Sc 166572

Per well, 8 × 10³ HDF were grown in 96-well plates and treated with the 0.002% of ObHEx or TGFβ 2.5 ng/mL. After 24 h, the cells were processed for ELISA using monoclonal primary antibody anti-procollagen type I (sc-166572, Santa-Cruz Biotechnology, Dallas, TX, USA), followed by incubation with the secondary anti-mouse antibody labeled with peroxidase (170-6516, Biorad, Hercules, CA, USA). The supernatants of the cells were coated on another plate for the detection of tropoelastin and periostin using the anti-tropoelastin rabbit antibody (ab21600, Abcam, Cambridge, UK) or anti-periostin mouse antibody (sc-398631, Santa-Cruz Biotechnology, Dallas, TX, USA), followed by incubation with secondary antibody anti-rabbit labeled with peroxidase (170-6515, Biorad, Hercules, CA, USA). The colorimetric reaction was developed by adding 100 μL of an aqueous solution of OPD (O-phenylenediamine), 0.35 mg/mL in 50 mM citrate buffer, and 0.012% hydrogen peroxide (H₂O₂). After 30 min, the absorbance was measured at 490 nm using the multiplate reader Victor Nivo (PerkinElmer, Waltham, MA, USA).

Western blot analysis was used to detect the protein expression levels in the cells and uterosacral ligaments. Briefly, the total cell protein was extracted using RIPA (Radio Immunoprecipitation Assay), the protein concentration was measured by the BCA (bicinchoninic acid) method, and 20 μg protein was subjected to gel electrophoresis. Following protein transfer, the membranes were incubated with anti-Mfn2 (1:500, catalog #AB56889, Abcam, Cambridge, UK), anti-procollagen 1A1/1A2/3A1 (anti-procollagen1A1: 1:1000, catalog #OM164818, OmnimAbs, Alhambra, CA, USA; anti-procollagen1A2: 1:2000, catalog #SC-166572, Santa Cruz, CA, USA; anti-procollagen3A1: 1:2000, catalog #SC-166333, Santa Cruz, CA, USA), and anti-β-actin (1:2000, catalog #TA-09, ZSGB-Bio, Beijing, China). The next day, after three washes with TBST, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:5000; catalog #ZB-2305, ZSGB-Bio, Beijing, China), and HRP-labeled goat anti-rabbit IgG (H + L) (1:5000; catalog #ZB-2301, ZSGB-Bio, Beijing, China) were incubated at room temperature for 1 h. Immunoblotting chemiluminescence imaging was performed using an HRP chemical substrate (Immobilon Western Chemilum HRP Substrate, WBKLS0100, Millipore, Darmstadt, Germany). ImageJ software (1.8.0., NIH, Bethesda, MD, USA) was used to calculate the grayscale ratio of the target protein bands to the β-actin bands.

Dimethyl sulfoxide (DMSO, A3672) was purchased from AppliChem GmbH (Darmstadt, Germany). TRIzol reagent and reverse transcription polymerase chain reaction polymer kit (AQ131-02) were purchased from Beijing Full-Type Gold Biotechnology Co., Ltd. (Beijing, China); besides, DMEM/F12 (11330-032), antibiotics-antimycotic (15240-062), fetal bovine serum (FBS; 10099141), 0.25% trypsin-EDTA digestive juices (25200-056) were purchased from Gibco (New York, NY, USA). The cell counting kit-8 (CCK-8) was provided by Dojindo Molecular Technologies, Inc. (Rockville, MD USA). Mfn2 monoclonal antibody (dilution, 1/1000; Ab56889) was purchased from Abcam (Cambridge, UK). Anti-procollagen 1A1/1A2/3A1 antibodies (dilution, 1/200; Sc-293182, Sc-166572, and Sc-166333, respectively) were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). Anti-ERα (D8H8) Rabbit mAb antibody(dilution, 1/1000) and Anti-cyclinD1 (92G2) Rabbit mAb antibody(dilution, 1/1000) were purchased from Cell Signaling Technology, Inc. (USA). Horseradish peroxidase (HRP) goat anti-mouse IgG (dilution, 1/5000, ab6789), Anti-ERβ(dilution, 1/1000, ab196787) and Anti-GPR30 (dilution, 1/1000, ab39742) were purchased from Abcam (Cambridge, UK). Procedural cooling box was purchased from Sigma-Aldrich (St. Louis, MO, USA).