anti mtorc1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Distinct sensing of BCAAs by mTOR and c-Myc governs T cell proliferation, independent of catabolism

doi: 10.64898/2026.01.16.699967

Figure Lengend Snippet: (A) Schematic of acute BCAA restriction and addback culture. Bulk CD8 T cells were isolated and purified from spleens of WT mice, activated with anti-CD3 and anti-CD28 antibodies (2ug/mL each) for 1 day in replete TCM followed by 2-4hr in BCAA-free TCM with addback of varying combinations of Leu, Ile and Val (0.8mM each). (B,C) Western blot for expression/activation of mTORC1 signaling pathway and c-Myc in acute BCAA culture. (D) Schematic of acute BCAA restriction (without addback) and subsequent addback rescue culture. Bulk CD8 T cells were isolated and purified from spleens of WT mice, activated with anti-CD3 and anti-CD28 antibodies (2ug/mL each) for 1 day in replete TCM followed by 2hr in BCAA-free TCM followed by 2hr in BCAA-free TCM with addback of varying combinations of Leu, Ile and Val (0.8mM each). (E) Western blot for c-Myc expression in acute rescue BCAA culture. (F) qPCR for Myc transcript during acute BCAA culture (as in A, 4hr). (G) Schematic and c-Myc expression (by flow cytometry, mean fluorescent intensity) over time following acute BCAA culture (2.4mM addbacks) with addition of cycloheximide (50ug/mL) or MG132 (10uM) at time of BCAA culture and with collections at 15, 30, 60 and 120 minutes following drug treatment/BCAA restriction and addback. All error bars are representative of 3 technical replicates. Half-lives in (G) calculated using one phase decay non-linear regression analysis.

Article Snippet: mTORC1 sensing of Leu – and to a lesser extent Ile – is the most established mechanism for how intracellular BCAAs can differentially regulate cell signaling ( Saxton et al ., 2016 ; Wolfson et al ., 2016 ).

Techniques: Isolation, Purification, Western Blot, Expressing, Activation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Distinct sensing of BCAAs by mTOR and c-Myc governs T cell proliferation, independent of catabolism

doi: 10.64898/2026.01.16.699967

Figure Lengend Snippet: (A) Schematic of acute BCAA restriction (without addback) and subsequent addback rescue culture with Torin/ISRIB treatment. Bulk CD8 T cells were isolated and purified from spleens of WT mice, activated with anti-CD3 and anti-CD28 antibodies (2ug/mL each) for 1 day in replete TCM followed by 2hr in BCAA-free TCM followed by 2hr in BCAA-free TCM with addback of varying combinations of Leu, Ile and Val (0.8mM each) as well as treatment with ISRIB (1uM), Torin (1uM) or vehicle control. (B) Western blot for expression of c-Myc as well as confirmation of ISR inhibition (ATF4 expression) and mTOR inhibition (phosphorylation of S6) in acute BCAA rescue culture with Torin/ISRIB. (C) Schematic of Day 2-3 BCAA restriction with rapamycin and addback culture. Bulk CD8 T cells were isolated and purified from spleens of WT mice, activated with anti-CD3 and anti-CD28 antibodies (2ug/mL each) for 2 days in replete TCM followed by 1 day in BCAA-free TCM with addback of varying combinations of Leu, Ile and Val (0.8mM each) and simultaneous treatment with 50nM rapamycin or vehicle control. Validation of mTOR inhibition by rapamycin in complete media control cultures via expression of CD98 (mTOR target). (D,E) Frequency and representative flow plot of highly dividing (>4 divisions) cells as measured by CTV dilution in Day 2-3 BCAA culture with rapamycin (CTV stain performed immediately after CD8 purification on Day 0 of culture). (F) Frequency of cells incorporating Edu in Day 2-3 BCAA culture with rapamycin (addition of 10uM Edu during last 4 hours of culture). (G) Western blot for expression/activation of cell cycle regulators (p21, p/Rb) and c-Myc after Day 2-3 BCAA culture with rapamycin. (H) Schematic of proposed model representing how Leu, Ile and Val sensing uncouples mTORC1 and c-Myc regulation to govern T cell proliferation. All error bars are representative of 3 technical replicates. Statistical significance in (C) was calculated using Welch’s t test. Statistical significance in (D,F) was calculated using two-way ANOVA with multiple comparisons and Sidak’s correction. ****p<0.0001.

Article Snippet: mTORC1 sensing of Leu – and to a lesser extent Ile – is the most established mechanism for how intracellular BCAAs can differentially regulate cell signaling ( Saxton et al ., 2016 ; Wolfson et al ., 2016 ).

Techniques: Isolation, Purification, Control, Western Blot, Expressing, Inhibition, Phospho-proteomics, Biomarker Discovery, Staining, Activation Assay

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