PIKFYVE regulates cell cycle and cell division during Th17 differentiation. (A) Label-free mass spectrometry determined the relative abundance of phosphopeptides for proteins in the cell cycle based on four biological replicates. (B) A flow cytometry–based assay was utilized to analyze the cell cycle in primary murine CD4⁺ T cells treated with vehicle or apilimod (AP) at 96 h of activation. (C–E) The percentage of cells in (C) G1, (D) G2/M, and (E) S were measured (N = 3 biological replicates). (F) Naive murine CD4⁺ T cells isolated by negative selection were stained with cell tracer dye and activated under Th17 conditions with vehicle or apilimod for 5 days and analyzed by flow cytometry. Panel F represents an example from three biological replicates. (G–J) CD4⁺ cells were gated and the cell count was plotted as a function of incorporated cell tracer and for (G and H) IL-17a–producing cells and (I and J) FoxP3-expressing cells. Three biological replicates were included in the cell proliferation assays depicted in panels G–J. Data shown depict ± SEM. P values were calculated with Student’s t test in panels C–E. ANOVA two-way followed by Sidak test was used to calculate P values in panels H and J. **P < 0.01, ****P < 0.0001.