Fig. 6: EsxB inhibits p38-mediated phosphorylation of METTL14.
a, b Immunofluorescence staining for Mettl14 in peritoneal macrophages from p38αfloxp/floxp (p38αf/f) or p38αfloxp/floxp; LysM-Cre (p38α−/−) mice stimulated with or without PGN for 2 h. Scale bar, 10 μm. Every point in b represents the number of Mettl14 foci in 30 cells. c Endogenous interaction of Mettl14 and p38. d Interaction of GST-p38 with GFP-METTL14 in vitro. e, f Endogenous interaction of Mettl14 and p38 in peritoneal macrophages infected with H37Rv for indicated times (MOI = 5). Relative gray intensity of p38 was shown in (f). g In vitro kinase assay of purified recombinant GST-p38 (active) with GFP-Mettl14-WT or GFP-Mettl14-T72A. h Mettl14-WT and Mettl14-T72A are subjected to in vitro kinase assay and then performed in vitro LLPS assay to test the ability of droplet formation. i IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). j IB and IP of HEK293T cells transfected with plasmids encoding FLAG-EsxB for 24 h and stimulated with TNF-α for indicated times. k–n Real-time fluorescence amplification curves and bar plot of the threshold cycle (CT) of qPCR showing SELECT results for detecting m6A3656 in Nox2 mRNA (k), qPCR analysis of Nox2 mRNA (l), Nox2 protein levels (m) and changes in the levels of ROS (DCF staining; green) (n) in peritoneal macrophages from p38αf/f or p38α−/− mice infected with H37Rv or H37Rv(ΔEsxB) for 2 h in k or 4–8 h in (l–n) (MOI = 5). Rn is the raw fluorescence for the associated well normalized to the fluorescence of the passive reference dye (ROX). o IB analysis of protein levels from mouse macrophages infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. p IB analysis of iBMDM overexpression with Flag-p38 together with vector (-) or TAB1 or MKK6(E) or EsxB. q In vitro kinase assay of purified recombinant 6× His-p38 with TAB1 and EsxB. r IB analysis of mouse peritoneal macrophages treated with DMSO or SB203580 (10 μM) and infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. s IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). All of the immunoblot data are representative images from one of three independent experiments. Results in h were repeated twice independently. Results in b, f, k, l, n reflect the mean ± SEM from three independent biological experiments. Two-tailed unpaired Student’s t-test were used.
