Journal: PeerJ

Article Title: Exosomes from adipose-derived stem cells alleviate premature ovarian failure via blockage of autophagy and AMPK/mTOR pathway

doi: 10.7717/peerj.16517

Figure Lengend Snippet: (A) Ovarian tissue samples were collected on the 15th day, and the apoptosis of ovarian GCs was detected by TUNEL staining. Scale bar: 20 µm. (B) The ultrastructure and intracellular autophagy of cells in ovarian tissue were observed by transmission electron microscope. Scale bar: 2 µm. (C) Levels of autophagy-related genes (Beclin-1 and LC3II/LC3I), apoptosis-related protein Bcl-2, and pathway-related proteins (p-AMPK/AMPK and p-mTOR/mTOR) were detected by western blotting. ** p < 0.01 compared with the WT group. # p < 0.05 and ## p < 0.01 compared with the POF group. Ovarian tissue samples were collected on the 15th day.

Article Snippet: The primary antibodies used in this study were the AMPK antibody (1:1,000, ab32047, Abcam, Cambridge, UK), p-AMPK antibody (1:1,000, ab32047, Abcam), mTOR antibody (1:1,000, CSB-PA208208; Cusabio, Hubei, China), p-mTOR antibody (1:1,000, CSB-PA271384; Cusabio), Beclin-1 antibody (1:1,000, ab210498; Abcam), LC3II/LC3I antibody (1:1,000, 12741T; CST), Bcl-2 antibody (1:1,000, ab182858; Abcam), CD63 antibody (1:1,000, ab217345; Abcam), CD81 antibody (1:1,000, DF2306; Affinity, Cincinnati, OH, USA), TSG101 antibody (1:1,000, DF8427; Affinity), HSP70 antibody (1:1,000, AF5466, Affinity) and the GAPDH antibody (1:1,000, ab245355; Abcam).

Techniques: TUNEL Assay, Staining, Transmission Assay, Microscopy, Western Blot

Journal: PeerJ

Article Title: Exosomes from adipose-derived stem cells alleviate premature ovarian failure via blockage of autophagy and AMPK/mTOR pathway

doi: 10.7717/peerj.16517

Figure Lengend Snippet: (A) ELISA was used to evaluate the levels of E2, FSH, MDA, ROS, and SOD. (B) Ovarian tissue samples were collected on the 15th day, and the apoptosis of ovarian GCs was detected by TUNEL staining. Scale bar: 20 µm. (C–D) The protein levels of Beclin-1, LC3II/LC3I, Bcl-2, p-AMPK, and p-mTOR were detected by western blotting. ** p < 0.01 compared with the WT group, # p < 0.05 and ## p < 0.01 compared with the POF group, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\hat {}\hat {}$\end{document} ˆ ˆ p < 0.05 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\hat {}\hat {}$\end{document} ˆ ˆ p < 0.01 compared with the ADSCs group.

Article Snippet: The primary antibodies used in this study were the AMPK antibody (1:1,000, ab32047, Abcam, Cambridge, UK), p-AMPK antibody (1:1,000, ab32047, Abcam), mTOR antibody (1:1,000, CSB-PA208208; Cusabio, Hubei, China), p-mTOR antibody (1:1,000, CSB-PA271384; Cusabio), Beclin-1 antibody (1:1,000, ab210498; Abcam), LC3II/LC3I antibody (1:1,000, 12741T; CST), Bcl-2 antibody (1:1,000, ab182858; Abcam), CD63 antibody (1:1,000, ab217345; Abcam), CD81 antibody (1:1,000, DF2306; Affinity, Cincinnati, OH, USA), TSG101 antibody (1:1,000, DF8427; Affinity), HSP70 antibody (1:1,000, AF5466, Affinity) and the GAPDH antibody (1:1,000, ab245355; Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Western Blot

Journal: PeerJ

Article Title: Exosomes from adipose-derived stem cells alleviate premature ovarian failure via blockage of autophagy and AMPK/mTOR pathway

doi: 10.7717/peerj.16517

Figure Lengend Snippet: The protein levels of p-AMPK/AMPK, p-mTOR/mTOR, Bcl-2, Beclin-1, and LC3II/LC3I were detected by western blotting. ** p < 0.01 compared with the PBS group, # p < 0.05 and ## p < 0.01 compared with the Exo group.

