Fig. 2. PKM2 activators alter differentiation of T_H17 cells in vitro.

(A) Proposed mechanism of action for the PKM2 activators TEPP-46 and DASA-58 in the oligomerization of PKM2. Cyto, cytoplasm. Nuc, nucleus. (B) Immunoblot for PKM2 oligomerization in cytoplasmic extracts of CD4⁺ T cells treated with TEPP-46 or vehicle. Blots are representative of N=2 independent experiments. (C) Intracellular flow cytometry for RORγT abundance in unstimulated CD4⁺ T cells or T_H17 cells differentiated with TEPP-46, DASA-58 or vehicle (DMSO) for 24 hours. Representative histograms (left) and quantification (right) (N=2 independent experiments). (D) Intracellular flow cytometry for IFN-γ-production in T_H1 cells differentiated with TEPP-46 or DMSO for 48 hours, then stimulated with PMA/ionomycin for 6 hours. Dot plots are representative of N=4 independent experiments with quantification of pooled data in the graph. (E) qRT-PCR analysis of T_H17 lineage mRNA expression in T_H17 cells differentiated with TEPP-46 or DMSO at indicated time points. Data are means ± SEM pooled from N=2 independent experiments on 2 mice. (F) Intracellular flow cytometry for IL-17A/GM-CSF production by T_H17 cells differentiated in the presence of DMSO, TEPP-46, or DASA-58. Dot plots are representative of N=2 independent experiments on 5 mice with quantification of pooled data in right panel as mean ± SEM. (G) Seahorse analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) in unstimulated CD4⁺ T cells (green) and T_H17 cells differentiated with TEPP-46 (red) or DMSO (blue) for 48 hours. Seahorse traces and OCR/ECAR quantification are representative of N=2 independent experiments on 6 mice and presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by one-way ANOVA with Tukey post test (C and F), paired Student’s t-test (D), or two-way ANOVA with Sidak post test (E).