Fig 2: Supplementation of 1,25(OH)2D3 or overexpression of Sirt1 alleviates hepatic glucose overproduction and glucose metabolism disorders caused by 1,25(OH)2D3 deficiency. (a) PTT performed in mice of different treatment groups. Blue line: RDWT group; red line: RDKO group; purple line: RD 1α(OH)ase-/-SIRT1Tg group; green line: ND 1α(OH)ase-/-+1,25(OH)2D3 group. (b) GTT performed in mice of different treatment groups. Blue line: RDWT group; red line: RDKO group; purple line: RD 1α(OH)ase-/-SIRT1Tg group; green line: ND 1α(OH)ase-/-+1,25(OH)2D3 group. (c) Expression levels of proteins including VDR, Sirt1, Rictor, PCK1, and G6PC collected from liver samples of mice of each group under normal feeding conditions. (d) Phosphorylation of AKT-S473 and FOXO1-S256 in the liver of mice stimulated with insulin. (e) VDR and Sirt1 protein expression levels in HepG2 cells treated with different concentrations of 1,25(OH)2D3 or Sirt1 agonists (SRT1720HCL) were determined by Western blotting. (f) Glucose production content of HepG2 cells with knockdown of VDR or treated with 1,25(OH)2D3 or SRT1720 HCL in response to pyruvate stimulation. ∗∗∗P < 0.001; blue bar: si-NC group; red bar: si-VDR group; purple bar: 1,25(OH)2D3 group; green bar: SRT1720HCL group. (g) Detection of mRNA expression of related genes in the liver of different treatment groups (qRT-PCR). ∗P < 0.05. (h) Western blotting was performed to detect the expression levels of related proteins in different treatment groups of HepG2 cells. (i) The phosphorylation of AKT-S473 and FOXO1-S256 in HepG2 cell treated with different concentrations of 1,25(OH)2D3 and Sirt1 agonist (SRT1720HCL) in response to insulin stimulation was measured by Western blotting.

Fig 3: Protein expression and phosphorylation state of the mechanistic target of rapamycin (mTOR) signalling after inhibition of Hedgehog and/or mTOR signalling. Primary hepatocytes from C57BL/6N mice were isolated and incubated for 24 h with 10 µM cyclopamine, 50 nM rapamycin, 5 µM Torin and combinations of them. (A,L): Densitometric evaluation of the band intensities of the Western blots normalized on the total protein staining, relative to DMSO control (dashed line). (A,B): mTOR and phosphorylated (p) mTOR (Ser2448) of male (A) and female (B) hepatocytes. (C,D): Raptor and pRaptor (Ser792) of male (C) and female (D) hepatocytes, (E,F): Pras40 and pPras40 (Thr246) of male (E) and female (F) mice. (G,H): Rictor and pRictor (Thr1135) of male (G) and female (H) hepatocytes. (I,J): p70S6 and pp70S6 (Thr389) of male (I) and female (J) hepatocytes. (K,L): 4E-BP1 and p4E-BP1 (Thr37/46) from male (K) and female (L) hepatocytes. Significance was calculated with one-way ANOVA for repeated measurements. Stars show the significance to Dimethyl sulfoxide (DMSO) control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; n = 3–5.

Fig 4: Effect of Raptor and Rictor siRNA on rickettsial replication. ECs were transfected with Raptor or Rictor siRNA (25 or 50 nM), along with control siRNA (50 nM), using Lipofectamine RNAiMAX 24 h prior to infection with R. rickettsii (SS) for 48 h. DNA was isolated, and rickettsial copy number was measured (A). Total protein lysates were prepared and subjected to Westen blot using antibodies against Raptor and Rictor to determine the extent of Raptor knockdown (B) and Rictor knockdown (C). The α-tubulin antibody was used as a loading control, and a representative blot (n ≥ 3) is shown. In some experiments, (C) statistically significant changes are shown as ** = p ≤ 0.001 and *** = p ≤ 0.0001.

Fig 5: Differential activation of mTORC1 and mTORC2 in PTENwt and PTENmu GBM cells along with downstream signaling. (a) Schematic representation of proposed hypothesis: role of PTEN in mTORC2 signaling pathways. (b–h) PTENmu and PTENwt cells were harvested and lysed and kept in phosphate-buffered saline (PBS) with protease and phosphatase inhibitors. Protein concentrations were measured by the Bradford's protein assay reagent. Proteins (50-100 μg) from each sample were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 5–12%) followed by electrotransfer into nitrocellulose/polyvinylidene difluoride (PVDF) membrane and probed with primary antibodies (1:1000–1:2000) and incubated overnight at 4 oC. Then the membrane was washed with Tween-20 (0.1%) containing Tris-buffered saline (TBS) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000). They were washed and target proteins were identified by the West Pico chemiluminiscence detector system or Biorad ChemiDoc MP System (Bio-Rad). Developed bands of corresponding proteins were densitometrially analyzed by ImageJ software. (b) Level of PTEN in PTENmu and PTENwt cells. (c) Status of mTOR, p-mTOR Ser2448, p-mTOR Ser2481, Rictor and Raptor. (d) AKT, the downstream substrate of mTORC2 was highly phosphorylated at Ser473 in PTENmu cells. (e) Status of AKT phosphorylation at the Thr450 and Thr308 positions. (f) Densitometric analysis showed phosphorylation of AKT at the Thr450, Thr308 and Thr 473 sites. Values were normalized against the relative expression of β-actin and total AKT, quantified by ImageJ software. Each value was the mean±s.d. of three independent experiments. *P<0.05, significant difference between two test groups. (g) Status of Ser9 phosphorylation of GSK3β, the substrate of activated AKT. (h) mTORC1 activity readout as identified by phosphorylation of S6K1 and 4E-BP1. β-Actin served as loading control for all the western blots.