Fig. 5.

Effect of suramin on interleukin (IL)-1β-induced mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kB signalling pathway activation in nucleus pulposus (NP) cells. a) Cells were treated with or without 10 μM suramin for one hour before IL-1β treatment (10 ng/ml) for ten minutes. NF-kB localization was determined by immunofluorescence analysis. b) The NP cells were treated with 10 ng/ml IL-1β, 10 μM suramin, or suramin plus IL-1β for ten, 20, or 30 minutes. The total protein and phosphorylation levels of NF-κB in NP cells were detected using western blotting (n = 3 to 5). c) NP cells cultured in the absence or presence of IL-1β, with or without the addition of suramin for ten, 30, or 60 minutes. Cells were then collected and total protein extracted. The expressions of phosphorylated extracellular signal-regulated kinase (p-ERK), p-p38, phosphorylated c-Jun N-terminal kinase (p-JNK), ERK, p38, and JNK were detected by immunoblotting. Antibody to β-Actin was used as loading control (n = 3 to 5). d) Cells were transfected with NF-κB luciferase plasmid by using lipofection method as described in the Methods section. Cells were pretreated with different doses of suramin for 60 minutes, and then incubated with 10 ng/ml IL-1β for four hours. The luciferase activity was determined and normalized with the amount of total protein. Values are means and standard deviations of triplicate measurements (**p < 0.01 compared with untreated control or IL-β treated group). e) Sub-confluent cells were pretreated with 10 μM of suramin for one hour, and then followed by treatment with 10 ng/ml IL-1β for 24 hours. Total cell lysates were analyzed by immunoblot using anti-Toll-like receptor 2 (TLR2) and MyD88 antibody (n = 3). f) Cells were pretreated with 10 μM of suramin for one hour followed by treatment with 10 ng/ml IL-1β for 24 hours. Total RNA were extracted for real-time polymerase chain reaction analysis of TLR2 messenger RNA (mRNA) expression. The level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA verified loading of an equivalent amount of RNA (n = 5) (***p < 0.001 compared with untreated control or IL-β treated group). g) NP cells were pretreated with 10 µM of suramin for one hour, followed by treatment with 400 ng/ml Pam3CSK4 for 24 hours. Total cell lysates were analyzed by immunoblot using TLR2, p65, p-p65(ser536), and MyD88 antibodies (n = 3). h) NP cells were pretreated with 10 µM of suramin for one hour, followed by co-treatment with 400 ng/ml Pam3CSK4, 50 μM C29, or 10 μM transforming growth factor-beta-activated kinase for 24 hours. Total cell lysates were analyzed by immunoblot using TLR2, p65, p-p65(ser536), and MyD88 antibodies (n = 3). DAPI, 4′,6-diamidino-2-phenylindole.