Fig 2: Plk2 presence in tubular adenomas coincides with the absence of activated mTOR. A) Plk2 expression in tubular adenoma (patient C), 200×, B) Phosphorylated mTOR ser2448 is negative in the same tumor area as panel A, 200×.

Fig 3: The effects of i.t. Ad-PTEN on CCI-induced upregulation of TNF-α and phospho-mTOR in spinal astrocytes. Spinal cord sections (10 μm) obtained 14 days post-surgery from sham-operated plus i.t. vehicle (A, D), CCI plus i.t. Ad-GFP (B, E), and CCI plus i.t. Ad-PTEN (C, F) groups. Confocal double-immunofluorescent staining of TNF-α (red; A-C) or phospho-mTOR (red; D-F) with GFAP (astrocyte specific marker; green) in the dorsal horn region of the lumbar spinal cord. The merged images (yellow; white arrow) indicate co-localization of TNF-α or phospho-mTOR with GFAP immunoreactive cells in the spinal cord. The confocal results showed that spinal TNF-α and phospho-mTOR were primarily co-localized with astrocytes in the CCI group, whereas i.t. Ad-PTEN significantly attenuated CCI-induced upregulation of spinal TNF-α and phospho-mTOR immunoreactivity in astrocytes. Scale bars are 50 μm for all images.

Fig 4: mTOR kinase activity remains high when IPF fibroblasts are cultured on collagen matrix.A) Left, 2×105 serum starved control lung (n = 11) and IPF fibroblasts (n = 10) were cultured on polymerized collagen in serum free medium for 24 h and ser 2448 p-mTOR (p-mTOR) levels were measured by Western analysis. Shown is a Western analysis for p-mTOR levels in all IPF and control fibroblasts grown in serum free medium on polymerized collagen. GAPDH was used as a loading control. Right, Shown is a box-and-whisker plot of p-mTOR protein expression in 11 control and 10 IPF fibroblasts normalized to GAPDH on collagen matrix. p<0.001 IPF versus control fibroblasts. B) Upper, randomly selected 2×105 of 3 control or of 3 IPF fibroblasts were cultured on polymerized collagen in serum free medium as a function of time and ser 2448 p-mTOR levels were measured. GAPDH was used as a loading control. Lower, shown is the p-mTOR/GAPDH protein expression ratio in randomly selected control and IPF fibroblasts as a function of time. p<0.01 IPF versus control fibroblasts at 24 h. C) Upper, control and IPF fibroblasts (n = 5 each) were cultured in the presence of various doses of rapamycin on collagen gel for 24 h and p-mTOR level was then measured. Shown is the representative p-mTOR protein level in control and IPF fibroblasts on polymerized collagen. GAPDH level was used as a loading control. Lower, p-mTOR/GAPDH expression ratio in control (n = 5) and IPF fibroblasts (n = 5) in the presence of various doses of rapamycin on collagen are shown. p = 0.01 versus DMSO control. p<0.01 versus DMSO control as indicated. Cells were randomly selected for this assay. D) IPF fibroblasts infected with adenovirus expressing dominant negative Akt (DA) or empty vector GFP were cultured on collagen gel for 24 h in serum free medium and p-mTOR, Akt, phosphorylated Akt and GAPDH levels were measured. IPF fibroblasts expressing enhanced p-mTOR level was selected to examine whether Akt inhibition suppresses p-mTOR level (lane 5 in upper panel of IPF in Fig 2A). This assay was repeated twice. E) Control fibroblasts infected with adenovirus expressing hyperactive Akt (HA) or empty vector (GFP) were cultured on collagen for 24 h in serum free medium and p-mTOR, total Akt, phosphorylated Akt and GAPDH levels were measured. Control fibroblasts expressing low p-mTOR level were selected to examine whether hyperactive Ak increases mTOR kinase activity (lane 3 in upper panel of control fibroblasts in Fig 2A). This assay was repeated twice.

Fig 5: Representative stainings for metabolic protein expression analyses in human glioma biopsies. Metabolic and mTOR pathway related protein expression patterns and evaluated staining scores were shown in representative human IDH wild-type and mutant gliomas. Stainings of normal brain tissues were added to the upper left corner. High mTOR activity—with tissue heterogeneity and characteristic CPT1a protein overexpression (17/18 3+ scores) were detected in almost all studied gliomas. The intra- and intertumoral heterogeneity appeared in e.g. p-S6, FASN and p-(Ser473)-Akt stainings, respectively. Significant differences between IDH wild-type and mutant cases were only observed in GLS expression scores, the IDH-mutant glioma tissues expressed higher amount of GLS protein (expression scores were 2/3+ in 6/8 mutant and 0/1+ in 8/10 studied IDH wild-type cases). Further explanation is in the text. Immunohistochemistry stainings of IDH wild-type (n = 10) and IDH-mutant (n = 8) cases were developed by DAB (brown) using haematoxylin counterstaining—the representative photos were taken by Panoramic Viewer (3D Histech—×40)