F actin

Human TGF-β1 and Smad4 was obtained from Sigma-Aldrich, Castle Hill, NSW, Australia; Signal inhibitors Smad4 siRNA and control siRNA were obtained from Calbiochem (Calbiochem, Cambridge, MA, USA). Epigallocatechin gallate (EGCG) (purity >98%), which was purchased from Chengdu Herbpurify Co., Ltd (Chengdu, Sichuan, China), was dissolved in PBS with 2% dimethyl sulfoxide (DMSO; Sigma‐Aldrich, St. Louis, MO, USA). Mouse monoclonal antibodies for cytokeratin, vimentin, F-actin, E-cadherin and a goat polyclonal antibody for phosphorylated (p)-Smad2/3/4 were used as primary antibodies were from Santa Cruz Biotechnology, Inc., Shanghai, China. All chemicals were obtained from Sigma-Aldrich unless otherwise indicated.

Ret was immunoprecipitated from control, DMSO-, 10 μM latrunculin B- (Lat B; QCC, CA, USA), 50 nM jasplakinolide- (Jas; QCC, CA, USA) or GDNF-treated MN9D cell lysates. After treatment, the cells were washed in cold PBS and harvested in lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM ZnCl₂, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM PMSF, and 5 mg/mL each of NA3VO4, leupeptin, aprotinin, and pepstatin). Cleared lysates were assayed for protein content and normalized to total protein. Then, the lysates were immunoprecipitated with 3 µg/100 µg anti-Ret antibody and a mixture of Protein A/Protein G agarose conjugates (Pierce Biotechnology, Thermo Fisher, Rockford, IL, USA), and then subjected to SDS-PAGE analysis. The protein was transferred onto a nitrocellulose filter (NC) membrane, and blots were probed with appropriate antibodies and detected with HRP-conjugated secondary antibodies. The antibodies used for western blotting were as follows: Ret and F-actin (1:100, Santa Cruz Biotechnology).

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L‐glutamine and penicillin/streptomycin were obtained from GIBCO BRL/Invitrogen (Carlsbad, CA, USA). Flavokawain A (FKA) was purchased from LKT Laboratories, Inc. (St Paul, MN, USA). TGF‐β1 was purchased from Pepro TechInc. (Rocky Hill, NJ, USA). Antibodies against phospho‐Smad3, Smad3, phospho‐Smad2 and Smad2 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against fibronectin, MMP‐9, MMP‐2, TIMP‐1, F‐actin, Nrf2, NQO‐1, Keap‐1, histone and β‐actin were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). 4′,6‐Diamidino‐2‐phenylindole dihydrochloride (DAPI) was obtained from Calbiochem (La Jolla, CA, USA). 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), dihydrofluorescein‐diacetate (DCFH₂‐DA) and N‐acetyl‐cysteine (NAC) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies against HO‐1 and α‐SMA were purchased from Abcam (Cambridge, UK). All other chemicals were purchased from either Merck & Co., Inc. (Darmstadt, Germany) or Sigma‐Aldrich (St. Louis, MO, USA).

Embryos were fixed with 4% PFA for 2 h at room temperature. DIG-labeled probes were synthetized using either SP6/T7/T3 RNA Polymerases (New England Biolabs) and DIG-Labeling Mix (Roche). Hybridization was carried out as described [58 (link)]. Nuclei were stained with DAPI (1:200, Thermo Fischer) and F-actin with Phalloidin-TRITC conjugate (1:200, Santa Cruz Biotech, Dallas, TX, USA). For F-actin staining, larvae were fixed in 4% PFA overnight at 4 °C, permeabilized for 2 h with PBS-Triton X-100 2% at room temperature. For immunostaining, additional permeabilization was performed by partial digestion with Proteinase K (10 µg/mL) at room temperature. ZN8, F59, S58, F310 and 4D9 (Developmental Studies Hybridoma Bank) were used 1:20 and Elp3 (1:200, Abcam). Alexa Fluor-conjugated secondary antibodies were used at 1:500 (Thermo Fischer). Confocal images were captured using a Zeiss LSM 710 Meta confocal microscope (Zeiss, Oberkochen, Germany).

The anti-ODAM antibody was generated in rabbits by immunization with ODAM peptides (22 (link)). Anti-RhoA, F-actin, GAPDH, HA, ROCK, His, lamin B, integrin β₁, integrin β₃, integrin β₆, HRP-conjugated goat anti-mouse, HRP-conjugated goat anti-rabbit-IgG, and HRP-conjugated rabbit anti-goat-IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-RhoA, E-cadherin, p-myosin, p-paxillin, and paxillin antibodies as well as integrin β₁ and integrin β₆ siRNA were obtained from Cell Signaling Technology (Beverly, MA). The anti-GTP-RhoA antibody was purchased from BIOSOURCE. Anti-Arhgef5 was obtained from Proteintech Group (Chicago, IL). Anti-FLAG and transglutaminase 2 (TG2) antibodies, fibronectin, laminin, collagen, and Porphyromonas gingivalis LPS were from Sigma-Aldrich (St. Louis, MO). The Alexa Fluor 488 phalloidin (rhodamine-phalloidin) antibody was obtained from Invitrogen. Anti-FITC or Cy3-conjugated anti-mouse, rabbit, or goat IgG antibodies were purchased from Life Technologies. Y-27632 for ROCK inhibition was obtained from Tocris Cookson (Avonmouth, UK).