Article Snippet: The primary antibodies used in this study were the AMPK antibody (1:1,000, ab32047, Abcam, Cambridge, UK), p-AMPK antibody (1:1,000, ab32047, Abcam), mTOR antibody (1:1,000, CSB-PA208208; Cusabio, Hubei, China), p-mTOR antibody (1:1,000, CSB-PA271384; Cusabio), Beclin-1 antibody (1:1,000, ab210498; Abcam), LC3II/LC3I antibody (1:1,000, 12741T; CST), Bcl-2 antibody (1:1,000, ab182858; Abcam), CD63 antibody (1:1,000, ab217345; Abcam), CD81 antibody (1:1,000, DF2306; Affinity, Cincinnati, OH, USA), TSG101 antibody (1:1,000, DF8427; Affinity), HSP70 antibody (1:1,000, AF5466, Affinity) and the GAPDH antibody (1:1,000, ab245355; Abcam).

Techniques: Western Blot

Journal: PeerJ

Article Title: Exosomes from adipose-derived stem cells alleviate premature ovarian failure via blockage of autophagy and AMPK/mTOR pathway

doi: 10.7717/peerj.16517

Figure Lengend Snippet: (A) IC50 value for ADSCs-Exo treating CTX-treated KGN cells was identified by cell counting kit (CCK)-8 assay. (B) Cell viability was detected by CCK-8 assay. (C) Cell apoptosis was assessed by flow cytometry. (D) The protein levels of Beclin-1, LC3II/LC3I, Bcl-2, p-AMPK/AMPK, and p-mTOR/mTOR were detected by western blotting. KGN cells were treated with 250 µM CTX, 10 µg/mL ADSCs-Exo, or/and 5 µM rapamycin (Rapa). ** p < 0.01 compared with the control group, ## p < 0.01 compared with the CTX group, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\hat {}$\end{document} ˆ p < 0.05 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\hat {}\hat {}$\end{document} ˆ ˆ p < 0.01 compared with the CTX + ADSCs-Exo group.

Article Snippet: The primary antibodies used in this study were the AMPK antibody (1:1,000, ab32047, Abcam, Cambridge, UK), p-AMPK antibody (1:1,000, ab32047, Abcam), mTOR antibody (1:1,000, CSB-PA208208; Cusabio, Hubei, China), p-mTOR antibody (1:1,000, CSB-PA271384; Cusabio), Beclin-1 antibody (1:1,000, ab210498; Abcam), LC3II/LC3I antibody (1:1,000, 12741T; CST), Bcl-2 antibody (1:1,000, ab182858; Abcam), CD63 antibody (1:1,000, ab217345; Abcam), CD81 antibody (1:1,000, DF2306; Affinity, Cincinnati, OH, USA), TSG101 antibody (1:1,000, DF8427; Affinity), HSP70 antibody (1:1,000, AF5466, Affinity) and the GAPDH antibody (1:1,000, ab245355; Abcam).

