Journal: Molecular Medicine Reports

Article Title: Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

doi: 10.3892/mmr.2016.5264

Figure Lengend Snippet: Mechanical strain induces hUSLF apoptosis and senescence and suppresses collagen anabolism. The hUSLF were exposed with or without mechanical strain of 5333 µ ε. (A) Cellular apoptosis was observed by staining with Annexin V/PI and detected by flow cytometry. (B) Cell senescence was evaluated by positive senescence-associated-β-galactosidase percentage. Scale bars, 100 µ m and arrows indicate examples of positive staining. (C and D) The expression of COL1A1 were assessed by western blotting. The level of COL1A1 protein concentration was calculated and normalized to the level of GAPDH protein concentration. All experiments were performed in triplicate. * P<0.05 vs. the control group. hUSLF, human uterosacral ligament fibroblasts; PI, propidium iodide; COL1A1, procollagen type 1 α1.

Article Snippet: The following primary antibodies were used: Rabbit polyclonal Akt (1:500; cat. no. 9272), rabbit monoclonal phosphorylated (p)-Akt (1:500; cat. no. 4058), mouse monoclonal FOXO1 (1:1,000; cat. no. 97635) and rabbit polyclonal p-FOXO1 (1:1,000; cat. no. 9461) all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal Mn-superoxide dismutase (Mn-SOD; 1:500; cat. no. sc-130345) and rabbit polyclonal procollagen type I α1 (COL1A1; 1:1,000; cat. no. sc-28657) all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); and rabbit polyclonal GPX1 (1:1,000; cat. no. ab22604) and rabbit polyclonal GAPDH (1:1,000; cat. no. ab9485) obtained from Abcam, Cambridge, UK).

Techniques: Staining, Flow Cytometry, Expressing, Western Blot, Protein Concentration, Control

Journal: Molecular Medicine Reports

Article Title: Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

doi: 10.3892/mmr.2016.5264

Figure Lengend Snippet: Mechanical strain induces hUSLF apoptosis and senescence and suppresses collagen I production via the PI3K/Akt signaling pathway. The hUSLF were treated with or without mechanical strain of 5333 µ ε for 4 h in the presence or absence of LY294002. Cells were incubated with LY294002 (20 µ M) for 30 min prior to the application of mechanical strain. (A and B) Cellular apoptosis was detected by Annexin V/PI double staining and flow cytometer. (C and D) Cell senescence was evaluated by positive SA-β-gal percentage. Scale bars, 100 µ m. (E–G) The mRNA and protein expression levels of COL1A1 were assessed by reverse transcription-quantitative polymerase chain reaction and western blotting. The level of COL1A1 mRNA and protein expression were calculated and normalized to the level of GAPDH. All experiments were performed in triplicate. * P<0.05, ** P<0.01, *** P<0.001 vs. other groups or groups indicated. hUSLF, human uterosacral ligament fibroblasts; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PI, propidium iodide; COL1A1, procollagen type 1 α1.

Article Snippet: The following primary antibodies were used: Rabbit polyclonal Akt (1:500; cat. no. 9272), rabbit monoclonal phosphorylated (p)-Akt (1:500; cat. no. 4058), mouse monoclonal FOXO1 (1:1,000; cat. no. 97635) and rabbit polyclonal p-FOXO1 (1:1,000; cat. no. 9461) all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal Mn-superoxide dismutase (Mn-SOD; 1:500; cat. no. sc-130345) and rabbit polyclonal procollagen type I α1 (COL1A1; 1:1,000; cat. no. sc-28657) all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); and rabbit polyclonal GPX1 (1:1,000; cat. no. ab22604) and rabbit polyclonal GAPDH (1:1,000; cat. no. ab9485) obtained from Abcam, Cambridge, UK).

Techniques: Incubation, Double Staining, Flow Cytometry, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine Reports

Article Title: Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

doi: 10.3892/mmr.2016.5264

Figure Lengend Snippet: Apoptosis level increased and collagen type I production was reduced in USLs of POP patients. (A) Cell apoptosis in USL tissue explants were detected using the TUNEL assay. TUNEL stain positive cells stain green and nuclei were stained blue by DAPI. Scale bars, 100 µ m. (B) Apoptosis levels were calculated by percentage of TUNEL positive cells. (C) USL RNA was collected and COL1A1 mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction. GAPDH mRNA served as an internal control. (D and E) COL1A1 concentration in USL was detected by western blotting. GAPDH concentration served as an internal control. The non-POP group served as the control and all experiments performed in triplicate. *** P<0.001 vs. the non-POP group. USL, uterosacral ligament; POP, pelvic organ prolapse; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole; COL1A1, procollagen type 1 α1.

Article Snippet: The following primary antibodies were used: Rabbit polyclonal Akt (1:500; cat. no. 9272), rabbit monoclonal phosphorylated (p)-Akt (1:500; cat. no. 4058), mouse monoclonal FOXO1 (1:1,000; cat. no. 97635) and rabbit polyclonal p-FOXO1 (1:1,000; cat. no. 9461) all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal Mn-superoxide dismutase (Mn-SOD; 1:500; cat. no. sc-130345) and rabbit polyclonal procollagen type I α1 (COL1A1; 1:1,000; cat. no. sc-28657) all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); and rabbit polyclonal GPX1 (1:1,000; cat. no. ab22604) and rabbit polyclonal GAPDH (1:1,000; cat. no. ab9485) obtained from Abcam, Cambridge, UK).

Techniques: TUNEL Assay, Staining, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Concentration Assay, Western Blot