C jun

Western blot was performed as described before (22 (link)). Primary antibodies were used as follows: Bcl2 (1:200, Cell Signalling Technology (Frankfurt am Main, Germany)), BAX(1:1000, Cell Signalling Technology), c-Jun(1:100, Santa Cruz Biotechnology (Dallas, USA)), p-c-Jun(1:100, Santa Cruz Biotechnology), Lamin B1 (0.1 µg/ml, Abcam). β-actin(1:5000, Sigma Aldrich) and GAPDH (1:5000, Sigma Aldrich) act as the internal control. Images were acquired by FUSION FX (Vilber Lourmat Deutschland GmbH, Weinheim, Germany).

U266 and U937 cells (1 × 10⁶ cells/mL) were treated with indicated concentrations of SM (25 or 50 µg/mL) for 24 h. Then, the cells were lysed with RIPA buffer (1 M EDTA, 1 mM Na₃VO₄, 1 mM NaF, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid) containing a protease inhibitors cocktail (Amresco, Solon, OH, USA). The protein supernatant was collected and quantified for protein concentration by using an RC DC protein assay kit II (Bio-Rad, Hercules, CA, USA). The proteins (30 µg) were separated via SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes that were blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 and incubated with the required antibodies. Primary antibodies, including cleaved PARP (1:1000, 89 kDa), CHOP (1:500, 27 kDa), P-eIF2α (1:500, 38 kDa) (Cell Signaling, Beverly, MA, USA), P-ATF4 (1:500, 39 kDa) (Thermo Fisher, Waltham, MA, USA), P-PERK (1:500, 125 kDa) (Thermo Fisher, Waltham, MA, USA), c-Jun (1:1000, 39 kDa), and β-actin (1:1000, 43 kDa) (Santa Cruz, Dallas, TX, USA), were used at a 1:500~1000 dilution (5% bovine serum albumin) and secondary antibodies at a 1:1000 dilution (5% skim milk) (Santa Cruz, Dallas, TX, USA). After detected protein bands were visualized by Hybond ECL (Amersham Pharmacia, Piscataway, NJ, USA), the blots were scanned.

Western blot analysis was performed as described previously [37 (link)]. Primary antibodies used were as follows: E-cadherin, phos-cJun and cJun, Ets1 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); CD44, ZEB1, Egr-1, phos-IGF1Rβ, IGF1Rβ, phos-EGFR, and EGFR were purchased from Cell Signaling Technology (Beverly, MA); Vimentin was from Life Technologies (Carlsbad, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences (Piscataway, NJ).

All cell culture reagents were purchased from Gibco-Invitrogen (Carlsbard, CA, USA). Cell culture dishes and other plasticware were obtained from Nalgene Nunc (Rochester, NY, USA). Salts and buffers were purchased Sigma (St. Louis, MO, USA). Leptomycin B (LMB), α-tocopherol, LPS O111:B4 and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) were obtained from Sigma-Aldrich. Primary antibodies used in protein detection of CALM, α-adaptin, β-adaptin, eps 15, c-Myc, c-Jun, c-Abl and SR-B1 as well as secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Cambridge, UK). Fluorescent conjugates of LPS BODIPY FL, and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate for ROS detection were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

LicA was purchased from Sigma (St. Louis, MO), and a 100 mM stock solution was prepared in dimethyl sulfoxide (DMSO) and stored at −80°C. DAPI and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were purchased from Sigma (St. Louis, MO). Antibodies against p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, JNK1/2, uPA, ATF-2, NF-κB (p65), c-jun, c-fos, β-actin, and si-MKK4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse, anti-goat and anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The JNK1/2 inhibitor, SP600125, was purchased from Calbiochem (San Diego, CA).