Sample buffer

Two days after treatment with Bangle extract or CHIR99021, the hfNSCs were first washed with cold PBS, and then hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 1.5 mM MgCl₂) was added. The nuclear extract was retrieved by centrifugation (800× g). Pellet samples (nuclei) and supernatant (almost pure cytoplasm), for immunoblotting, were prepared as follows. Pellets were lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100) that included a protease inhibitor cocktail (Nacalai Tesque, Inc.) and a phosphatase inhibitor cocktail (Nacalai Tesque, Inc.) for 30 min on ice. Then, sample buffer (Nacalai Tesque, Inc.) was added to the pellet lysed samples and supernatant samples, and these samples were incubated for 5 min at 95 °C. Aliquots of total cell lysate were separated by 5–20% SuperSep (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA). After blocking with BlockingOne (Nacalai Tesque, Inc.) containing 0.1% Tween 20, the membranes were incubated with appropriate primary antibodies (Table S3). After washing with TBST (Tris-buffered saline, 0.1% Tween 20), the membranes were then incubated with the appropriate HRP-conjugated secondary antibodies (Abcam), washed, and developed with ECL Prime reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

Cells were seeded and incubated overnight, and then divided into 2 groups and switched to media with or without 5 ng/mL TGF-β. After incubation for 24 h, nanaomycin K (5 µg/mL or 50 µg/mL) or 0.05% DMSO was added to the cultures. After incubation for an additional 48 h, cells were washed and lysed in 8 M urea buffer. Each sample was added into sample buffer (Nacalai Tesque, Kyoto, Japan) and heated at 95 °C for 5 min. The samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with Blocking One or Blocking One-P (Nacalai Tesque) followed by washing, the membranes were incubated overnight at room temperature with anti-E-cadherin (Biolegend, San Diego, CA), anti-N-cadherin (Biolegend), anti-vimentin (Biolegend), anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology: CST, Danvers, MA), anti-phospho-SAPK/JNK (Thr183/Tyr185) (CST), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST), anti-Snail (CST), anti-Slug (CST) or anti-β-actin (Santa Cruz Biotechnology, Dallas, TX), respectively. After another washing, membranes were incubated for 1 h with HRP-conjugated secondary antibodies. Antibody binding to proteins was detected by enhanced chemiluminescence.

Proteins were extracted from HeLa cells, immortalized DMD cells, and mouse muscle tissue using CelLytic M (Sigma-Aldrich, St. Louis, MO, USA), 1× Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA), and CelLytic MT (Sigma-Aldrich), respectively. All buffers were supplemented with protease and phosphatase inhibitor cocktail (#25955 and #0757461, Nacalai). Protein concentrations were quantified with Protein Assay Reagent (Pierce by Thermo Fisher Scientific). The HeLa cell and mouse tissue lysates were mixed with Sample buffer (#0949914, Nacalai), denatured at 95 °C for 5 min, and then electrophoresed in a 10% SuperSep^TM Ace gel (Wako). The lysates of immortalized cells were mixed with NuPAGE LDS Sample buffer (Thermo Scientific Fisher), denatured at 70 °C for 10 min, and electrophoresed in a 3–8% NuPAGE Novex Tris-Acetate gels (Thermo Scientific Fisher). The samples were then transferred to PVDF membranes (Pall Corporation, Port Washington, NY, USA). Antibody reactions were performed with Can Get Signal^® Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan). Peroxidase activities were visualized with ImmunoStar^® LD (Wako) and a ChemiDoc^TM MP Imaging System (Bio-Rad).

Cells were lysed in lysis buffer A (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 20 mM MgCl₂, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40). After centrifugation, the supernatants were mixed with sample buffer (Nacalai Tesque). Then, the samples were separated on polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred onto polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt, Germany) and blocked with Blocking One, then immunoblotted with primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase. The bound antibodies were incubated with ImmunoStar Zeta (Fujifilm) and detected by X-ray film (Fujifilm) exposure. Images were captured as TIFF files using LiDE scanners (Canon, Tokyo, Japan) and processed using the accompanying LiDE driver software (Canon). The band pixels were measured in Image J software. Each image in each figure is representative of at least 3 independent experimental results.

One day after treatment with FAD, the hfNSCs were washed with cold PBS and hypotonic buffer (20 mm Tris/HCl pH 7.4, 10 mm NaCl, 1.5 mm MgCl₂) added. The nuclear extract was retrieved by centrifugation (800 g). Pellet samples (nuclei) and supernatant (without nuclei; almost purely cytoplasm) for immunoblotting were prepared as follows. Pellets were lysed with lysis buffer (50 mm Tris/HCl pH 7.4, 150 mm NaCl, 1% Triton X‐100) that included a protease inhibitor cocktail (Nacalai Tesque, Inc.) and a phosphatase inhibitor cocktail (Nacalai Tesque, Inc.) for 30 min on ice. Then, sample buffer (Nacalai Tesque, Inc.) was added to the lysed samples, and the samples were incubated for 5 min at 95 °C. Aliquots of total cell lysate were separated by 5–20% SuperSep (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and transferred onto polyvinylidene fluoride membranes (EMD Millipore). After blocking with BlockingOne (Nacalai Tesque, Inc.) containing 0.1% Tween 20, the membranes were incubated with appropriate primary antibodies. After washing with TBST (Tris‐buffered saline, 0.1% Tween 20), the membranes were then incubated with the appropriate HRP‐conjugated secondary antibodies (Abcam), washed, and developed with ECL Prime reagents (GE Healthcare Bio‐Sciences, Pittsburgh, PA, USA).

A rabbit anti-T7 tag monoclonal antibody (T7 tag, D9E1X XP rabbit mAb, #13246, Cell Signaling, MA, USA) and rabbit anti-alpha-tubulin monoclonal antibody (11H10 mAb, #2125, Cell Signaling) were used as primary antibodies for immunoblotting. Groups of 10 pairs of testes or accessory glands were homogenized by a plastic homogenizer in 100 µl of sample buffer (Nacalai) containing 5% 2-mercaptoethanol and were then boiled at 95 °C for 3 min. Ten microliters of each sample (equivalent to 1 pair of testes or accessory glands) was separated on a 4–12% NuPAGE gel (Thermo Fisher Scientific) and then transferred to an Immobilon-P Membrane (Merck). Membranes were blocked with T-TBS (20 mM Tris-HCl, 137 mM NaCl, pH 7.4, 0.05% Tween-20) containing 5% skimmed milk (Megmilk Snow Brand, Tokyo, Japan). Membranes were incubated with primary antibodies. The polypeptides recognized by primary antibodies were detected with either horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074, Cell Signaling). The detection of HRP-labelled antibodies was performed by exposing membranes on Hyperfilm-ECL using ECL Prime Western Blotting Detection Reagents (all GE Healthcare UK Ltd.) according to the supplier’s protocol.

The cells were collected, lyzed in radio-immunoprecipitation assay (RIPA) buffer (Wako, Osaka, Japan), and ultrasonic treated three times (15 s/time). The supernatant was centrifuged at 15,000 g × 10 min as the whole cell protein extract. The EVs were lyzed in five times concentration of RIPA buffer, ultrasonically treated for 5 min, incubated on ice for 15 min, and boiled with six times volume of sample buffer (Nacalai tesque, Germany) to use as the EVs protein extract.