α sma

For western blot analysis, the visceral peritoneum and cells were homogenized in RIPA buffer and then centrifugated to get proteins in the supernatant. The proteins were subjected to SDS‐PAGE gel and transferred to a nitrocellulose membrane. Protein expression was analysed by western blot analysis with primary antibody against human/mouse vimentin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Zo‐1 (Santa Cruz Biotechnology, Inc.), α‐SMA (Santa Cruz Biotechnology, Inc.), E‐cad (Santa Cruz Biotechnology, Inc.), Col I (Cell Signaling, Inc., Danvers, MA, USA) or CTGF (Santa Cruz Biotechnology, Inc.) and then incubated with an appropriate secondary antibody. After washing, the protein was visualized and quantified by BIO‐RAD ChemiDoc™ XRS+ with Image Lab™ Software. The relative protein levels of vimentin, Zo‐1, Col I, E‐cad, α‐SMA and CTGF were normalized to β‐actin.

The cells or tissues were fixed with 4% paraformaldehyde in PBS for 1 h and permeabilized with 0.1% Triton X-100 for 5 min. After blocking in 5% BSA for 2 h at room temperature, the cells were incubated with the primary antibodies against p53 (Santa Cruz Biotechnology, USA), α-SMA (Santa Cruz Biotechnology, USA) or STAT3 (Santa Cruz Biotechnology, USA) and then incubated with the second antibodies. The cells were also stained with Hoechst 33342 (Sigma, USA) for 15 min and examined by confocal laser-scanning or fluorescent microscopy.

A549 cells were seeded in 6‑well plates. After the indicated treatment, the cells were fixed and incubated with primary antibodies against α-SMA (Santa Cruz Biotechnology, Inc.) and E‑cadherin (Santa Cruz Biotechnology, Inc.) overnight at 4 °C, after which the cells were washed three times with PBS. The cells were stained as described in previous studies [28 ].

Human fetal lung fibroblast (MRC5) cell lines were propagated in EMEM media (Gibco, Rockville, IL, USA) with 10% FBS (Hyclone, Logan, UT, USA), and 1% penicillin/streptomycin antibiotic mix (Lonza, Allendale, NJ, USA). The cells were kept in a 37 °C humidified incubator with 5% carbon dioxide. Immobilized protein A/G beads, FN, α-SMA, TβRI, HA tag, USP11, V5 tag, TβRII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-SMAD2, phospho-SMAD3, total SMAD2, SMAD3, and ubiquitin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Bleomycin, leupeptin, CHX, MTX, and antibodies against Flag-tag and β-actin were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG-132 was from Calbiochem (KGaA, Darmstadt, Germany). DAPI was purchased from ThermoFisher Scientific (Waltham, MA, USA). All materials in highest grades used in the experiments are commercially available.

Recombinant Human TGF‐β₁(100‐21) was obtained from PeproTech (Rocky Hill, NJ, USA). The antibodies used were as follows: E‐cadherin (1:1000, #14472), N‐cadherin (1:1000, #14255), Vimentin (1:1000, #5741), Smad2/3(1:1000, #5678), p‐Smad2/3 (Ser465/467/ser423/425, 1:1000, #8828), beta‐catenin (1:1000, #9562) were purchased from Cell Signaling Technology (CST, Danvers, MA). Transforming growth factor‐β (1:1000, sc‐146) and α‐SMA (1:1000, sc‐53015) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). FITC Mouse Anti‐Human CD51/CD61 (555505; BD biosciences, San Jose, CA, USA) and PE Mouse Anti‐Human CD106 were obtained from BD biosciences. Tetraspanin 1 (1:200, NBP2‐33867) was purchased from Novus Biologicals (LLC, Littleton Co., Centennial, CO, USA). Bleomycin was obtained from Sigma (St. Louis, MO).

Western blot analysis was carried out as previously described [24 (link)]. The primary antibodies against Snail, α-SMA, COL1A1, IL-6 and vimentin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). GADPH was used as an internal control.