Mki67

RNA extractions, cDNA synthesis and quantitative PCR (qPCR) analysis were performed as described previously [14 (link)]. For qPCR, all samples were analyzed in duplicate with relative expression levels determined using the dCt method with actin beta (ACTB) as the endogenous control. The following PrimePCR gene-specific primers were purchased from Bio-Rad: ACTB (qHsaCED0036269), SCGB1A1 (qHsaCID0018013), KRT5 (qHsaCED0047798), MUC5AC (qHsaCID0017663), FOXJ1 (qHsaCID0016777), NOTCH3 (qHsaCID0006529), HEYL (qHsaCID0006092), CLDN3 (qHsaCEP0032466), OCLN (qHsaCEP0041012), TJP1 (qHsaCIP0031627), TJP2 (qHsaCIP0026125), TJP3 (qHsaCIP0030881), PARD3 (qHsaCIP0039350), PARD6B (qHsaCEP0051277) and MKI67 (qHsaCID0011882). The assays were performed using the manufacturer’s recommended cycling parameters.

The CV extract was prepared from commercially available CV capsules (MycoMedica Company, Police nad Metují, Czech Republic) following previously described protocols [38 (link),40 (link),41 (link)]. The protein and carbohydrate contents in this extract were measured using phenol sulfuric acid and bicinchoninic acid assays, respectively. d-pen, KA (5-hydroxy-2-hydroxymethyl-1,4-pyrone), melanin, Nec-1, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (Darmstadt, Germany). GSK’872 was purchased from R&D Systems (Minneapolis, MN, USA), and necrosulfonamide (NSA) was purchased from Tocris Bioscience (Bristol, UK). An EXTRACTME Total RNA Plus kit was obtained from Blirt company (Gdansk, Poland), and iScript Reverse Transcription Supermix for RT-qPCR, SSoAdvanced Universal SYBR^® Green Supermix, the PrimePCR™ SYBR^® Green Assay, human IL-1β (qHsaCID0022272), IL-2 (qHsaCID0015409), IL-6 (qHsaCID0020314), IL-12 (qHsaCID0007212), MKI67 (qHsaCID0011882), ITGA5 (qHsaCED0045939), ITGB3 (qHsaCED0045078) and ACTB (qHsaCED0036269) were purchased from Bio-Rad (Hercules, CA, USA). Ficoll–Paque Plus was bought from Amersham Biosciences (Piscataway, NJ, USA). All reagents for cell culture were purchased from Sigma-Aldrich unless stated otherwise.