Journal: Molecular Medicine Reports

Article Title: Expression of TGF-β1/mTOR signaling pathway in pathological scar fibroblasts

doi: 10.3892/mmr.2017.6437

Figure Lengend Snippet: PI3K, Akt, mTOR gene primer sequences and amplification fragment length of the product.

Article Snippet: D-Hank's liquid (Shanghai Kang Lang Biological Technology Co., Ltd., Shanghai, China), 0.25% trypsin (Shanghai Bioleaf Biotech Co., Ltd., Shanghai, China), fetal bovine serum (Shanghai Bioleaf Biotech Co., Ltd.), Triton X-100 (Shanghai Lianshuo Biological Technology Co., Ltd., Shanghai, China), reverse transcription kit superscript III (Invitrogen, Carlsbad, CA, USA), SDS-PAGE gel preparation kit (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China), rabbit anti-human TGF-β1 monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China), rabbit anti-human monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human protein kinase B (Akt) monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.); rabbit anti-human mTOR monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), and goat anti-rabbit secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) labeled by fluorescein isothiocyanate were used in the study.

Techniques: Amplification, Sequencing

Journal: Molecular Medicine Reports

Article Title: Expression of TGF-β1/mTOR signaling pathway in pathological scar fibroblasts

doi: 10.3892/mmr.2017.6437

Figure Lengend Snippet: mRNA relative expression quantities of TGF-β1, PI3K, Akt, and mTOR in the two groups.

Article Snippet: D-Hank's liquid (Shanghai Kang Lang Biological Technology Co., Ltd., Shanghai, China), 0.25% trypsin (Shanghai Bioleaf Biotech Co., Ltd., Shanghai, China), fetal bovine serum (Shanghai Bioleaf Biotech Co., Ltd.), Triton X-100 (Shanghai Lianshuo Biological Technology Co., Ltd., Shanghai, China), reverse transcription kit superscript III (Invitrogen, Carlsbad, CA, USA), SDS-PAGE gel preparation kit (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China), rabbit anti-human TGF-β1 monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China), rabbit anti-human monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human protein kinase B (Akt) monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.); rabbit anti-human mTOR monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), and goat anti-rabbit secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) labeled by fluorescein isothiocyanate were used in the study.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Expression of TGF-β1/mTOR signaling pathway in pathological scar fibroblasts

doi: 10.3892/mmr.2017.6437

Figure Lengend Snippet: The protein relative expression quantities of TGF-β1, PI3K, Akt, and mTOR in the two groups.

Article Snippet: D-Hank's liquid (Shanghai Kang Lang Biological Technology Co., Ltd., Shanghai, China), 0.25% trypsin (Shanghai Bioleaf Biotech Co., Ltd., Shanghai, China), fetal bovine serum (Shanghai Bioleaf Biotech Co., Ltd.), Triton X-100 (Shanghai Lianshuo Biological Technology Co., Ltd., Shanghai, China), reverse transcription kit superscript III (Invitrogen, Carlsbad, CA, USA), SDS-PAGE gel preparation kit (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China), rabbit anti-human TGF-β1 monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China), rabbit anti-human monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human protein kinase B (Akt) monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.); rabbit anti-human mTOR monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), and goat anti-rabbit secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) labeled by fluorescein isothiocyanate were used in the study.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Expression of TGF-β1/mTOR signaling pathway in pathological scar fibroblasts

doi: 10.3892/mmr.2017.6437

Figure Lengend Snippet: Protein expression of electrophoresis of transforming growth factor-β1 (TGF-β1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), β-actin of the two groups.

Article Snippet: D-Hank's liquid (Shanghai Kang Lang Biological Technology Co., Ltd., Shanghai, China), 0.25% trypsin (Shanghai Bioleaf Biotech Co., Ltd., Shanghai, China), fetal bovine serum (Shanghai Bioleaf Biotech Co., Ltd.), Triton X-100 (Shanghai Lianshuo Biological Technology Co., Ltd., Shanghai, China), reverse transcription kit superscript III (Invitrogen, Carlsbad, CA, USA), SDS-PAGE gel preparation kit (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China), rabbit anti-human TGF-β1 monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China), rabbit anti-human monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human protein kinase B (Akt) monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.); rabbit anti-human mTOR monoclonal antibody (Wuhan Boster Biological Technology Co., Ltd.), and goat anti-rabbit secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) labeled by fluorescein isothiocyanate were used in the study.

