Fig. 4: NSF-951 induces the pro-inflammatory cytokines via MyD88/TRIF signalling through p38 and JNK-dependent MAPK pathway.

A Analysis of adapter molecule involved in signalling through NSF-951. WT, MyD88^−/−, TRIF^−/− and TRIF^−/−/MyD88^−/− mouse macrophage cell lines were treated with MPLA (2 µg/mL) or NSF-951 (2 µM) for 24 h at 37 °C in the presence of 5%CO2 and levels of IL-6 and TNF-α were measured in culture supernatant using a sandwich ELISA kit. B qRT-PCR analysis of TRIF signalling-associated cytokines and chemokines in NSF-951 stimulated macrophages. WT and TRIF^−/− cells were stimulated with MPLA (2 µg/mL) or NSF-951 (2 µM) for 24 hrs at 37 °C/5%CO₂. Cells were recovered and RNA was isolated and converted to cDNA and IFN-1 pathway gene expression was analysed by qRT-PCR as described in the Materials and Methods. C Western blot analysis of phosphorylation of mediators of MAP kinase pathway in macrophages stimulated with NSF-951. RAW264.7 cells were stimulated with MPLA (2 µg/mL) or 2 µM of NSF-418 or NSF-501 or NSF-951 for 24 h at 37 °C in 5%CO2. Levels of p38, JNK, and ERK1/2 and respective phosphorylated forms were analysed by western blot as described in materials and methods. D Analysis of MAP kinase signalling pathway in mouse macrophages stimulated with NSF-951. RAW264.7 cells pre-treated with pharmacological inhibitors of NF-kB (SN50; 20 μM), JNK (SP600125; 40 μM) or p38 (SB203580; 30 μM) or ERK (U0126; 50 µM) or combined together (All 4) for 30 min and then stimulated with MPLA (2 µg/mL) or NSF-951 (2 µM) for 24 h at 37 °C in the presence of 5%CO₂ and supernatant was collected to measure levels of IL-6 by ELISA. All data represent the mean ± SD of triplicates (n = 3) and are representative of three independent biological experiments. Significant differences were analysed using one-way analysis of variance (ANOVA), followed by the Dunnett post hoc test. The value of p < 0.05 was considered statistically significant and noted as ***p < 0.001, ****p < 0.0001 and “ns” as not significant.