Interleukin 6 (il 6)

Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY, USA). The antibodies for iNOS, COX-2, p65, PARP, β-actin, normal rabbit IgG and normal mouse IgG_2b were obtained from Santa Cruz Biotechnology Inc. (Santa cruz, CA, USA). The enzyme immunoassay (EIA) kits for PGE₂, IL-6, IL-1β, and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA). TOPscript™ RT Dry MIX was purchased from Enzynomics Co. Ltd. (Daejeon, Korea). iNOS, COX-2, TNF-α, IL-6, IL-1β, and β-actin oligonucleotide primers were purchased from Bioneer (Seoul, Korea). MTT, l-N⁶-(1-iminoethyl)lysine (l-NIL), NS-398, Escherichia coli LPS and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade acetonitrile was purchased from Fisher Scientific Korea (Seoul, Korea) and HPLC-grade formic acid was purchased from Wako (Osaka, Japan).

LPS, Griess reagent, Tween 20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and 4,6-diamidino-2-phenyllindile (DAPI) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies against iNOS, COX-2, NF-κB p65, inhibitor kappaB (IκB), nucleolin and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The peroxidase-labeled donkey anti-rabbit immunoglobulin, peroxidase-labeled sheep anti-mouse immunoglobulin and enhanced chemiluminescence (ECL) detection kit were purchased from Amersham Corp. (Arlington Heights, IL, USA). Dulbecco’s modified Eagle’s minimum essential medium (DMEM) containing l-glutamine (200 mg/L), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco-BRL (Grand Island, NY, USA). The enzyme-linked immunosorbent assay (ELISA) kits for PGE2, TNF-α, and IL-1β were obtained from R&D Systems (Minneapolis, MN, USA). COX-2, iNOS, TNF-α, IL-6, and GAPDH oligonucleotide primers were purchased from Bioneer (Seoul, Korea). Fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG and Fluoromount-G were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA) and Southern Biotechnology Associates Inc. (Birmingham, AL, USA), respectively. All other chemicals were purchased from Sigma-Aldrich.

Roswell Park Memorial Institute medium (RPMI-1640) for culturing macrophage, penicillin–streptomycin, fetal bovine serum, and phosphate-buffered saline were acquired from Welgene (Gyeongsan-si, Korea). Oligo-dT and primer sequences, including COX-2, iNOS, IL-1β, IL-6, and TNF-α, were obtained from Bioneer (Daejeon, Republic of Korea). Other reagents were purchased from Invitrogen, Sigma-Aldrich, and Thermo Fisher.

For this study, roxatidine (≥98% purity, Fig. 1A), primary antibodies for p65, p–IκBα, IκBα, p-ERK, p-p38, p-JNK, ERK, p38, JNK, PARP, α-tubulin, caspase-1, MKK3/6, MAPK-activated protein kinase 2 (MK2), and β-actin and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). p-IKK, IKK, p-Akt, Akt, p-MKK3/6, and p-MK2 primary antibodies were obtained from Cell Signaling Technology (Danvers, MA) and M5/114.15.2 (MHC class II) antibody was purchased from eBioscience (eBioscience, Affimatrix Phorbol 12-myristate 13-acetate (PMA), calciumionophore A23187 (Calcymycin; C29H37N3O6), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tertazolium bromide (MTT), fluorescein isothiocyanate (FITC) isomer-I, 2,4-dinitrochlorobenzene (DNCB) and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Iscove’s modified Dulbecco’s medium (IMDM) fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY). The enzyme immunoassay (EIA) kits for TNF-α, IL-6, and IL-1β were obtained from R&D Systems (Minneapolis, MN, USA). SYBR Premix Ex Taq was purchased from Takara (Shuzo, Shiga, Japan). TNF-α, IL-6, IL-1β, and GAPDH oligonucleotide primers were purchased from Bioneer (Daejeon, Chungbuk, South Korea). SB203580 was purchased from Sigma Chemical Co. (St. Louis, MO).

Based on Lee et al.’s paper [27 (link)], with the total RNAs isolated from CK-treated DU145 cells by using RNeasy mini kit (Qiagen), quantitative reverse transcription PCR (RT-qPCR) was carried out under the LightCycler TM instrument (Roche Applied Sciences, Indianapolis, IN, USA) by using the following primers, IL-6- forward: 5′-CCACCGGGAACGAAAGAGAA−3‘; reverse-: 5′-GAGAAGGCAACTGGACCGAA–3′ (Bioneer, Daejeon, Korea); IL-10 forward: 5′-TCTCCGAGATGCCTTCAGCAGA-3′; reverse-: 5′-TCAGACAAGGCTTGGCAACCCA−3′ (Bioneer, Daejeon, Korea); hGAPDH-forward: 50 -CCA CTC CTC CAC CTT TGA CA-30; reverse-: 50 -ACC CTG TTG CTG TAG CCA −3 0 (Bioneer, Daejeon, Korea).