Techniques: Expressing, Electrophoresis

Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: Effect of MTOR inhibition by rapamycin on HCC cell proliferation. Four different HCC cell lines (HepG2, Huh7, Hep3B and SNU3160) were cultured in conditional media (1% FBS) with or without rapamycin (RAPA). Proliferation of Huh7 or Hep3B cells was inhibited by 10 nM rapamycin for 48 h ( A ). Proliferation of Huh7 or Hep3B cells was regulated by rapamycin in a dose-dependent manner (within 0.1~20 nM) ( B ). PROX1 expression was increased by rapamycin in Huh7 or Hep3B cells. The inhibitory effect of rapamycin was confirmed by detecting the phosphorylated form of MTOR (p-MTOR). MTOR and β-actin were detected as quantitative controls ( C ). Relative expression of PROX1 were analyzed image J ( D ). PROX1 expression was also assessed by immunofluorescence microscopy ( E ). Cell nuclei were observed by DAPI staining. D: DMSO-treated cells, R: rapamycin-treated cells. Scale bars represent 100 μm. * p < 0.05; ** p < 0.01, *** p < 0.001.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Inhibition, Cell Culture, Expressing, Immunofluorescence, Microscopy, Staining

Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: PROX1 expression was increased by rapamycin in Huh7 or Hep3B cells. PROX1 expression was elevated in Huh7 ( A ) or Hep3B cells ( E ) after 12~36 h of rapamycin treatment. The inhibitory effect of rapamycin was confirmed by detecting the phosphorylated form of MTOR (p-MTOR). MTOR and β-actin were detected as quantitative controls. The relative analysis comparing PROX1 expression with β-actin shows that PROX1 expression was significantly up-regulated by rapamycin in Huh7 ( B ) or Hep3B cells ( F ). Western blot analysis was performed after Huh7 ( C ) or Hep3B cells ( G ) were treated with rapamycin at concentrations of 0.1~20 nM. Rapamycin activity was confirmed by detecting p-MTOR. MTOR and β-actin were measured as quantitative controls. PROX1 expression in Huh7 ( D ) or Hep3B cells ( H ) was analyzed and compared with β-actin. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Expressing, Western Blot, Activity Assay

Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: Rapamycin increased the intracellular half-life of PROX1 protein. The mRNA expression of PROX1 was analyzed by semi-quantitative RT-PCR using total RNAs prepared from Huh7 ( A ) or Hep3B cells ( E ) treated with or without rapamycin. Quantitative RT-PCR results showed that the expression of PROX1 mRNA in Huh7 ( B ) or Hep3B cells ( F ) was not affected by rapamycin. The half-life of PROX1 protein in Huh7 ( C ) or Hep3B cells ( G ) was measured by treating cells with or without rapamycin for 24 h and then exposing them to cycloheximide (50 µg/mL). Phosphorylated MTOR (p-MTOR) was detected to monitor the inhibitory effect of rapamycin. MTOR and β-actin were measured as quantitative controls. Relative analysis showed that PROX1 expression in Huh7 ( D ) or Hep3B cells ( H ) began to decrease after 2 h of cycloheximide treatment in DMSO-treated control cells, but it was maintained after rapamycin treatment for up to 6 h. ** p < 0.01. DMSO: DMSO-treated cells, RAPA: rapamycin-treated cells.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: PROX1 played a key role in the anti-proliferative effect of rapamycin. Huh7 ( A ) or Hep3B cells ( E ) were transfected with a plasmid expressing human PROX1 or an empty plasmid, and 24 h later were treated with or without rapamycin for an additional 24 h. Western blot analysis showed that PROX1 expression was increased by the over-expression system and by rapamycin, and markedly by rapamycin treatment plus PROX1 over-expression. D: cells treated with DMSO, R: cells treated with rapamycin. The effect of PROX1 on the proliferation of Huh7 ( B ) or Hep3B cells ( F ) was also investigated using the over-expression system. After 24 h of transfection, cells were treated with or without rapamycin for an additional 48 h. Cell proliferation was inhibited by rapamycin and by PROX1 over-expression. Huh7 ( C ) or Hep3B cells ( G ) were transfected with siRNA targeting PROX1 mRNA (siPROX1) or control siRNA (siCTR). After 24 h, cells were treated with or without rapamycin for an additional 24 h. Western blot analysis clearly showed the knock-down effect of siPROX1. The inhibitory effect of rapamycin was confirmed by determining phosphorylated MTOR (p-MTOR). MTOR and β-actin were detected as quantitative controls. To investigate the effect of siPROX1 on cell proliferation, Huh7 ( D ) or Hep3B cells ( H ) were transfected with siPROX1 and treated with or without rapamycin for an additional 48 h. siPROX1treatment was found to increase the proliferation of Huh7 or Hep3B cells and to decrease the anti-proliferative effect of rapamycin. **, p < 0.01; ***, p < 0.001. DMSO: DMSO-treated cells, RAPA: rapamycin-treated cells.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Over Expression, Control, Knockdown

