Figure 2

RSV blocks LPS-induced NFκB activation, SphK activity and ERK1/2 phosphorylation. (A) NF-κB activity in untreated cells or following LPS (100 ng/ml) stimulation for 30 min (LPS) was assessed using the NF-κB kit (***p < 0.001, ****p < 0.0001); n = 5 for each condition and two-way ANOVA followed by post hoc Sidak’s multiple comparisons test. (B) SphK activity of cells without LPS stimulation or stimulated with 100 ng/ml LPS for 5, 10, 30 and 45 min in PBS, RSV or DMS pre-treated groups (**p < 0.01, ***p < 0.001, ****p < 0.0001); n = 3 for each condition and statistical analysis was performed using two-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. (C) Immunoblot analysis of expression of phosphorylated ERK1/2 in human primary monocytes treated with or without 40 µM RSV and then stimulated with 100 ng/ml LPS for 5, 10 and 30 min, total ERK1/2 and α-tubulin serve as loading controls. (D) Immunoblot analysis of expression of phosphorylated ERK1/2 in human primary monocytes treated with or without 10 µM DMS and then triggered with 100 ng/ml LPS for 5, 10 and 30 min, total ERK1/2 and α-tubulin serve as loading controls. Note: western blots were performed on the same membranes cut in different strips based on molecular weight to probe for the desired targets. (E) SphK activity of cells without LPS stimulation or stimulated with 100 ng/ml LPS for 5, 10, 30 and 45 min in both untreated group or PD98059 pre-treated group (n = 3 for each condition). Statistical comparison performed for each time point between untreated and treated conditions. Data were derived from Dr. Wang Binbin PhD Thesis ³⁶.