Sb203580

Anti-Bcl-xL (sc-634, 1:700), anti-p53 (sc-126, 1:1000), anti-p21 (sc-6246, 1:700), anti-Mcl-1 (sc-819, 1:1000), and anti-c-FLIP (sc-8347, 1:700) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-β-actin (A5441, 1:2000) antibody and glutathione ethyl ester (GEE) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anti- poly (ADP-ribose) polymerase (PARP) (#9542, 1:1000) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-caspase-3 (610322, 1:1000) antibody was purchased from BD Biosciences (Bedford, MA, USA). Benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was purchased from R&D Systems (Minneapolis, MN, USA). PD-98059 (a MEK inhibitor; PD), SP600125 (a JNK inhibitor; SP), and SB-203580 (a p38 MAP Kinase inhibitor; SB) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). N-acetylcysteine (NAC) was purchased from Calbiochem (San Diego, CA, USA).

BEAS-2B cells (2 × 10⁵ cells/mL) were treated with 0–30 μM fisetin at 37 °C for 1 h and then incubated with 10 ng/mL TNF-α or 10 ng/mL TNF-α/IL-4 at 37 °C for 24 h. The supernatants were collected to detect the levels of intercellular adhesion molecule-1 (ICAM-1), IL-8, IL-6, MCP-1, and c-c motif chemokine ligand CCL5, CCL11, and CCL24 using sandwich ELISA kits (R&D, Minneapolis, MN, USA). Specific protein levels were detected using a microplate reader (Thermo) at an OD of 450 nm as described previously [25 (link)]. Fisetin (10 μM) was combined with MAPK specific inhibitors, including 10 μM SP600125 (JNK inhibitor), 10 μM SB203580 (p38 inhibitor), and 10 μM PD98059 (ERK inhibitor) (Enzo Life Sciences, Inc., Farmingdale, NY, USA), to detect the ICAM-1 protein level by ELISA.

Inhibitors were used for the following: intracellular Ca²⁺ increases: BAPTA-AM (EMD4 Biosciences), P38MAPK: SB203580 (Enzo Life Sciences), Src family protein tyrosine kinase: PP1 (Sigma), neuraminidase: N-acetyl-2,3-dehydro-2-deoxy neuraminic acid sodium salt (DANA) (EMD4 Biosciences) and oseltamivir phosphate (Santa Cruz), pan-caspases: Q-VD-OPh (Sigma), cAMP: prostacyclin (PGI₂) (Cayman) and AMR: asialofetuin and its control fetuin (Sigma). OSGE (Cedarlane) was used to cleave GPIbα.

IKKβ inhibitor IV and MG-132 were purchased from EMD Millipore. CAPE, SB203580 were purchased from Enzo Life Sciences. Curcumin was purchased from Sigma. Antibodies for p-IKKα/β (#2697), p-p38 (#9211), p38 (#9212), anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were purchased from Cell Signaling Technology. Antibodies for IKKα/β (sc-7607), p65 (sc-8008), MKP-1 (sc-370), α-tubulin (sc-69969), c-Myc (sc-40), anti-mouse FITC-conjugated antibody (sc-2010), anti-rabbit Rhodamine-conjugated antibody (sc-2091) were purchased from Santa Cruz Biotechnology.

Cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in appropriate medium containing 10% fetal bovine serum (FBS; Welgene, Gyeongsan, Korea), 1% penicillin-streptomycin, and 100 μg/mL gentamycin (Thermo Fisher Scientific, Waltham, MA, USA). All cell lines tested negative for mycoplasma contamination. The lines were authenticated by standard morphologic examination using microscopy. FTY720 was purchased from Echelon Biosciences (Salt Lake City, UT, USA). N-acetyl-l-cysteine was obtained from Calbiochem (San Diego, CA, USA). E64D and NBD-FTY720 were purchased from Cayman Chemical (Ann Arbor, MI, USA). TNF-α and z-VAD-fmk were purchased from R&D Systems (Minneapolis, MN, USA). PD98059, SB203580, SP600125, NecroX-5, and pepstatin A were obtained from Enzo Life Science (Farmington, NY, USA). Anti-PARP, anti-Cathepsin D, anti-LAMP1, and anti-Beclin 1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-ERK, phosho-p38, anti-phospho-JNK, and anti-Alix were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-ATG7 antibody was obtained from ProSci Inc. (Poway, CA, USA). The cycloheximide, H₂O₂, 3-methyladenine, actinomycin D, glutathione ethyl ester, and anti-actin antibody were obtained from Sigma (St. Louis, MO, USA).