Bay 11 7085

Total RNA for detection of A549-related cytokines, chemokines, and ICAM-1 was extracted after stimulation and treatment with casticin. cDNA was synthesized using a cDNA synthesis kit (Life Technologies), and gene expression was measured by incorporation of fluorescent SYBR Green using a spectrofluorometric thermal cycler (iCycler; Bio-Rad Laboratories, Hercules, CA, USA). Specific primers were designed as shown in Table 1. PI3K inhibitor (AS604850) and AKT inhibitor (Akt1/2 kinase inhibitor) (Sigma), NF-κB inhibitor (BAY11-7085), and MAPK inhibitors (JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580) (Enzo Life Sciences, Inc., Farmingdale, NY, USA) were cultured with casticin for detection of ICAM-1 gene expression.

Migration assays of WT BMDMs toward SFM or CM from R7 cells with or without shRNA targeting of HGFL (1:1 dilution ratio of BMDM CM to SFM), or co-culture with R7 or R7 shHGFL cells (1:10 ratio of R7 cells to WT BMDM cells) using 4000 R7 cells and 40,000 BMDM cells were performed for up to 6 h using Boyden chambers [5 (link)]. For studies involving inhibitors, R7 cells were pretreated with inhibitors including 20 µM Ravoxertinib (ERK1/2 inhibitor; MedChemExpress, Monmouth Junction, NJ, USA, Cat#50-187-3397), 20 µM Afuresertib (Akt inhibitor; MedChemExpress Cat#50-187-3338), 20 µM STAT3 Inhibitor VII, S3I-201 (Calbiochem Cat#573103), or 10 µM Bay 11-7085 (EnzoLifeSciences, Farmingdale, NY, USA, Cat#573103) for 6 h prior to washing out and addition of fresh media, and addition of BMDM-containing Boyden chambers. The number of BMDMs that migrated toward SFM was used as a reference control. Following incubation, chambers containing migrated cells were removed, and the cells were fixed and stained using 0.5% w/v Crystal Violet (Sigma, Sofia, Bulgaria, Cat#C6158) in methanol. The upper part of the chamber was gently wiped clean using a cotton tip applicator to remove non-migrated cells prior to imaging and counting transmigrated cells.

White, solid bottom tissue culture treated 96-well microplates were purchased from E&K Scientific (Santa Clara, CA). CellTiter-Glo Luminescent Cell Viability Assay was purchased from Promega (Madison, WI); dimethyl sulfoxide (DMSO) and amphotericin B were purchased from Sigma-Aldrich (St. Louis, MO); ebselen (2-phenyl-1,2-benzoisoselenazol-3(2H)-one), BAY 11-7082 or (E)-3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile and BAY 11-7085 or (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile were purchased from Enzo Life Sciences (Farmingdale, NY).