Inhibition of JNK signaling leads to increased GATA3 accumulation in Jurkat T cells. (A) Schematic of RNA-seq experimental design. Created in BioRender. Bauman, B. (2025) https://biorender.com/u68a150. (B) Venn diagram demonstrating the overlap between P/I-stimulated WT Jurkat T cells (blue), P/I-stimulated CARD11 KO Jurkat T cells (green), and P/I-stimulated WT Jurkat T cells preincubated with JNKi (orange). (C) Heatmap of the CARD11-JNK transcriptome, demonstrating the relative expression of each transcript between conditions, represented as scaled Z scores for each gene. (D) Volcano plot showing DEGs from the comparison of WT Jurkat T cells stimulated with 20 ng/ml PMA and 1 μg/ml ionomycin (P/I) for 2 h after a 2-h pre-treatment with JNKi versus those stimulated with P/I for 2 h after pre-treatment with DMSO. The upregulation of GATA3 is highlighted, and the expanded plot shows normalized counts for GATA3 across experimental conditions. (E) Representative immunoblot analysis of GATA3 and c-Jun accumulation in WT versus CARD11 KO Jurkat T cells stimulated for 2 h with P/I in the presence or absence of JNKi. (F) Quantitation of the fold change of GATA3 (left) and c-Jun (right) over baseline in both WT and CARD11 KO Jurkat T cells from immunoblots in E; n = 3. (G and H) Flow cytometric analysis of WT and CARD11 KO Jurkat T cells stimulated for 2 h with P/I in the presence or absence of JNKi. (G) Representative histograms of the data summarized in H, demonstrating the intracellular staining of GATA3 with the mean fluorescence intensity (MFI) annotated (numbers). (H) Quantitation of the fold change in MFI of GATA3 measured by intracellular flow cytometry (n = 3). Immunoblots are representative of three independent experiments. Error bars indicate the SEM. Asterisks denote significance by two-way ANOVA followed by Šídák’s multiple comparisons test. **P < 0.01; ***P < 0.001; and ****P < 0.0001. NS/No stim., no stimulus. Source data are available for this figure: SourceData F4.