mtorc1 cell signaling (2972)  (Cell Signaling Technology Inc)

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 Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: ORP5 alleviates pathological cardiac hypertrophy via mTORC1 pathway. (A) ORP5 and MTOR protein binding conformation: cartoon diagram (left) and surface diagram (right) with yellow indicating the binding area. (B) Immunoprecipitation and Western Blot (WB) analysis of proteins from cultured NRVMs transfected with vector or Flag-ORP5 plasmids using Flag magnetic beads. (C, E) Immunoblotting and semi-quantification of the mTORC1 pathway in LentiNC- and LentishORP5-infected NRVMs, 24 h post-PBS or AngII treatment (n = 6 per group). (D, F) Immunoblotting and semi-quantification of the mTORC1 pathway in AAV9-Veh and AAV9-sh-ORP5 mice, 4 weeks post-sham or TAC surgery (n = 6 per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The mTORC1 inhibitor Rapamycin (MCE, #HY-102190) was obtained from MCE (New Jersey, USA) and dissolved in dimethyl sulfoxide.

Techniques: Protein Binding, Binding Assay, Immunoprecipitation, Western Blot, Cell Culture, Transfection, Plasmid Preparation, Magnetic Beads, Infection

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: Pathological cardiac hypertrophy was exaggerated by ORP5 overexpression and reversed by rapamycin. (A, B) Immunoblotting and semi-quantification of the mTORC1 pathway in NRVMs infected with Lenti-Veh or Lenti-OE-ORP5, 24 h post PBS or AngII treatment (n = 6 per group). (C, D) Fluorescence imaging of α-actinin and cell surface area measurement in the same infected NRVMs, 24 h post PBS or AngII treatment (n ≥ 20 cells per group). (E) Relative mRNA levels of Nppa, Nppb, and Myh7 in NRVMs infected with Lenti-Veh or Lenti-OEORP5, 24 h post PBS, AngII, or Rapamycin treatment (n = 4 per group). (F) Experimental protocol for AAV9 administration, TAC, and rapamycin treatment. (G) Echocardiography images (left) and measurements of HW/BW, EF, FS and HR of AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin treatment (n = 6 per group). (H) Representative images of gross view and WGA staining of AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin administration (n = 6 per group). (I) Cardiomyocyte cross-sectional area measurement from (H) (n ≥ 20 fields from 6 mice per group). (J) Representative Masson staining images of AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin administration (n = 6 per group). (K) Left ventricular collagen volume measurement from (J) (n ≥ 20 fields from 6 mice per group). (L, M) Immunoblotting and semi-quantification of the mTORC1 pathway in AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin treatment (n = 6 per group). (N) Relative mRNA levels of Nppa, Nppb, and Myh7 in these mice under the same conditions (n = 4 per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance.

Article Snippet: The mTORC1 inhibitor Rapamycin (MCE, #HY-102190) was obtained from MCE (New Jersey, USA) and dissolved in dimethyl sulfoxide.

Techniques: Over Expression, Western Blot, Infection, Fluorescence, Imaging, Staining

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: ORP5 enhances mTORC1-mediated cardiac hypertrophy by promoting its translocation to the lysosome. (A, C) Immunoblotting and semi-quantification of the mTORC1 pathway in Lenti-Veh, Lenti-OEORP5, and si-mTOR infected NRVMs 24 h post-PBS or AngII (n = 6 per group). (B, D) Fluorescence of α-actinin and cell surface measurement in the same groups (n ≥ 20 cells per group). (E) Immunoblotting and semi-quantification of mTOR and ORP5 in plasma and lysosomes of Lenti-Veh and Lenti-OE-ORP5 infected NRVMs, 24 h post PBS or AngII treatment, using GAPDH and Lamp-2 as housekeeping genes for cytoplasm and lysosome, respectively (n = 3 per group). (F) Immunofluorescence and intensity analysis of mTOR and Lamp-2 in Lenti-Veh and Lenti-OE-ORP5 infected HL-1 cells, 24 h post PBS or AngII treatment (n ≥ 20 cells per group). (G, I) Immunoblotting and semi-quantification of the mTORC1 pathway in Lenti-Veh and Lenti-OEORP5 infected NRVMs 24 h post PBS, AngII, or lonafanib treatment (n = 6 per group). (H, J) Fluorescence of α-actinin and cell surface measurement in Lenti-Veh and Lenti-OEORP5 infected NRVMs 24 h post PBS, AngII, or lonafanib treatment (n ≥ 20 cells per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance.