Techniques: Cell Counting, CCK-8 Assay, Flow Cytometry, Western Blot, Control

Journal: World Journal of Gastroenterology

Article Title: Antagonizing adipose tissue-derived exosome miR-103-hepatocyte phosphatase and tensin homolog pathway alleviates autophagy in non-alcoholic steatohepatitis: A trans-cellular crosstalk

doi: 10.3748/wjg.v29.i29.4528

Figure Lengend Snippet: The capacity of miR-103 in targeting phosphatase and tensin homolog gene and affecting autophagy. A: The luciferase reporter assay results verified the interaction between miR-103 and phosphatase and tensin homolog; B: Western blotting showed the differential expression of the autophagy-related protein; C: Transmission electron microscopy images of autophagosomes (red arrowhead) in the liver; D: The results of immunofluorescence staining showed liver autophagosomes in different groups. a P < 0.01 vs control; b P < 0.01 vs model; c P < 0.01 vs miR-103; d P < 0.05 vs model. PTEN: Phosphatase and tensin homolog; WT: Wild type; MUT: Mutant; DAPI: 4’,6-diamidino-2-phenylindole; mTOR: Mammalian target of rapamycin; NC: Negative control.

Article Snippet: The membranes were blocked and incubated overnight with antibodies against PTEN (9188T, CST), p-AMPK (ab32047, Abcam), p-mammalian target of rapamycin (mTOR) (CSB-PA271384, Cusabio), LC3 (12741T, CST), p62 (ab91526, Abcam), and GAPDH (ab245355, Abcam) at 4 °C.

Techniques: Luciferase, Reporter Assay, Western Blot, Quantitative Proteomics, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Control, Mutagenesis, Negative Control

Journal: World Journal of Gastroenterology

Article Title: Antagonizing adipose tissue-derived exosome miR-103-hepatocyte phosphatase and tensin homolog pathway alleviates autophagy in non-alcoholic steatohepatitis: A trans-cellular crosstalk

doi: 10.3748/wjg.v29.i29.4528

Figure Lengend Snippet: The effect of adipose tissue-derived exosomes miR-103 on autophagy in mice. A and B: Western blotting detected the expression of the autophagy-related protein; C: The results of immunofluorescence staining to observe autophagosomes in the liver from different groups; D: Transmission electron microscopy images of autophagosomes (red arrowhead) in the liver from different groups. a P < 0.05 vs control; b P < 0.01 vs control; c P < 0.05 vs exosomes; d P < 0.01 vs exosomes. Exo: Exosomes; PTEN: Phosphatase and tensin homolog; DAPI: 4’,6-diamidino-2-phenylindole; mTOR: Mammalian target of rapamycin; NC: Negative control.

Article Snippet: The membranes were blocked and incubated overnight with antibodies against PTEN (9188T, CST), p-AMPK (ab32047, Abcam), p-mammalian target of rapamycin (mTOR) (CSB-PA271384, Cusabio), LC3 (12741T, CST), p62 (ab91526, Abcam), and GAPDH (ab245355, Abcam) at 4 °C.

Techniques: Derivative Assay, Western Blot, Expressing, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Control, Negative 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: 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: Animal Nutrition

Article Title: MRCKα is a novel regulator of prolactin-induced lactogenesis in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2022.06.001

Figure Lengend Snippet: Immunocytochemical characterization of the primary culture of bovine mammary epithelial cells (BMEC). (A) Isolated BMEC were cultured on coverslips for 24 h and analyzed by immunocytochemistry using anti-CK18 monoclonal antibody. Images show CK18 (green), nuclear DNA (DAPI, blue) and merged fluorescence confocal microscope emissions. (B) In parallel, cultured BMEC were lysed after 24 h and subjected to Western blot analysis using anti-CK18 monoclonal or β-casein polyclonal antibody. Equal amounts of total protein (25 μg) were loaded in each lane. BMEC-1 and BMEC-2 represent 2 independent batches of BMEC. DAPI, 4′,6-diamidino-2-phenylindole, a blue-fluorescent DNA stain. CK18 = cytokeratin 18.

Article Snippet: Rabbit polyclonal anti-human phosphorylated Ser2448-mTOR antibody (YT2913; 1:1,000) and rabbit polyclonal anti-human mTOR antibody (YP1134; 1:1,000) were obtained from ImmunoWay Biotechnology Company (Plano, TX).

