Smad5

HEK 293T cells were seeded into 6-well plates at a density of 1000,000 cells/well in DMEM. The following day, cells were stimulated for 1 h with 30 ng/ml BMP-4 or 100 ng/ml BMP-7 in the presence or absence of 28-fold molar excess of Tsg. After stimulation cell layers were incubated with lysis buffer (10 mM NaCl, 1.5 mM MgCl₂, 20 mM HEPES pH 7.4, 20% glycerol, 0.1% triton X-100, and 1 mM DTT), centrifuged at 2000 rpm for 5 min at 4 °C, and washed with PBS. Aliquots of the supernatant were supplemented with 1 × PhosSTOP (Roche) and western blotted. Phosphorylated Smad was detected using pooled anti-phosphosmad1 (1:1250) and Smad5 (1:1000) antibodies (Abcam). Band intensities were quantitated with ImageJ and plotted against concentration.

After deparaffinization, dehydration, and antigen retrieval, the slides were immersed in 3% hydrogen peroxide to block endogenous peroxidase activity, and then they were blocked in goat serum for 1 h. The slides were then incubated overnight at 4 °C with primary antibodies against α-smooth muscle actin (α-SMA, Abcam, 1:2000), TGF-β1 (Abcam,1:500), BMP7 (Abcam, 1 μg/ml), and Smad5 (Abcam, 1:800). The sections were then incubated with appropriate secondary antibodies for 60 min at room temperature. The slides were then stained with 3, 3-diaminobenzidine (DAB) at room temperature, lightly counterstained with hematoxylin, dehydrated, and covered with glass cover slips. Quantification of immunoreactivity was performed using Image Pro-Plus 6.0, and 3–5 fields were randomly selected from each slide to determine the mean optical density (MOD).

For identification of vesicular markers, protein extracts were isolated from EVs using RIPA protein lysis buffer containing 1 mM PMSF. Equal amounts of protein (10 μg/lane) were subjected to SDS-PAGE under non-reducing conditions, and the gels were blotted onto nitrocellulose membranes (Pierce, Waltham, MA, United States) in an electrophoretic transfer cell (Bio-Rad). The membrane was blocked with 5% non-fat milk at 4°C overnight and then probed with anti-CD63 (1:800 dilution; Abcam), anti-CD9 (1:800 dilution; Abcam), and anti-ALIX (1:800 dilution; Abcam) antibody for 1 h. The membrane was incubated with sheep anti-mouse IgG conjugated to HRP (NeoBioscience, Shenzhen, China) for 1 h. Band density was determined using the Bio-Rad Quality One Software. To detect the expression of key proteins in ORSCs and MxCs, total protein was extracted from human HF using RIPA lysis buffer and subjected to western blot analysis as described above. Antibodies against the following proteins were obtained from Abcam (Cambridge, United Kingdom): β-catenin (1:100 dilution), Lef-1 (1:100 dilution), Shh (1:100 dilution), Gli1 (1:100 dilution), Cyclin D1 (1:400 dilution), Cyclin E (1:400 dilution), BMP2 (1:800 dilution), and SMAD5 (1:400 dilution). p-SMAD5 was obtained from Millipore (1:800 dilution).

Immunofluorescence was performed as previously described (16 (link)). Cells were cultured on coverslips overnight and fixed with 4% paraformaldehyde for 20 min at room temperature. Samples were then incubated with 0.3% Triton X-100 for 10 min and blocked for 2 h with the blocking solution (Beyotime), after which cells were probed overnight at 4°C with a diluted primary antibody, followed by a secondary antibody for 2 h. The primary antibodies used were: a mouse monoclonal antibody for Pan-ck (1:300, Abcam), Vimentin (1:150, Santa Cruz), and rabbit polyclonal antibody against E-cadherin (1:100, Abcam), α-SMA (1:100, Abcam), and Smad5 (1:100, Abcam); the secondary antibodies used were Alexa 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) and Alexa 594-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA).

For the western blot analysis, the antibodies used were as follows: E-cadherin (AF0131, 1:1000, Affinity), VEGFA (sc-57496, 1:200, Santa Cruz), SMAD5 (ab40771, 1:5000, Abcam), MMP1 (A0568, 1:1000, Boster), HIF1α (sc-13515, 1:200, Santa Cruz), Snail (3099-1-AP, 1:1000, Proteintech), and Twist1 (5465-1-AP, 1:1000, Proteintech), with GAPDH (AB0037, 1:5000, Abways) used as an internal control.

Western blot was performed as previously described (14 (link)). Total proteins were extracted from HK-2 cells using RIPA lysis buffer (Beyotime, Haimen China) and separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein lysates were transferred onto a 0.22-μm PVDF membrane (Millipore). After blocking in 5% non-fat milk at room temperature for 2 h, the membranes were incubated with primary antibodies specific for E-cadherin (Abcam), α-SMA (Abcam), Vimentin (Santa Cruz), Pan-ck (Abcam), Smad5 (Abcam), and GAPDH (Cell signaling) at 4°C overnight. After three washes, the blots were incubated with IRDye 700/800-conjugated secondary antibodies. The results were visualized and analyzed using an Odyssey infrared scanner.

For the western blot analysis, each protein extract (30 μg of total protein) was resolved by SDS-PAGE using 10% polyacrylamide gels and subsequently transferred to Immun-Blot^® PVDF membranes (Bio-Rad, Hercules, CA, USA). The primary antibodies and their dilution factors were as follows BST2, 1:1000 (Santa Cruz Biotechnology, Dallas, TX, USA); RUNX2, 1:1000 (Santa Cruz Biotechnology); SMAD1, 1:1000 (Abcam, Cambridge, UK); SMAD4, 1:500 (Abcam); SMAD5. 1:500 (Abcam); SMAD 8, 1:500 (Abcam); T-SMAD1/5/8, 1:500 (Santa Cruz Biotechnology); p-SMAD1/5/8, 1:500 (Santa Cruz Biotechnology), and β-actin, 1:1000 (Sigma-Aldrich). Secondary antibodies were used at a 1:5000 dilution. The detection of protein bands was facilitated by an Enhanced Chemiluminescence Kit (Elpis Biotech) and membranes exposures to X-ray film (Amersham, Buckinghamshire, UK).