Fig. 5: Cul5-deficient CD4⁺ T cells show an increased Th9 gene signature.

Naïve CD4⁺ T cells from 8–10-week-old control and Cul5^fl/flCD4-Cre mice were isolated and cultured in Th2 and Th9 polarising conditions. a Left panel shows the representative flow plot for IL-4⁺ and IL-5⁺ cells under Th2 polarizing condition. The bar graph on right shows the consolidated data. For IL-4⁺ cells, n = 3 control, n = 6 Cul5^fl/flCD4-Cre, for IL-5⁺ cells, n = 3 control, n = 5 Cul5^fl/flCD4-Cre, examined in two independent experiments. b Left panel shows the representative flow plot for IL-9⁺ cells in control and Cul5^fl/flCD4-Cre CD4⁺ T cells cultured under Th9 polarizing conditions. The bar graph on the right shows the consolidated data for n = 8 control, n = 10 Cul5^fl/flCD-Cre, examined in four independent experiments. c–h RNASeq analysis was used to measure the transcript levels in control and Cul5 deleted CD4⁺ T cells cultured for 2 days in Th9 polarizing condition. n = 3 control and Cul5^fl/flCD4-Cre CD4⁺ T cells examined in one experiment. c Ontological analysis of the differentially regulated genes, calculated by GOplot algorithm is shown. Circle plots depict the enrichment of genes associated with particular Th subtype. Greater the size of the inner trapezoids represents greater -log10 value (adj. p-value). Heatmap of Th subtypes. To generate heat maps, we first selected genes that were enriched in different Th subtype compared to naïve CD4 T cells from publicly available dataset³⁹. We selected 200 most differentially regulated genes for each subtype and classified these as top hit genes. We then compared these top hits genes to the differentially regulated genes from our RNAseq dataset and to generate heatmaps. Heatmap of top hit genes identified in Th9 (d), Th2 (e), and Th1 (f) subtypes that were also differentially regulated in WT and Cul5 deficient CD4⁺ T cells. g Heatmap of top hit genes which were identified exclusively in Th9 subtype. h The red circle in the table indicates the binding sites for the respective transcription factors present in the particular gene. Data is presented as Mean ±SEM in panels a, b. p-value was calculated using unpaired two tailed t-test. Source data are provided as a Source Data file.