Article Snippet: The mTORC1 inhibitor Rapamycin (MCE, #HY-102190) was obtained from MCE (New Jersey, USA) and dissolved in dimethyl sulfoxide.

Techniques: Translocation Assay, Western Blot, Infection, Fluorescence, Clinical Proteomics, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: The ORD domain of ORP5 is indispensable for ORP5-mediated mTORC1-dependent cardiac hypertrophy (A) Diagram showing ORP5 and its deletion mutants missing PH, ORD, and Tm domains. (B) Proteins from NRVMs transfected with vector, ORP5-Flag, ΔORD, ΔPH, and ΔTm plasmids were immunoprecipitated using Flag magnetic beads, followed by Western blot. (C, D) Representative Western blots and semi-quantification of the mTORC1 pathway in wild-type and sh-ORP5 NRVMs infected with vector, ΔORD, ΔPH, and ΔTm plasmids 24 h after PBS or AngII treatment (n = 6 per group). (E) Representative fluorescence of α-actinin (left) and measurement (right) of cell surface of the same cells 24 h after PBS or AngII treatment (n ≥ 20 cells per group). (F) Protocol for AAV-9 administration and TAC. (G) Echocardiography images (left) and measurements of HW/BW, EF, FS and HR of AAV9-Veh and AAV9-sh-ORP5 plus AAV9-ΔORD or AAV9-OE-ORP5 mice 4 weeks post-sham or TAC surgery (n = 6 per group). (H) Gross view and WGA staining images of the same mice 4 weeks post-sham or TAC surgery (n = 6 per group). (I) Measurement of the cardiomyocyte cross-sectional area in (H) (n ≥ 20 cells per group). (J) Masson staining images of the same mice 4 weeks post-sham or TAC surgery (n = 6 per group). (K) Measurement of left ventricular collagen volume in (I) (n ≥ 20 cells per group). (L, M) Immunoblotting (L) and semi-quantification (M) of mTORC1 pathway in the same mice 4 weeks post-sham or TAC surgery (n = 6 per group). (N) Relative mRNA levels of Nppa, Nppb, and Myh7 in the same mice 4 weeks post-sham or TAC surgery (n = 4 mice per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance.

Article Snippet: The mTORC1 inhibitor Rapamycin (MCE, #HY-102190) was obtained from MCE (New Jersey, USA) and dissolved in dimethyl sulfoxide.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Western Blot, Infection, Fluorescence, Staining

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Changes in the phosphorylation of mTORC1-mediated proteins and in the expression of synaptic proteins after DEX exposure. Beginning on DIV 10, the neural spheroids were exposed to 100 μM DEX for 5 days. Western blotting and immunofluorescence were performed using each primary antibody (n = 4 biological replicates). (A) Western blotting revealed the levels of phospho-Ser 2448 -mTORC1 (a), phospho- Thr 37/46 -4E-BP1 (b), and phospho-Thr 389 -p70S6K (c). (B) Western blot analysis and representative images of immunoblots of PSD-95 (a) and GluA1 (b) are shown. Synaptic markers: PSD-95 (green) costained with the neuronal marker MAP-2 (red); GluA1 (green) costained with MAP-2 (red). Scale bar: 100 μm. The data are presented as the means ± standard deviations. mTORC1, mechanistic target of rapamycin complex I; DEX, dexamethasone; DIV, days in vitro; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1; PSD-95, postsynaptic density protein-95; GluA1, AMPA receptor subunit glutamate receptor 1; MAP-2, microtubule-associated protein-2; CON, controls. * p < 0.05 or ** p < 0.01 vs. CON.

Article Snippet: mTORC1 , Cell Signaling Technology (2972) , AB_330978 , WB (1:1,000).

