Ab7778

All the antibodies were purchased from Abcam Company: anti‐collagen I (ab34710), anti‐collagen III (ab7778), anti‐fibronectin (ab2413), anti‐CD31 (ab24590), anti‐SMAD7 (ab216428), anti‐p‐p65 (ab86299), anti‐p‐SMAD2 (ab53100), anti‐p‐SMAD3 (ab52903) and anti‐α‐SMA (ab5694).

Scoparone (E‐0358) was purchased from Tauto (Shanghai, China). Ang II (A9525) was purchased from Sigma‐Aldrich (St. Louis, MO, USA), Alzet osmotic minipump (model 2004) was purchased from DURECT (California, USA), tail‐cuff instrument was purchased from IITC Inc (Woodland Hills, California, USA), and the ultrasound imaging system(Vevo2100) was purchased from VisualSonics (Toronto, Canada). Antibodies against 4HNE (ab46545), RAC1‐GTP (ab203884), α‐actinin (ab9465), vimentin (ab8978), collagen I (ab34710), collagen III (ab7778) and connective tissue growth factor (CTGF, ab6992) were purchased from Abcam (Cambridge, MA, USA). Antibody against α‐smooth muscle actin (α‐SMA, A2547, for Western blot use) was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies against total‐RAC1 (66122‐1‐lg), NOX2 (19013‐1‐AP), NOX4 (14347‐1‐AP), α‐SMA (14395‐1‐AP, for immunofluorescence stain use), cardiac troponin I (CTNI, 66376‐1‐Ig) and β‐actin (60008‐1‐Ig) were purchased from Proteintech (Hubei, China). ³H‐leucine (NET135H) was purchased from Perkin Elmer (Waltham, MA, USA), ROS assay (S0033) was purchased from Beyotime (Shanghai, China), and the Cytotoxicity Detection Kit (11644793001) was purchased from Roche (Hoffmann, Germany).

Protein was extracted from frozen distal vaginal segments (n=6 animals/group) and quantified using a bicinchoninic acid protein assay. The samples were separated by SDS-PAGE (a-SMA, collagen I, and β-actin on a 10% gel; collagen III on an 8% gel), and protein was transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Massachusetts). Membranes were blocked with 5% nonfat milk for one hour in 0.5% TBS-T prior to incubation with antibodies. Anti-a-SMA primary antibody (1:2000, ab5694, Abcam), anti-collagen I primary antibody (1:1000, ab34710, Abcam), anti-collagen III primary antibody (1:7000, ab7778, Abcam), anti-β-actin primary antibody (1:3000, GB12001, Servicebio), horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3000, GB23303, Servicebio), and horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:3000, GB23301, Servicebio) were diluted in TBS-T. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). The densitometry of the immunoreactive bands on the Western blot was calculated using Image J software.

The target cell protein was obtained by RIPA method, and the protein concentration was determined by BCA kit (Solarbio, Beijing, China). Then, prepared protein was taken for gel electrophoresis separation, and the isolated protein was electrically transferred to PVDF membrane. After blocked with 5% skim milk, the PVDF membrane was subjected to primary antibodies: connective tissue growth factor (CTGF, #ab6992, 1:1000, Abcam, Cambridge, UK), collagen I (#ab270993, 1:1000, Abcam, Cambridge, UK), collagen III (#ab7778, 1:1000, Abcam, Cambridge, UK), Bcl-2 (#ab196495, 1:1000, Abcam, Cambridge, UK), Bax (#ab32503, 1:1000, Abcam, Cambridge, UK), NOD-like receptor protein 3 (NLRP3, #ab263899, 1:1000, Abcam, Cambridge, UK), ASC (rat: #ab180799, 1:1000, Abcam, Cambridge, UK; mouse: #67824S, Cell Signaling, Danvers, MA), caspase-1 (#83383, 1:1000, Cell Signaling, Danvers, MA), IL-β (#ab254360, 1:1000, Abcam, Cambridge, UK), IL-18 (#ab191860, 1:1000, Abcam, Cambridge, UK) and GAPDH (#ab9485, 1:1000, Abcam, Cambridge, UK). The next day, the PVDF membrane was incubated with secondary antibody (#ab288151, 1:5000, Abcam, Cambridge, UK) at room temperature. An enhanced chemiluminescence kit (ECL, #P0018S, Beyotime Biotechnology, Beijing, China) was added and exposed in the gel imaging system. The protein content was analysed using Quantity-One software (Bio-Rad, Hercules, CA).

Paraffin- or optimal cutting temperature compound-embedded tissues were sectioned and stained6 (link)7 (link) using the following primary antibodies (all diluted 1:100 unless stated otherwise) for immunofluorescence labelling: Lrig: R&D Systems, FAB3688G; CD26: R&D Systems, AF954; Sca1: R&D Systems, AF1226; PDGFRa: R&D Systems, AF1062; Collagen III: Abcam, ab7778, Collagen11a1: Abcam, ab64883; Elastin: Abcam, ab21610; Caveolin: Cell Signaling Technology, 3267; phospho-Histone H3 (Ser10) antibody: Cell Signalling Technology, 970; Active Caspase-3: RnD Systems, AF835; K14: Covance, PRB-155P, 1:500; GFP: Abcam, ab13970, 1:500; RFP: Rockland, 600-401-379, 1:300. EdU staining was performed with a Click-it EdU imaging kit (Invitrogen) according to the manufacturer's recommendations. Images were acquired with a Nikon A1 Upright Confocal microscope. Images of H&E- and Herovici-stained sections were acquired with a Hamamatsu slide scanner. Representative images of skin from two to three independent experiments with at least three biological replicates per group are shown.

THC was purchased from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, China) with a purity of 98% tested by high-performance liquid chromatography (HPLC). STZ was purchased from Sigma-Aldrich (St. Louis, MO, USA). The fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) used to detect intracellular ROS generation was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Dihydroethidium (DHE; for detecting ROS generation in cardiac tissues) were purchased from Invitrogen (Carlsbad, CA, USA). A primary antibody against SOD2 (sc-30080) was obtained from Santa Cruz Biotechnology (CA, USA). Primary antibodies against T-Smad3 (SC101154), p-Smad3 (8760), and β-actin (4970) were all purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against SIRT1 (ab110304), acetylated SOD2 (Ac-SOD2, ab137037), α-SMA (ab5694), TGFβ1 (ab92486), collagen І (ab90395), and collagen III (ab7778) were purchased from Abcam (Cambridge, MA, USA). The rabbit anti-goat and goat anti-mouse secondary antibodies were obtained from the Zhongshan Company (Beijing, China).