Journal: Scientific reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p.

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: Fig. 4. miR-199b-3p regulate the expression of mTOR (A) Quantitative analysis of mTOR in fibroblasts using qRT-PCR. (B) Quantitative analysis of mTOR in fibroblasts using western blot and the statistics. The originals gels can be seen in the supplementary figure S5. (C) Predicted miR-199b-3p target sequence of the mTOR 3’UTR using miRBase. (D) Quantification of miR-199b-3p and mRNA using qRT-PCR, the WT represents the mixture of the equal amount total RNA from the two healthy fibroblasts. Relative expression levels were normalized with GAPDH. (E). Luciferase reporter assay. (F) Quantitative analysis of mTOR in HEK293T cells at 48 h post-treatment with miR-199b-3p mimics (low panel) and the statistics (up panel). The originals gels can be seen in the supplementary figure S5. (G) Western bolt analysis of p-AKT, AKT, p-p70S6K, p70S6K in HEK293T cells at 48 h post-treatment with miR-199b-3p mimics,β-actin served as a control of total protein. (H) Quantitative analysis of mTOR in HEK293T cells at 48 h post-treatment with the inhibitor specific to miR- 199b-3p (low panel) and the statistics (up panel). (I) Western bolt analysis of p-AKT, AKT, p-p70S6K, p70S6K in HEK293T cells at 48 h post-treatment with the inhibitor specific to miR-199b-3p, β-actin served as a control of total protein. The originals gels can be seen in the supplementary figure S5.

Article Snippet: HEK293T cells were maintained in DMEM (Boster) supplemented with 10% FBS (BI).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Sequencing, Luciferase, Reporter Assay, Control

Journal: Scientific reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p.

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: Fig. 5. TSC complex regulates the endogenous content of miR199b-3p, *p < 0.05, **p < 0.01,***p < 0.001 comparison with WT. (A) The efficiency of knockdown TSC1 in shTSC1-HEK293T cell line. The originals gels can be seen in the supplementary figure S6. (B) The expression of miR-199b-3p in shTSC1-HEK293T cells and overexpression TSC1-WT or TSC1-N837fs in HEK293T cell line. (C) Quantitative analysis of mTOR in shTSC1-HEK293T cell line and overexpression TSC1-WT or TSC1 N837fs in HEK293T cell line using western blot (low panel) and the statistics (up panel). The originals gels can be seen in the supplementary figure S6. (D) The efficiency of knockdown TSC2 in shTSC2-HEK293T cell line. The originals gels can be seen in the supplementary figure S6. (E) The expression of miR-199b-3p in shTSC2-HEK293T cell line and overexpression TSC2-WT or TSC2- Q371fs in HEK293T cell line. (F) Quantitative analysis of mTOR in shTSC2-HEK293T cell line (low panel) and overexpression TSC2-WT or TSC2- Q371fs in HEK293T cell line using Western blot (low panel) and the statistics analysis (up panel). The originals gels can be seen in the supplementary figure S6.

Article Snippet: HEK293T cells were maintained in DMEM (Boster) supplemented with 10% FBS (BI).

Techniques: Comparison, Knockdown, Expressing, Over Expression, Western Blot

Journal: Theranostics

Article Title: Ubiquitin specific peptidase 5 regulates colorectal cancer cell growth by stabilizing Tu translation elongation factor

doi: 10.7150/thno.33803

Figure Lengend Snippet: USP5 interacts with TUFM. A. HEK293T cells transfected with vector or Myc-USP5 plasmid were lysed and immunoprecipitated with anti-Myc antibody. The immunoprecipites were separated on SDS-PAGE and visualized with silver staining. Differential bands indicated were cut for LC-MS/MS analysis. B. Twelve peptides identified by LC-MS/MS (highlighted in yellow) were fragments of TUFM. C & D. Myc-USP5 and Flag-TUFM-expressing plasmids were transfected into HEK293T for 24 hours. Reciprocal co-immunoprecipitation and immunoblotting were performed by using anti-Flag (C) and anti-Myc antibodies (D). E & F. Whole cell lysates of HCT116 cells were subjected to reciprocal co-immunoprecipitation assays were performed by using anti-TUFM (E) or anti-USP5 antibody (F). G. The correlation between USP5 and TUFM based on data retrieved from GEPIA ( http://gepia.cancer-pku.cn ).

Article Snippet: HEK293T cell line was kindly provided by Dr. Huashun Li from Tongji University, Shanghai, China.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot

Journal: Theranostics

Article Title: Ubiquitin specific peptidase 5 regulates colorectal cancer cell growth by stabilizing Tu translation elongation factor

doi: 10.7150/thno.33803

Figure Lengend Snippet: USP5 stabilizes TUFM through deubiquitination. A. Myc-USP5-WT, Myc-USP5-C335A and Flag-TUFM were co-transfected into HEK293T cells. Twenty-four hours later, cells were prepared for immunoblotting analysis using anti- Flag, Myc and GAPDH antibodies. B. HCT116 cells were transfected with increased amounts of plasmids Myc-USP5-WT and Myc-USP5-C335A, and then analyzed by immunoblotting with antibodies against TUFM, Myc and GAPDH. C. After infected with lentiviruses expressing shUSP5#1, shUSP5#2, shUSP5#3 or control for 3 days, HCT116 cells were lysed and analyzed by immunoblotting against USP5 and TUFM. GAPDH was used as a loading control. D. After transfected with Myc-USP5 or control vector for 24 hours, HCT116 cells were exposed to CHX for indicated time and analyzed for TUFM and Myc-USP5 levels by immunoblotting. GAPDH was used as a loading control. E. Quantitative and statistical analyses of data from Figure D (mean +/- SD). F. Following infected with lentiviruses expressing shUSP5#3 or shNC for 36 hours, HCT116 cells were treated with 10 μM of MG132 for 12 hours and then analyzed for the levels of TUFM and USP5 by immunoblotting. GAPDH was used as a loading control. G. Quantitative and statistical analyses of data from Figure F (mean +/- SD). H. Plasmids expressing Myc-USP5-WT, Myc-USP5-C335A, Flag-TUFM or HA-Ub-K48 were transfected into HEK293T cells for 24 hours. The cells were then lysed, immunoprecipitated with anti-Flag antibody, and immnublotted with anti-HA antibody as indicated. The cell lysates were also directly immunoblotted with anti- Flag and Myc antibodies. GAPDH was used as a loading control. I. HCT116 cells were transfected with plasmids Flag-TUFM, HA-Ub-K48, or infected with lentiviruses expressing shUSP5#1, shUSP5#3 or shNC for 48 hours. After treated with 20 μM of MG132 for 6 hours, the cells were lysed, immunoprecipitated with anti-Flag antibody, and immunoblotted with anti-HA antibody to detect the ubiquitination of TUFM. The cell lysates were also directly immunoblotted with antibodies against Flag, USP5 and GAPDH.

Article Snippet: HEK293T cell line was kindly provided by Dr. Huashun Li from Tongji University, Shanghai, China.

Techniques: Transfection, Western Blot, Infection, Expressing, Control, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics