Duox1

Cells cultured in 24-well culture plates were lysed using 100 μl of 1 × western solubilization lysis buffer (1% Triton, 50 mM HEPES, 250 mM NaCl, 10% glycerol, 1.5 mM MgCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 2 mM Na₃VO₄, 10 μg/ml aprotinin and 10 μg/ml leupeptin; pH 7.4) per well. Equal amounts of protein (20–25 μg; determined using bicinchoninic acid protein assay) were separated on Novex 10% Tris-Glycine gels (Life Technologies, Grand Island, NY, USA), transferred to nitrocellulose membranes and blotted using antibodies against the following: vimentin (#5741; 1:500; Cell Signaling, Danvers, MA, USA); Akt (#9272; 1:1000; Cell Signaling); p-Akt (#4060X; 1:1000; Cell Signaling); β-actin (#MA5-15739; 1:10  000; Invitrogen, Waltham, MA, USA); Collagen (#ab34710; 1:500; Abcam, Cambridge, MA, USA); E-cadherin (#3195; 1:1000; Cell Signaling); DUOX1 (1:1000; kindly provided by F. Miot^{3 (link), 54 (link)}); and DUOXA1 (#H00090527-B01P; 1:1000; Abnova, Jhongli, Taiwan), and detected using enhanced chemiluminescence (Pierce, Rockfort, IL, USA).

Coronarin D (purity > 95%) was purchased from ChemFaces (Wuhan, Hubei, China). It was dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium to the final concentration on the experimental day. The final concentration of DMSO for all treatments was consistently less than 0.1%. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), RNase A, DAPI dye, protease inhibitor cocktail, phosphatase inhibitor cocktail, monodansylcadaverine (MDC), acridine orange (AO), and 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibody against cleaved caspase-3, -8, and -9, cleaved poly (ADP-ribose) polymerase (PARP), LC3B, p62, NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2 TSC1, TSC2, Rheb, p-mTOR (Ser2448), mTOR, p-AKT, AKT, p-ERK1/2, ERK1/2, p-p38, p38, p-JNK1/2, JNK1/2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Specific inhibitors for N-Acetyl-L-cysteine (NAC) AKT inhibitor (LY294002), ERK1/2 (U0126), p38 MAPK (SB203580), JNK (SP600125) Wortmannin and Bafilomycin A1 (Baf A1), and z-VAD-FMK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).