mtorc1 activity  (Cell Signaling Technology Inc)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Mitochondrial impairment and mTORC1 signalling exhaustion define NK Cell dysfunction progression in melanoma

doi: 10.1007/s00262-026-04323-0

Figure Lengend Snippet: mTORC1 signalling is defective in melanoma patient NK cells but be rescued by an mTORC1 activator in LN+ patients. PBMC from HD, LN+ or Met melanoma patients were analysed ex vivo or following stimulation with IL-12/15. (A,B) MFI of cMyc on CD56 dim (left) and CD56 bright (right) NK cells A ex vivo and B following stimulation with IL-12/15 represented as a fold change relative to unstimulated cells. Representative histograms of HD, LN+ and Met patient NK cells stained with cMyc following stimulation with IL-12/15. ( C ) MFI of pS6 + CD56 dim (left) and CD56 bright (right) NK cells ex vivo. Representative plot showing pS6 MFI in HD, LN+ and Met melanoma patients. Representative histograms of HD, LN+ and Met melanoma patient NK cells stained with pS6 ex vivo. D MFI of pS6 + CD56 dim (left) and CD56 bright (right) NK cells following IL-12/15 stimulation. E Frequency of pS6 + CD56 dim (left) and CD56 bright (right) NK cells following stimulation with IL-12/15. F MFI pS6 CD56 dim NK cells following 4 h treatment with 10 µM MHY1485 represented as a fold change relative to untreated (UT) cells. Representative plot showing pS6 MFI in untreated and MHY1485 treated HD cells. G MFI of pS6 expressing CD56 dim NK cells following 4 h pre-treatment with 10 µM MHY1485 and stimulation with IL-12/15 for 18 h. Data represented as a fold change relative to IL-12/15 stimulated cells. H CD71 + CD56 bright NK cells from Met patients following stimulation with IL-12/15 with and without pre-treatment with 10µM MHY1485. I IFN-γ + CD56 bright NK cells from LN+ and Met melanoma patients following stimulation with IL-12/15 with and without pre-treatment with 10 µM MHY1485. Representative dot plot showing IFN-y + NK cells in LN+ patients. * p < 0.05, ** p < 0.01, *** p < 0.01 by Kruskal–Wallis test with post-hoc Dunn’s test or one sample Wilcoxon test

Article Snippet: For experiments using mTORC1 activator MH1485 (MedChem Express), cells were treated for 4 h with 10 μM MH1485, with and without subsequent stimulation with 30 ng/ml IL-12 (Miltenyi Biotec) and 100 ng/ml IL-15 (Miltenyi Biotec).

Techniques: Ex Vivo, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: TLR4 modulates simvastatin’s impact on HDL cholesterol and glycemic control

doi: 10.3389/fphar.2025.1655873

Figure Lengend Snippet: Simvastatin administration during fasting activates SREBP-2 and autophagy and augments FFA-induced APOA1 expression. (A) Huh7 cells cultured under serum-depleted conditions were treated with simvastatin (1, 5, or 10 μM) for 24 h. APOA1, PLTP, and GK mRNA levels were quantified by qPCR, normalized to the geometric mean of HPRT and B2M , and expressed as fold change relative to untreated controls. (B) Simvastatin (10 μM) enhances sodium oleate (50 μM)–induced APOA1 expression in serum-depleted Huh7 cells. Gene expression was measured by qPCR and normalized to HPRT and B2M . (C) Fasting-phase simvastatin treatment upregulates hepatic Srebf2 and its target genes in vivo . A/J mice received simvastatin by gavage for 5 weeks and were sacrificed 5 h after the final dose (ZT09); hepatic mRNA levels were normalized to Hprt . (D) Expression of autophagy-related genes ( Atg7 , Atg12 , Becn1 and others) in liver following fasting-phase simvastatin treatment. (E) Pharmacologic inhibition of autophagy attenuates simvastatin-induced APOA1 expression in Huh7 cells. Serum-starved cells were treated with simvastatin in the presence or absence of 500 µM leucine (mTORC1 activator) or 50 µM leupeptin (lysosomal protease inhibitor); APOA1 mRNA was measured by qPCR and normalized to HPRT1 and B2M . Data are mean ± SEM with individual data points overlaid (n indicated in each panel). Statistical significance: p < 0.05; * p < 0.01; ** p < 0.001 (two-way ANOVA with Tukey’s post hoc test).

Article Snippet: For fasting-mimetic conditions ( ; ), cells were washed twice with 1× PBS, incubated overnight in serum-free DMEM, and then treated for 24 h with 5 μM simvastatin alone or in combination with one or more of the following: 50 μM oleic acid (MilliporeSigma; C18:1, #O1383, purity >99% by GC); 10 μM GW6471 (MedChemExpress; #HY-15372, purity 99%), a selective PPARα antagonist; 500 μM leucine (MedChemExpress; #HY-N0486, purity 98%), which suppresses autophagy initiation via mTORC1 activation; or 50 μM leupeptin (MedChemExpress; #HY-18234A, purity 99.39%), a cysteine/serine/threonine protease inhibitor that blocks autophagic flux ( ; ; ).

