Fig 1: Blockade of the S100B receptor and PI3K decreases C. difficile toxin-induced IL-6 upregulation in EGCs. (A) RAGE gene expression (mean ± s.e.m) by qPCR in enteroglial cells (EGC/PK060399) challenged with TcdA and TcdB (n = 4). (B) Analysis of IL-6 gene expression (mean ± s.e.m) by qPCR in enteroglial cells (EGC/PK060399) challenged with TcdA and TcdB for 18 h in the presence or absence of 30 µM FPSZM1 (a RAGE antagonist) which was added 1 h prior to C. difficile toxin challenge. (C) Representative photomicrographs of NF?Bp65 (green) immunostaining and DAPI (blue) nuclear staining in enteroglial cells (EGC/PK060399) exposed to TcdA and TcdB after 18 h of incubation. (D) Percentages of cells (mean ± s.e.m) with positive nuclear NF?Bp65 staining under different experimental conditions at 18 h of incubation with TcdA and TcdB. (E) Western blot (WB) bands of NF?B p65 and PCNA in nuclear extract fraction of enteroglial cells (EGC/PK060399) exposed to TcdA and TcdB at 18 h of incubation. (F) Analysis of IL-6 gene expression (mean ± s.e.m) by qPCR in enteroglial cells (EGC/PK060399) challenged with TcdA and TcdB for 18 h in the presence or absence of 10 µM LY294002 (a PI3K inhibitor) which was added 1 h prior to C. difficile toxin challenge. (A–F) Cells receiving only supplemented DMEM was applied as a control. (B, F) Experiments were performed with the same negative (Control) and positive controls (TcdA and TcdB). **p < 0.0001. ANOVA followed by (B, D, F) Turkey test was used. *p < 0.01.