocytes were pretreated with or without 10 μg/ml PHDi for 24 h. Cells were washed and treated with or without glucagon for 7 min. Total (tCREB) and phosphorylated (pCREB) CREB levels were determined by Western blots. Samples from 3 independent experiments were ran in a single gel and the pCREB:tCREB ratios were calculated and graphed. (J) mRNA expression of Pepck and G6pase in primary hepatocytes treated with or without 10 μg/ml PHDi for 24 h. ∗P < 0.05 by the Student's t tests (F, J) or by the 1-way ANOVA with post hoc t tests between the individual groups (C, D, and H).

Fig 5: The apoptotic molecular mechanism was investigated by Human Apoptosis Antibody Array. (A) The expression of apoptosis-related proteins in pancreatic cancer (PC) cells infected with shEIF3B was measured by enhanced chemiluminescence (ECL) with Human Apoptosis Antibody Array. The results circled in red indicated that the protein expression was up-regulated and P < 0.05. (B) Protein expression was presented in grayscale and visualized by R studio. (C) The expression levels of apoptosis-related proteins were analyzed in PC cells with shEIF3B. (D) The expression of Akt, p-Akt, CCND1, CDK6, and PIK3CA was detected by western blot in SW1990 cells of the shCtrl and shEIF3B groups. Results are presented as mean ± SD. *P < 0.05, **P < 0.01.