Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. #siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.