Fig 2: Targeting AKT2 enhances antiviral defense in mice AFACS analysis of the percentage of macrophages (CD11b+ F4/80+ cells) in WT and Akt2 KO BM and spleen.BSurvival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 h (1 × 107 PFU/g, i.v.).CqRT–PCR analysis of the Ifnb1 mRNA expression in the liver (n = 8), spleen (n = 7) and lung (n = 7) of WT or Akt2 KO mice 6 h after the injection of VSV (1 × 106 PFU/g, i.v.).DIFNβ1 concentrations from WT (n = 8) and Akt2 KO (n = 7) serum were detected by ELISA 3 h after VSV infection (1 × 106 PFU/g, i.v.).EWT and Akt2 KO mice were injected with VSV for 24 h (1 × 106 PFU/g, i.v.). Representative images of the VSV‐infected cells (GFP) were collected by fluorescence microscope (left panel) and the VSV copies (WT, n = 6; Akt2 KO n = 7) were measured by qRT–PCR (right panel) in the liver of mice. Bar, 100 μm.F–HBone marrow cells from WT and Akt2 KO mice (CD45.2) were adoptively transferred to lethally irradiated CD45.1 mice and infected with VSV. Survival rates (F left panel), serum IFNβ1 concentrations (F right panel, n = 5), VSV copies (G, n = 9) and representative images (H) were analyzed as Fig 5B, D and E, respectively. Bar, 100 μm.ISurvival rates of WT mice that were intraperitoneal injected with CCT128930 (20 mg/kg) twice every other day before VSV infection (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a paired t‐test (C and F right panel), or unpaired t‐test (D, E right panel and G) or log‐rank (Mantel–Cox) test (B, F left panel and I). Data are representative of three independent experiments, respectively (A–I), in which the different groups of mice were sacrificed and quantified for indicated experimental purposes (C–E, F right panel, G). Error bars (D, E right panel and G, mean ± SD). Source data are available online for this figure.

Fig 3: Loss of TBK1 kinase activity diminishes insulin receptor interaction and enhances insulin sensitivity on HFD. (A) Liver lysates from ND or HFD-fed Tbk1Δ/Δ and Tbk1+/+ mice were probed for pIRβ(S994) and IRβ. GAPDH is shown as a loading control. (B) Liver lysates from ND- or HFD-fed Tbk1+/+ and Tbk1Δ/Δ mice were immunoprecipitated with an antibody against TBK1 and assayed for IRβ and pIRβ(S994). Normal rabbit IgG was used as a control for specificity (IgG lane). Lysates for immunoprecipitation were immunoblotted with antibodies against pIRβ(S994), IRβ, and TBK1 for input. Liver (C) and WAT (D) lysates from ND- or HFD-fed Tbk1Δ/Δ and Tbk1+/+ mice injected intraperitoneally with insulin (1 U/kg of body weight) or water as a control were probed for indicated proteins. All mice in these experiments were fasted overnight before sacrifice. (E) Total IRS1 protein levels in liver, WAT and muscle tissue lysates from 14-week-old HFD-fed Tbk1Δ/Δ and Tbk1+/+ mice were detected by immunoblot and quantified. GAPDH, IRβ and AKT2 were used as internal loading controls. All mice are from 129S5 background. Statistical analysis by Student's t-test. *p < 0.05, ***p < 0.001.

Fig 4: Signaling pathway activation by hotspot and non-hotspot Akt1 and Akt2 mutantsImmunoblotting was performed on lysates from: A. IL-3-deprived BaF3 cells. B. Serum-starved Rat1a cells. Note, Akt T308/9 western shown is from the same gel; however samples were in a different order from the other blots and Akt2 D32H was not run. C. MCF-10A cells cultured in the absence of EGF. EV, empty vector control. WT, wild type. Note that all images are cropped to show only the relevant bands.

Fig 5: Alteration of cellular bioactivies with the changes of rpS6 phosphorylation from Akt2 interference or phosphorylation inhibition. a CCK-8 assay revealed rpS6 hyphosphorylation from Akt2 overexpressing in HBE greatly promoted cells proliferation (left; P < 0.05), and p-rpS6 reducing in H1650 and SK-MES-1 cells from Akt2 knockdown or phosphorylation inhibition markedly deteriorated cells proliferation (middle and left; P < 0.05). b Influence of p-rpS6 on cell cycles in HBE and NSCLC cell lines (P < 0.05). c Wound-healing assay was performed after rpS6 hyphosphorylation in HBE (left; P < 0.05), and cell migration assay was carried out after p-rpS6 reduce in H1650 and SK-MES-1 cell lines (middle and left; P < 0.05). CCK-8 experiments were carried out in sextuplicate, and the remaining tests were triplicate. Data are presented as mean ± SD. oe-Akt1: overexpression of Akt1; oe-NCa1: negative control for oe-Akt1; oe-Akt2: overexpression of oe-Akt2; oe-NCa2: negative control for oe-Akt2; oe-Akt3: overexpression of oe-Akt3; oe-NCa3: negative control for oe-Akt3. sh-Akt1: knockdown for Akt1; sh-NCa1: negative control for sh-Akt1; sh-Akt2: knockdown for Akt2; sh-NCa2: negative control for sh-Akt2; sh-Akt3: knockdown for Akt3; sh-NCa3: negative control for sh-Akt3. *: vs the corresponding blank control cell lines without any transfection (HBE, H1650 and SK-MES-1 respectively), P < 0.05; #: vs the corresponding control cell lines with the negative control (NC) vectors transfection (HBE + oe-NC, H1650 + sh-NC, SK-MES-1 + sh-NC, respectively), P < 0.05