mtorc1 specific inhibitor rapamycin  (MedChemExpress)

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