Techniques: Phospho-proteomics, Expressing, Western Blot, Immunofluorescence, Marker, In Vitro, Binding Assay

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Schematic diagram showing the molecular mechanisms underlying DEX-induced impaired neuroplasticity. Exposure to DEX downregulates signaling pathways that affect neuroplasticity, including BDNF, sirtuin 1, and mTORC1 signaling. Activation of the mTORC1 signaling pathway induces the synthesis of synaptic proteins as well as BDNF. Secreted BDNF interacts with its receptor TrkB, further activating mTORC1 signaling via PI3K/Akt and MEK/ERK1/2. Therefore, synaptic plasticity is enhanced. Sirtuin 1 is also involved in the regulation of neuroplasticity. The original illustration was created using BioRender (biorender.com). Akt, protein kinase B; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BDNF, brain-derived neurotrophic factor; DEX, dexamethasone; ERK1/2, extracellular signal-regulated kinase 1/2; GluA1, AMPA receptor subunit glutamate receptor 1; MEK, mitogen‑activated protein kinase; mTORC1, mechanistic target of rapamycin complex I; p70S6K, p70S6 kinase; PI3K, phosphatidyl inositol-3 kinase; PSD-95, postsynaptic density protein-95; TrKB, tropomyosin receptor kinase B; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.

Article Snippet: mTORC1 , Cell Signaling Technology (2972) , AB_330978 , WB (1:1,000).

Techniques: Protein-Protein interactions, Activation Assay, Derivative Assay, Binding Assay

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Changes in the phosphorylation of mTORC1-mediated proteins and in the expression of synaptic proteins after DEX exposure. Beginning on DIV 10, the neural spheroids were exposed to 100 μM DEX for 5 days. Western blotting and immunofluorescence were performed using each primary antibody (n = 4 biological replicates). (A) Western blotting revealed the levels of phospho-Ser 2448 -mTORC1 (a), phospho- Thr 37/46 -4E-BP1 (b), and phospho-Thr 389 -p70S6K (c). (B) Western blot analysis and representative images of immunoblots of PSD-95 (a) and GluA1 (b) are shown. Synaptic markers: PSD-95 (green) costained with the neuronal marker MAP-2 (red); GluA1 (green) costained with MAP-2 (red). Scale bar: 100 μm. The data are presented as the means ± standard deviations. mTORC1, mechanistic target of rapamycin complex I; DEX, dexamethasone; DIV, days in vitro; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1; PSD-95, postsynaptic density protein-95; GluA1, AMPA receptor subunit glutamate receptor 1; MAP-2, microtubule-associated protein-2; CON, controls. * p < 0.05 or ** p < 0.01 vs. CON.

Article Snippet: p-Ser 2448 -mTORC1 , Cell Signaling Technology (2971) , AB_330970 , WB (1:1,000).

Techniques: Phospho-proteomics, Expressing, Western Blot, Immunofluorescence, Marker, In Vitro, Binding Assay

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Schematic diagram showing the molecular mechanisms underlying DEX-induced impaired neuroplasticity. Exposure to DEX downregulates signaling pathways that affect neuroplasticity, including BDNF, sirtuin 1, and mTORC1 signaling. Activation of the mTORC1 signaling pathway induces the synthesis of synaptic proteins as well as BDNF. Secreted BDNF interacts with its receptor TrkB, further activating mTORC1 signaling via PI3K/Akt and MEK/ERK1/2. Therefore, synaptic plasticity is enhanced. Sirtuin 1 is also involved in the regulation of neuroplasticity. The original illustration was created using BioRender (biorender.com). Akt, protein kinase B; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BDNF, brain-derived neurotrophic factor; DEX, dexamethasone; ERK1/2, extracellular signal-regulated kinase 1/2; GluA1, AMPA receptor subunit glutamate receptor 1; MEK, mitogen‑activated protein kinase; mTORC1, mechanistic target of rapamycin complex I; p70S6K, p70S6 kinase; PI3K, phosphatidyl inositol-3 kinase; PSD-95, postsynaptic density protein-95; TrKB, tropomyosin receptor kinase B; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.

Article Snippet: p-Ser 2448 -mTORC1 , Cell Signaling Technology (2971) , AB_330970 , WB (1:1,000).

Techniques: Protein-Protein interactions, Activation Assay, Derivative Assay, Binding Assay