Fig. 4: The efficacy of MEK inhibitors on Jurkat cells overexpressing MAP3K8, HCT-4 cells derived from a patient with HAM, and activated CD4⁺ T cells.

A Inhibitory effect of trametinib (1000 nM) on ERK phosphorylation in MAP3K8-overexpressing Jurkat cells. β-actin was used as a loading control. B Dose-dependent inhibitory effect of trametinib on ERK phosphorylation in HCT-4 cells. β-actin was used as a loading control. C IFNG relative expression levels (upper panel) and IFN-γ production levels (lower panel) in HCT-4 cells treated with trametinib. Data are presented as the mean +SD. n = 3 biologically independent experiments, each performed on separate days using independently cultured HCT-4 cells. D Percentages of cell proliferation in HCT-4 cells treated with trametinib. Data are presented as the mean +SD. n = 3 biologically independent experiments, each performed on separate days using independently cultured HCT-4 cells. E Inhibitory effect of MEK inhibitors on ERK phosphorylation in HCT-4 cells. β-actin was used as a loading control. F Downregulated gene expression levels in HCT-4 cells treated with trametinib compared with DMSO-treated cells. The plot shows fold changes (Trametinib/DMSO) and their respective ranking order. G Gene ontology (GO) analysis for the 452 genes downregulated in HCT-4 cells treated with trametinib compared with those treated with DMSO. GO terms enriched in the trametinib-treated group are displayed in order of P value (−log10). Statistical analysis was performed using a two-sided Fisher’s exact test. Nominal P values are reported. H Flow cytometric plots of IFN-γ and T-bet in normal CD4⁺ T cells stimulated with α-CD3/28 antibodies and treated with trametinib (1000 nM). The numbers in the plots indicate the percentage of positive cells. I Bar graphs indicating the percentage of T-bet-positive cells in normal CD4⁺ cells stimulated with α-CD3/28 antibodies and treated with trametinib (1000 nM). Data are presented as mean values + SD. n = 3 biologically independent experiments, each performed using CD4⁺ T cells isolated from different healthy donors. Asterisks indicate statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001). Statistical analysis was performed using a two-sided unpaired t-test (Fig.4C) and a one-way ANOVA (Fig.4D and Fig.4I). Representative immunoblot results from two to three independent experiments using cultured cell lines are shown (Fig. 4A, B, and E).