Figure 3: Acetylation of α-tubulin regulates LPS-induced p38 signalling.

(a,b) Control and MEC17 KD macrophages (RAW264.7) were treated with TBSA or DMSO for 12 h followed by LPS treatment for indicated time. p38 phosphorylation (a) and ERK1/2 and JNK phosphorylation (b) were determined by immuno-blotting. (c) Control and HDAC6 KD macrophages were pretreated with a p38 specific inhibitor, SB202190 (2 μM) or DMSO for 4 h and followed by 18 h of LPS treatment. p38 inhibitor treatment inhibited LPS-induced IL-10 production in control and HDAC6 KD macrophages in a dose-dependent manner. The graph shows the means with s.e.m. (error bars) from three experiments. Asterisks indicate statistical significance (***P<0.001, Student’s t-test). (d) A JNK specific inhibitor, SP600125, had very limited effect on LPS-induced IL-10 production. The graph shows the means with s.e.m. (error bars) from three experiments. (e) Control and HDAC6 KD macrophages were treated with LPS for the indicated time and samples were subjected to IL-10 mRNA expression analysis by real-time PCR. HDAC6 KD significantly enhanced IL-10 mRNA upon LPS stimulation. The graph shows the means with s.e.m. (error bars) from three experiments. Asterisks indicate statistical significance (*P<0.05 and **P<0.005, Student’s t-test). (f) HDAC6 KD and control macrophages were pretreated with the Sp1-specific inhibitor, mithramycin, for 6 h followed by 18 h of LPS treatment. ELISA analysis showed mithramycin treatment dramatically inhibited LPS-induced IL-10 production in control macrophages and HDAC6 KD macrophages. The graph shows the means with s.e.m. (error bars) from three experiments. Asterisks indicate statistical significance (***P<0.001, Student’s t-test).