Gapdh

Recombinant human TGF-β1 was obtained from R&D systems (Minneapolis, MN, USA). Bleomycin sulfate was obtained from Hisun Pharm (Taizhou, China). Sodium orthovanadate (SOV) was obtained from Sigma-Aldrich (Shanghai, China). Wiskostatin (Wis) was obtained from Abcam (Shanghai, China). N-WASP was obtained from Proteintech (Wuhan, China). Goat anti-rabbit IgG of β-actin, Alpha-smooth muscle actin (α-SMA), p^Y256N-WASP and GAPDH were purchased from Affinity Biosciences (Changzhou, China). Goat anti-mouse IgG of Vimentin and E-cadherin were purchased from Cell Signaling Technology (Shanghai, China), Goat anti-rabbit IgG of N-cadherin, Fibronectin and Collagen I were purchased from Cell Signaling Technology (Shanghai, China), TUFT1 was purchased from Thermo Fisher (Suzhou, China).

IPTG was purchased from Gibco (Gand Island, NY, USA). Ni-agarose affinity chromatography columns were purchased from Invitrogen (Cambridge, UK). Bleomycin hydrochloride (BLM) was purchased from Maclean (Shanghai, China). DMEM was supplied by Gibco (Gand Island, NY, USA); Fetal bovine serum was purchased from PAN-Biotech (Heilbronn, Germany); Recombinant human TGF-β1 was purchased from PEPROTECH (Rocky Hill, USA); GAPDH, α-SMA and FN antibodies were purchased from Affinity (Cincinnati, OH, USA); Col-I ELISA kit was purchased from MEIMIAN (Jiangsu, China); Hydroxyproline kit was purchased from Nanjing Jiancheng (Nanjing, China).

Western blotting analysis was conducted using NUSAP1 (1:1000; #12024-1-AP; Proteintech, Rosemont, IL, USA), YAP1 (1:1500; #14074; Cell Signaling Technology, Danvers, USA), LATS1 (1:1500; #3477; Cell Signaling Technology, Danvers, USA), LATS2 (1:2000; #5888; Cell Signaling Technology, Danvers, USA), CTGF (1:2000; #86641; Cell Signaling Technology, Danvers, USA), CYR61 (1:2000; #14479; Cell Signaling Technology, Danvers, USA), anti-HA (1:2500; #ab9110; Abcam, Cambridge, USA), anti-Flag (1:2500; #A2220; Sigma-Aldrich, USA), β-actin (1:3000; #AF7018; Affinity, Jiangsu, China) and GAPDH (1:3000; #A2220; Affinity, Jiangsu, China). Human GC samples originally obtained from the First Affiliated Hospital of Nanchang University, along with the xenograft tumors, were ground and lysed in lysis buffer before Western blotting analysis. The Western blotting experiments were performed as previously described (28 (link)). GAPDH or β-actin was used as an internal control, and all the protein bands were analyzed using ImageJ software.

The total proteins, nuclear proteins or cytoplasmic proteins were extracted from the exosomes or cortex tissues of the infarcted cerebral hemisphere according to the manufacturer's instructions (Beyotime). The amount of protein was quantified by the BCA assay. Equal amounts of protein were electrophoresed by 10% SDS‐PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, membranes were incubated with the following primary antibodies, including CD63 (1:500; Abcam), CD9 (1:500; Abcam), CD81 (1:500; Abcam), TLR4 (1:1000; Affinity Biosciences), NF‐κB p65 (1:1000; Affinity Biosciences), HMGB1 (1:1000; Abcam), GAPDH (1:2000; Affinity Biosciences), MyD88 (1:1000; Affinity Biosciences), β‐actin (1:2000; Proteintech), Histone H3 (1:2000; Affinity Biosciences) and the secondary HRP‐labelled antibodies (1:5000; Proteintech). Band visualization was enhanced with chemiluminescence (ECL; Biological Industries), and the results were processed with a Chemiluminescent Imaging and Analysis System (Biolight Biotechnology Co., Ltd.).

Cells were lysed in a RIPA lysis buffer (Beyotime, Haimen, China) to extract total protein. Protein was separated on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Skimmed milk was used for blocking PVDF membranes. Immunoblotting of the membranes was conducted by incubation with primary antibodies: P27, CyclinD1 (CST, Danvers, MA, USA) and GAPDH (Affinity Biosciences, Cincinnati, OH, USA). Immunoblotting was carried out on a shaking table at 4 °C overnight. After incubation with a second antibody (CST) for one hour at room temperature and washing with TBS-T, a Western Bright ECL HRP substrate (Advansta, Menlo Park, CA, USA) was added to the membrane and it was visualized by an automated chemiluminescence image analysis system (Tanon 5200, Shanghai, China). Densities of protein bands were measured using ImageJ software (NIH, Bethesda, USA).

Puerarin was purchased from Macklin (Art. No. P816259, China) with purity ≥ 98% (HPLC). Antibodies against P53, P21, PTEN, BRCA1, BIRC5, CTGF, Bax, Bcl-2, caspase 3, cleaved caspase 3, GAPDH, Ki67, and PCNA were purchased from Affinity Biosciences (USA); IgG H&L (Alexa Fluor® 488) was from Abcam (UK); CD3, CD4, and CD8 were from BD Biosciences Pharmingen (USA). The CCK-8 kit was obtained from MedChemExpress (USA); the cell apoptosis kit was obtained from BD Biosciences Pharmingen (USA); the EdU-594 cell proliferation detection kit was obtained from Beyotime Biotechnology (China); the TUNEL staining kit was obtained from Servicebio (China); ELISA kits for IL-6 and TNF-α were purchased from MEIMIAN (China); the ELISA kit for CA125 was purchased from ZSGB-BIO (China).

Global brain tissue was dissected from treated mice (purchased from the Beijing HFK Bioscience Co., LTD [Certificate No: SCXK (Jing) 2014-0004]) and proteins extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Scientific^TM T-PER^TM Tissue Protein Extraction Reagent, 78510). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% nonfat dry milk in Tris-buffered saline (20 mM Tris-HCl, 500 mM NaCl, pH 7.4) with 0.2% Tween-20 (Aladdin, T104863), the membranes were probed with antibodies overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse (Servicebio, G2211-1-A) or goat anti-rabbit (Servicebio, G2210-2-A) IgG secondary antibody (1:2000). The antibodies were as follows; anti-Abeita A4, -APOE, -BACE1, -p53 (abcam, 66064), -p16 (Affinity, AF0228), -p21, -p-Tau-396, -TNF-α (abcam, 9739), -p-Tau-202, obtained from Affinity as well as GAPDH (CST, 2118L) and β-Actin (CST, 4970S). Band intensity was quantified using ImageJ software (NIH).