P erk

Gln, H₂O₂, LPS, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies against iNOS, Nrf2, HO-1, ERK, phospho-ERK (p-ERK), and β-actin, and silencing RNA (siRNA) for Nrf2 (siNrf2), ERK (siERK), and control siRNA (siCON) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-labeled goat anti-rabbit immunoglobulin was purchased from KOMA Biotechnology (Seoul, Korea). Cobalt protoporphyrin (CoPP) and zinc protoporphyrin (ZnPP) were obtained from Tocris Bioscience (Bristol, UK) and PD98059 was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and antibiotics were obtained from WelGENE Inc. (Daegu, Korea). Other chemicals were purchased from Sigma-Aldrich Chemical Co.

RPMI-1640, fetal bovine serum (FBS), trypsin, 12-O-tetradecanoylphorbol-13-acetate (TPA), penicillin G, and streptomycin were purchased from Fisher Scientific (Waltham, MA, USA). Cleaved caspase-3, p-IκBα, p-p38, p-NF-κBp65, p-ERK, p-JNK, ERK, JNK, p38, IκBα, NF-κBp65, Bax and β-actin antibodies were obtained from Santa Cruz Biotechnology (Paso Robles, CA, USA). Anti-rabbit/mouse IgG secondary antibodies were obtained from Abcam (Cam-bridge, MA, USA). N-acetylcysteine (NAC) was obtained from Tocris (Minneapolis, MN, USA). Bay 11-7082, and SP600125 were purchased from Med Chem Expresss (Monmouth Junction, NJ, USA). Kaempferol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and had a purity of over 98%.

Whole-cell lysates were prepared as previously described^{55 (link)}. Antibodies specific for p53 (1:2000, sc-126), p-EGFR (1:1000, Tyr1173, sc-101668), EGFR (1:2000, sc-373749), p-Erk (1:1000, Thr202/Tyr204, sc-16982), Erk (1:3000, sc-94), Akt (1:3000, sc-5298), AXL (1:1000, sc-1096), E-cadherin (1:1000, sc-71008), EpCAM (1:1000, sc-71059), desmoplakin (1:1000, sc-390975), cytokeratin-8/18 (1:1000, sc-70939) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); those for p-Akt (1:1000, Ser473, #4060), β-catenin (1:1000, #8480), p-AXL (1:1000, #4060), and vimentin (1:1000, #5741) were obtained from Cell Signaling Technology (Beverly, MA, USA). To assess the level of p-AXL, lysates were immunoprecipitated with an anti-AXL antibody and immunoblotted with an anti-phosphotyrosine (p-Tyr, 1:1000, sc-7020, Santa Cruz) antibody. The immunoblotting is representative of three independent experiments.

Cell lysates were prepared by treating cells with ice-cold lysis buffer (20 mM Tris pH 8.0, 1% NP40, 10% glycerol, 137 mM NaCl, 10 mM EDTA pH 8.0, and Roche Applied Science (Roche Applied Science, Monza, Italy) protease inhibitor cocktail “Complete”) for 20 min, followed by high-speed centrifugation at 4 °C for 15 min [59 (link),60 (link)]. Proteins (50 µg) were separated on 12% SDS-PAGE gels and then transferred to polyvinylidene difluoride membrane.
The blots were blocked with 5% non-fat dry milk in 20 mM Tris/HCl, pH 7.5, 500 mM NaCl plus 0.1% Tween 20 (TBS-T). The membranes were subsequently incubated in agitation at 4 °C overnight in 1% BSA-TBS-T buffer containing the specific antibody against: p-ERK (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Erk 1/2 (cell Signaling cat n°9102 1:1000 3%BSA), EGFR (Santacruz sc-03 1:500 3%Milk O.N.) and tubulin (Santacruz sc-5286 1:1000 3%milk O.N.).
After washing four times with TBS-T, the blots were incubated for 1 h at room temperature (RT) with the second antibody conjugated to peroxidase, washed four times with TBS-T, developed with ECL detection reagents (Amersham, Little Chalfort, Buckinghamshire, UK) for 1 min and exposed to X-Omat film (Eastman Kodak Co., Rochester, NY, USA). The whole western blot figures can be found in the supplementary figures.

In this study, the antibodies respectively against p38, p-p38, ERK, p-ERK, Akt1/2/3, p-Akt1/2/3, OGT, OGA, CD68 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal Anti-β-O-Linked N-Acetylglucosamine Clone CTD110.6 was obtained from Sigma (St. Louis, MO, USA). Other antibodies including anti-JNK, anti-p-JNK, Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).
Chemicals such as 1-phenyl-3-methyl-5-pyrazolone (PMP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris, Tween 20, trifluoroacetic acid, LPS (from Escherichia coli 055:B5), 6-diazo-5-oxo-L-norleucine (DON) and bovine serum albumin were obtained from Sigma. The ABC kit and Agarose wheat germ agglutinin (WGA) were purchased from Vector Laboratories (Lowellville, OH, USA). Thiamet G (Thi G) was obtained from Selleck Chemicals (Houston, TX, USA). RPMI 1640 was purchased from Corning Incorporated (Corning, NY, USA), penicillin, streptomycin and heat-inactivated fetal bovine serum (FBS) was from Gibco (Grand Island, NY, USA). All the other chemicals including sodium dodecyl sulfonate, ammonium persulfate, isopropanol, hydrochloric acid, glycine, sodium chloride and ammonia were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

PHS was purchased from Avanti Polar lipids (Alabaster, AL). Antibodies specific to p-STAT3 (Tyr705, ##4113), p-AKT (Ser473, #4060), p-AKT (Thr308, #9275), p-P38 MAPK (Thr180/Tyr182, #9211), p38 MAPK (#9212), p-SAPK/JNK (Thr183/Tyr185, #4668), p-EGFR (Tyr1068, #2234), JNK (#9252), and SNAIL (#3879) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody specific to NOTCH2 (07-1234) was purchased from Millipore Corp (Billerica, MA, USA). Antibodies specific to SOX2 (sc-20088), OCT4 (sc-9081), AKT (sc-7126), STAT3 (sc-7179), p-ERK (sc-7383), p-JAK1 (Tyr1022/Tyr1023, sc-101716), EGFR (sc-03, sc-120), Fibronectin (sc-69681), SLUG (sc-10437), TWIST (sc-15393), E-cadherin (sc-8426), and p-Tyrosine (sc-508) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Vimentin (ab8978), Ki67 (ab15580), CD44 (ab157107) and CD24 (ab31622) were obtained from Abcam whereas anti-ZEB1 (HPA027524) and β-actin (#A1978) antibody were purchased from Sigma-Aldrich. Antibodies specific to β-catenin (#610153) and N-cadherin (#610921) were purchased from BD Transduction Laboratories. EGF, bFGF, and accutase were obtained from Sigma (St. Louis, MO, USA).