Techniques: Isolation, Cell Culture, Immunocytochemistry, Fluorescence, Microscopy, Western Blot, Staining

Journal: Animal Nutrition

Article Title: MRCKα is a novel regulator of prolactin-induced lactogenesis in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2022.06.001

Figure Lengend Snippet: Prolactin stimulates the expression of both β-casein and MRCKα. (A) Confluent bovine mammary epithelial cells (BMEC) were stimulated with prolactin (PRL, 0.6 μg/mL) or left untreated (Control) for 24 h, lysed and subjected to Western blot analysis using polyclonal antibodies against β-casein, MRCKα or β-actin. (B) Immunopositive bands were quantified by densitometry, normalized to β-actin and presented as protein expression levels relative to their corresponding control. (C) β-casein protein expression in BMEC tended to correlate positively with that of MRCKα. (D) Abundance of β-casein and MRCKα mRNA measured by RT-qPCR in BMEC upon stimulation with prolactin (PRL, 0.6 μg/mL) or vehicle (Control) for 24 h. (E) β-casein mRNA expression in BMEC correlated positively with that of MRCKα. Values are presented as mean ± SEM of 3 independent experiments. Means without a common letter differ, P < 0.05. MRCKα = myotonic dystrophy-related Cdc42-binding kinase alpha.

Article Snippet: Rabbit polyclonal anti-human phosphorylated Ser2448-mTOR antibody (YT2913; 1:1,000) and rabbit polyclonal anti-human mTOR antibody (YP1134; 1:1,000) were obtained from ImmunoWay Biotechnology Company (Plano, TX).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: Animal Nutrition

Article Title: MRCKα is a novel regulator of prolactin-induced lactogenesis in bovine mammary epithelial cells

doi: 10.1016/j.aninu.2022.06.001

Figure Lengend Snippet: MRCKα positively regulates the synthesis of β-casein in bovine mammary epithelial cells (BMEC). (A) Confluent BMEC were transfected with MRCKα siRNA (SI), scrambled siRNA (control, SC) or left untreated (control, Blank) for 24 h, lysed and subjected to Western blot analysis using polyclonal antibodies directed against MRCKα, β-casein and β-actin. Immunopositive bands were analyzed by densitometry. The resulted protein levels were normalized to β-actin and the average fold-change over Blank for MRCKα (B) and β-casein (C) protein expression are presented of 3 independent experiments. (D) Confluent BMEC were transfected with empty vector pcDNA3.1 (EV), MRCKα-FLAG (OE), or left untreated (control, Blank). After 24 h, cells were lysed and analyzed by immunoblotting using polyclonal antibodies against MRCKα and β-casein. Immunopositive bands were analyzed by densitometry. Protein levels were normalized to β-actin and the average fold-change over Blank for MRCKα (E) and β-casein (F) protein expression is presented of 3 independent experiments. Means without a common letter differ, P < 0.05. MRCKα = myotonic dystrophy-related Cdc42-binding kinase alpha.

Article Snippet: Rabbit polyclonal anti-human phosphorylated Ser2448-mTOR antibody (YT2913; 1:1,000) and rabbit polyclonal anti-human mTOR antibody (YP1134; 1:1,000) were obtained from ImmunoWay Biotechnology Company (Plano, TX).

Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation, Binding Assay

Journal: Molecular and Cellular Biochemistry

Article Title: Low glucose dependent decrease of apoptosis and induction of autophagy in breast cancer MCF-7 cells

doi: 10.1007/s11010-016-2711-4

Figure Lengend Snippet: Western blot ( a ) and densitometric analysis ( b ) of mTOR kinase expression in MCF-7 cells incubated in high glucose (H) and low glucose (L) DMEM for 12, 24, and 48 h. Samples containing 30 μg of protein were submitted to electrophoresis and immunoblotting. Densitometric analysis was presented as relative protein expression. The expression of β-tublin served as a control for protein loading. A representative Western blot from one of three independent experiments is presented ( a ). Mean values of densitometric analysis from three independent experiments ± SD are presented ( b )