Techniques: Expressing, Cell Culture, Gene Expression, In Vivo, Inhibition, Protease Inhibitor

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Mechanosensitive Ion Channel Piezo1 Regulates Chondrocyte Homeostasis Through the PI3K / AKT / mTORC1 Pathway in Osteoarthritis

doi: 10.1111/jcmm.70734

Figure Lengend Snippet: Activation of the Piezo1 channel induces the PI3K/AKT/mTORC1 pathway. (A) RNA sequencing was performed on chondrocytes treated with DMSO or 10 μM Yoda1 for 2 h, and comparative analysis of the data was conducted ( n = 3). The bar graph illustrates the most significantly up‐regulated biological processes in the GO functional enrichment analysis. (B) Bubble plots demonstrating significantly altered signalling pathways in Reactome enrichment analysis. (C–F) After treatment of chondrocytes with 10 μM Yoda1 for 0, 2, 6 and 12 h, western blot analysis was used to assess expression of p‐PI3K, PI3K, p‐AKT, AKT, p‐mTOR, mTOR and RAPTOR, and protein bands were quantified ( n = 3). (G, H) The levels of p‐PI3K, PI3K, p‐AKT, AKT, p‐mTOR, mTOR and RAPTOR expression were analysed by western blot after siNC and si Piezo1 ‐treated chondrocytes ( n = 3). All data were presented as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: To investigate the role of the PI3K/AKT/mTOR pathway, chondrocytes were pretreated for 2 h with either 25 μM LY294002 (MedChemExpress, HY‐10108) to inhibit PI3K/AKT signalling or 100 nM rapamycin (MedChemExpress, HY‐10219) to suppress mTORC1 activity.

Techniques: Activation Assay, RNA Sequencing, Functional Assay, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: The Mechanosensitive Ion Channel Piezo1 Regulates Chondrocyte Homeostasis Through the PI3K / AKT / mTORC1 Pathway in Osteoarthritis

doi: 10.1111/jcmm.70734

Figure Lengend Snippet: Piezo1 regulates chondrocyte homeostasis through the PI3K/AKT/mTORC1 pathway. (A, B) Chondrocytes were pretreated with LY294002 or rapamycin for 2 h, followed by stimulation with Yoda1 for 12 h. Protein levels of COL2, ACAN, MMP13, MMP3 and ADAMTS5 were analysed by western blotting and protein bands were quantified ( n = 3). (C) A schematic diagram illustrating the Piezo1‐mediated PI3K/AKT/mTORC1 signalling pathway. All data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To investigate the role of the PI3K/AKT/mTOR pathway, chondrocytes were pretreated for 2 h with either 25 μM LY294002 (MedChemExpress, HY‐10108) to inhibit PI3K/AKT signalling or 100 nM rapamycin (MedChemExpress, HY‐10219) to suppress mTORC1 activity.

Techniques: Western Blot

Journal: Animal Nutrition

Article Title: Propionate promotes gluconeogenesis by regulating mechanistic target of rapamycin (mTOR) pathway in calf hepatocytes

doi: 10.1016/j.aninu.2023.07.001

Figure Lengend Snippet: Role of mammalian target of rapamycin complex 1 (mTORC1) and PGC1α in propionate-mediated regulation of the expression of gluconeogenesis-related genes in calf hepatocytes. (A–C). Calf hepatocytes were treated with NaP and rapamycin (100 nM). The expression levels of FBP1 (A), PCK1 (B), and G6PC (C) were detected by RT-qPCR. (D–F). Calf hepatocytes were treated with NaP and MHY1485 (2 μM). The expression levels of FBP1 (D), PCK1 (E), and G6PC (F) were detected by RT-qPCR. (G–I). Calf hepatocytes were treated with NaP and SR18292 (20 μM). The expression levels of FBP1 (G), PCK1 (H), and G6PC (I) were detected by RT-qPCR. Data were analyzed by two-way ANOVA. a, b, c Bars with a different letter mean a significant difference ( P < 0.05).

Article Snippet: Cells were maintained in RPMI 1640 basic medium containing 2% BSA and treated with different concentrations of PA (0, 100, 200, or 400 μM) and NaP (0, 1, 2.5, or 5 mM), alone or in combination, for 12 h. A 2 × 2 factorial arrangement was applied for the experiments: primary hepatocytes were treated with NaP (2.5 mM), the mTORC1 inhibitor rapamycin (100 nM) (V900930; Sigma Aldrich, MO, USA), the mTORC1 activator MHY1485 (2 μM) (S7811; Selleck, Shanghai, China), and the PGC1α inhibitor SR-18292 (20 μM) (S8528; Selleck) for 12 h to observe the effect of mTORC1 and PGC1α on the mRNA expression of gluconeogenic genes.

Techniques: Expressing, Quantitative RT-PCR