Fig 2: BX-795 inhibits AKT pathway. (A) Gene expression analysis of PDK1, AKT1, MTOR, TBK1, and MYCN in response to BX-795 treatments in SH-SY5Y cells. (B) Western blot analysis of different AKT pathway proteins in response to increasing concentrations of BX-795 treatments. CyPB is used as loading control. * p < 0.05; ** p < 0.01.

Fig 3: CUDC-907 inhibits PI3K/AKT pathway: (A) Gene expression analysis of PIK3C2A, AKT1, MTOR, TBK1, and MYCN in response to CUDC-907 treatments in SH-SY5Y cells. * p < 0.05, ** p < 0.01, and *** p < 0.001. (B) Western blot analysis of different PI3K pathway proteins in response to increasing concentrations of CUDC-907 treatments. CyPB was used as a loading control. Full blots and densitometric data are available in Supplementary Figure S2.

Fig 4: Glucagon-induced gluconeogenesis strongly correlates with changes in enzyme expression. mRNA levels (normalized to Actb) of G6pc (A), Pck1 (B), and Pcx (C) in mouse primary hepatocytes treated with increasing concentrations of glucagon. n = 6 biological replicates per group. D, Western blot for G6PC, PCK1, and PCX proteins treated with increasing concentrations of glucagon. CYPB is a loading control. E, glucose production rate (nmol/h) in mouse primary hepatocytes treated with increasing concentrations of glucagon. n = 6 biological replicates per group. F, a model of glucagon action on the GNG pathway showing the stimulation of PCK1 and G6PC expression. All data are expressed as mean ± SEM. ∗∗p < 0.01; ∗p < 0.05; ns = not significant. Statistical analysis was performed using one-way ANOVA. All comparisons are against the no-glucagon and no-inhibitor control groups. CYPB, cyclophilin b; GNG, gluconeogenesis; PCX, pyruvate carboxylase.

Fig 5: NO production and NOS expression in CD8+ T cells. A, CD8+ T cells were activated with anti-CD3/CD28 Dynabeads (or SIINFEKL peptide, when using OT-I CD8+ T cells) for 1 to 4 days in 21%, 5%, and 1% O2. After activation, NO production and NOS expression were analyzed. B, NO production determined by extracellular quantification of nitrites (NO2-, a NO byproduct) in mouse CD8+ T cells activated for 3 days (N = 11–13). C, Western blot analysis of NOS using a panNOS antibody (all isoforms detected) in lysates of mouse CD8+ T cell activated for 3 days. Results normalized to total protein stain (TPS, top) and representative blot (bottom; N = 3). D, Time course qRT-PCR analysis of Nos2 and Nos3 mRNA expression in activated mouse CD8+ T cells (N = 3–9). E, Western blot analysis of NOS2 protein levels in mouse OT-I CD8+ T cells treated or untreated with 50 µmol/L FG-4592 and activated for 3 days in 1% O2 with increasing amounts of SIINFEKL peptide (0.001, 0.1, 1, and 1000 ng/mL). Quantification normalized to TPS (top) and representative blot (bottom). BMDMs polarized to M1 with 100 U/mL LPS were used as positive control for NOS2 expression (N = 3). F, NO production as determined by extracellular quantification of nitrites in human T cells cultured for 1 day in 21% and 1% O2 with or without anti-CD3/CD28 beads (N = 4). G, Time course qRT-PCR analysis of NOS2 and NOS3 mRNA levels in activated human CD8+ T cells. NOS1 mRNA levels were under the detection limit (N = 5–8). H, Western blot analysis of panNOS (antibody detecting all NOS isoforms) and NOS3 in human CD8+ T cells. HUVEC cells were used as positive control and PPIB was used as loading control (representative of N = 3). Apart from panel D and G, each data point represents an independent animal and results are shown as median ± interquartile range (IQR). *, P < 0.05; Wilcoxon matched-pairs signed-rank test.