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 4.5 mg/ml (25 mM), Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 0.5 mg/ml (2.8 mM), l -glutamine, penicillin, streptomycin, trypsin–EDTA, FBS Gold, trypsin–EDTA were provided by Gibco (San Diego, USA), passive lysis buffer by Promega (Madison, USA), BCA Protein Assay Kit by Thermo Scientific (Rockford, USA), PE Annexin V Apoptosis Detection Kit I by BD PharmingenTM (CA, USA), Sigma-Fast BCIP/NBT reagent, 4′,6-diamidino-2-phenylindole dihydrochloride—DAPI, donkey serum, acridine orange, ethidium bromide, camptothecin by Sigma (St Louis, MO, USA), monoclonal (mouse) anti-human ORP150 antibody by IBL (Gunma, Japan), medium coverquick by Hygeco (USA), polyclonal (rabbit) anti-human NF-κB2 p100/p52 antibody, monoclonal (rabbit) anti-human CHOP antibody, monoclonal (rabbit) anti-human β-tubulin antibody, alkaline phosphatase-labeled anti-rabbit immunoglobulin G were provided by Cell Signaling Technology (Boston, USA), monoclonal (mouse) anti-human mTOR antibody (Merck-Millipore, USA), alkaline phosphatase-labeled anti-mouse immunoglobulin G by Rockland (PA, USA), polyclonal (rabbit) anti-human LC3 by Abgen, donkey anti-rabbit IgG conjugated with Alexa Fluor 488 by Molecular Probes (USA).

Techniques: Western Blot, Expressing, Incubation, Electrophoresis, Control

Journal: Molecular and Cellular Biochemistry

Article Title: Low glucose dependent decrease of apoptosis and induction of autophagy in breast cancer MCF-7 cells

doi: 10.1007/s11010-016-2711-4

Figure Lengend Snippet: The effect of glucose shortage on apoptosis, autophagy, and their mechanism in breast cancer MCF-7 cell line. ORP150 oxygen-regulated protein 150, UPR unfolded protein response, NF-κB nuclear factor-κB, mTOR mammalian target of rapamycin, ATP adenosine triphosphate, CHOP CCAAT/enhancer-binding protein (C/EBP) homologous protein, P53 protein p53, P27 protein p27

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 4.5 mg/ml (25 mM), Dulbecco’s modified Eagle’s medium (DMEM), containing glucose at 0.5 mg/ml (2.8 mM), l -glutamine, penicillin, streptomycin, trypsin–EDTA, FBS Gold, trypsin–EDTA were provided by Gibco (San Diego, USA), passive lysis buffer by Promega (Madison, USA), BCA Protein Assay Kit by Thermo Scientific (Rockford, USA), PE Annexin V Apoptosis Detection Kit I by BD PharmingenTM (CA, USA), Sigma-Fast BCIP/NBT reagent, 4′,6-diamidino-2-phenylindole dihydrochloride—DAPI, donkey serum, acridine orange, ethidium bromide, camptothecin by Sigma (St Louis, MO, USA), monoclonal (mouse) anti-human ORP150 antibody by IBL (Gunma, Japan), medium coverquick by Hygeco (USA), polyclonal (rabbit) anti-human NF-κB2 p100/p52 antibody, monoclonal (rabbit) anti-human CHOP antibody, monoclonal (rabbit) anti-human β-tubulin antibody, alkaline phosphatase-labeled anti-rabbit immunoglobulin G were provided by Cell Signaling Technology (Boston, USA), monoclonal (mouse) anti-human mTOR antibody (Merck-Millipore, USA), alkaline phosphatase-labeled anti-mouse immunoglobulin G by Rockland (PA, USA), polyclonal (rabbit) anti-human LC3 by Abgen, donkey anti-rabbit IgG conjugated with Alexa Fluor 488 by Molecular Probes (USA).

Techniques: Binding Assay