Fig. 5: MAPK4 deficiency activates the PI3K-AKT-mTOR pathway, enhancing B cell proliferation.

A, B Splenic B cells from WT and MAPK4 KO mice were incubated with sAg at different time points, then fixed, permeabilized, and stained for pPI3K (p85). Cell images (A) were captured using a Leica confocal microscope, and Pearson’s correlation coefficients (B) were calculated to evaluate the colocalization of pBTK with the BCR. Scale bar: 2.5 μm. Correlation coefficients were quantified for > 50 cells. C, D Phospho-flow cytometry (C) and western blot (D) analysis of pPI3K (p85) expression levels in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg at the indicated time points. E Western blot analysis of pAKT, AKT, pIKKβ, IKKβ, pmTOR, mTOR, pS6, S6, pFOXO1 and FOXO1 expression levels in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg for 0, 5, 10, and 30 min. Representative blots from three independent experiments are presented. F–H Flow cytometric analysis (F) and comparison of cell proliferation of naive B cells from WT and MAPK4 KO mice, labeled with CFSE and stimulated with CpG (G) or LPS (H) in vitro for 72 h (n = 3). I Flow cytometric analysis of IL-6, IL-10, and IFN-γ expression in B cells from WT and MAPK4 KO mice after stimulation with CPG + anti-CD40, CPG, and PMA + Ionomycin, respectively. J Comparison of the proportions of gated populations derived from (I) (n = 5). K Flow cytometric analysis of Eα52–68 peptide presentation by Y-Ae staining in B cells from WT (Cyan) and MAPK4 KO (Red) mice. L Comparison of the MFIs of Y-Ae in B cells from WT and MAPK4 KO mice (n = 7). *P < 0.05; **P < 0.01; ***P < 0.001.