Fig. 8

rNPM induces NFκB translocation to the nucleus via TLR4 activation and induces inflammation. a Representative image of immunocytochemistry of NFkB (green) with an α-p65rel NFkB antibody, nuclei were counter-stained with DAPI (blue). NFκB translocated in the nucleus in hCmPCs treated for 8h with rNPM (0.5μg/ml) compared to untreated cells (w/o NPM). Scale bar 5μm. b hCmPCs were infected for 48h with lentiviral particles bearing the NFkB-GFP reporter gene (Cignal Lenti Reporter Assay, Qiagen). Then cells were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8h with rNPM (0.5μg/ml). Histogram representing the Cignal Reporter Assay. NFkB activation was assessed by relative fluorescence units (RFU). Fluorescence was increased by rNPM treatment, whereas TLR4i inhibited it (n = 6; ^*P < 0.05). c hCmPCs were pre-treated with 5μM NFkB inhibitor (NFkBi) for 30 min and then treated or not for 8h with rNPM (0.5μg/ml). qRT-PCR of COX-2 and IL-6 was significantly reduced by NFkBi upon rNPM treatment (n = 6; ^*P < 0.05). d hCmPCs were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8h with rNPM (0.5μg/ml). qRT-PCR of COX-2 and IL-6 was significantly reduced by TLR4i upon rNPM treatment (n = 6; ^*P < 0.05)