Journal: Journal of Biomedical Science

Article Title: ZNF322A-mediated protein phosphorylation induces autophagosome formation through modulation of IRS1-AKT glucose uptake and HSP-elicited UPR in lung cancer

doi: 10.1186/s12929-020-00668-5

Figure Lengend Snippet: Silencing of ZNF322A induces autophagosome formation in lung cancer cells. a - b Protein expression of mTOR and p-mTOR S2448 was determined and normalized by immunoblotting. c - d Induction of autophagy related proteins including LC3B-I, LC3B-II, and SQSTM1 were analyzed and quantified by immunoblotting. Earle’s balanced salts solution (EBSS) was used as positive control to induce autophagy. e - f Electron microscopy of A549 lung cancer cells after 24 or 48 h of siControl or ZNF322A siRNA transfection was analyzed. Double- and multi-membrane autophagosomes were clearly observed in ZNF322A-silenced cells. Final stages of autophagic cell death were accompanied by ballooning of the perinuclear space and disappearance of cellular organelles such as autophagosomes, autolysosomes, and ER. Arrows: diverse autophagosomes, N: nuclear. β-actin is the internal control to normalize protein expression. The bars represent densitometric analysis of three biological replicates and the data are shown as mean ± SD. * p < 0.05; ** p < 0.01

Article Snippet: The membrane was blocked in 5% non-fat milk/PBST or 5% bovine serum albumin/PBST for 1 h, then incubated overnight with the following primary antibodies diluted in 5% non-fat milk/PBST or 5% bovine serum albumin/PBST at 4 °C: rabbit anti-ZNF322A (GTX121644, GeneTex, Irvine, CA 92606 USA), mouse anti-actin (MAB1501, Millipore, Billerica, Massachusetts, USA), rabbit anti-HSP27 S82 (MDBio, Taipei, Taiwan), rabbit anti-HSP27 (GTX101145, GeneTex), rabbit anti-eIF2α S51 (ab32157, Abcam, Cambridge, UK), rabbit anti-eIF2α (PA5–27366, Thermo Fisher Scientific), rabbit anti-IRS1 S1101 (A0446, Assay Biotech, Sunnyvale, CA, USA), rabbit anti-IRS1 (#2382, Cell signaling technology, Danvers, MA, USA), rabbit anti-AKT S473 (#9271, Cell signaling technology), rabbit anti-AKT (#9272, Cell signaling technology), rabbit anti-SQSTM1 (GTX100685, GeneTex), rabbit anti-LC3B/MAP1LC3B (NB6001384, Novus Biologicals, CO, USA) and mouse anti-actin (MAB1501, Millipore), rabbit anti-Pim-3 (#4165, Cell signaling technology), Rabbit anti-mTOR (GTX132803, GeneTex), Rabbit anti-mTOR S2448 (GTX101557, GeneTex).

Techniques: Expressing, Western Blot, Positive Control, Electron Microscopy, Transfection