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. 2021 Jul 26;118(31):e2010053118. doi: 10.1073/pnas.2010053118

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Fig. 3. — PIP4K2B and PIP4K2C regulate Treg cells transcriptomic profile. (A) Volcano plots (log fold change against the −log of the adjusted P value) of expressed genes in Tregs after depletion of PIP4K2B (Left) or PIP4K2C (Right). The numbers and dots in blue and red refer to genes that are significantly down-regulated or up-regulated, respectively (P < 0.05 and absolute log2fold change > 0.6). (B) Same as A, except PIP4K depletions were in Tconv cells. In both A and B, the x- and y-axis have the same minimum and maximum to illustrate the difference in effects of PIP4K depletions in Tregs compared to Tconv. (C) Scatter plots to show the changes in the expression of genes from the Treg_shPIP4K-DEG gene set after depletion of PIP4K in either Tregs or Tconvs as indicated. (Left) Depletion of PIP4K2B (log(sh_2B/sh_Ctrl) plotted on the x-axis and PIP4K2C depletion (log(sh_2C/sh_Ctrl) on the y-axis. (Middle) Depletion of PIP4K2B in Tregs against depletion of PIP4K2B in Tconv cells. (Right) Depletion of PIP4K2C in Tregs against depletion of PIP4K2C in Tconv cells. The data depict the average of three RNA-seq data sets for each knockdown in Treg and two in Tconvs. (D) The Treg_shPIP4K-DEG gene set was used for PCA. (E, Left) The Treg_shPIP4K-DEG (P-adjusted < 0.05) was used to generate a heat map with scaled rows and three hierarchical clusters. (Right) Gene sets from each cluster were extracted, and the average expression of genes within each cluster in Tregs and Tconv before and after depletion of PIP4Ks were quantitated. (F) Genes from cluster 3 (E) were used to interrogate their expression in an unrelated dataset comprising Tconv and Tregs before (Tconv_NS, Treg_NS) and after CD3/CD28 activation (Tconv_ST, Treg_ST) isolated from four separate donors. The heat map shows the average expression of each gene within cluster 3, while the graph depicts the average expression of all genes from cluster 3. The data illustrate that cluster 3 genes are up-regulated upon T cell activation. (G) Gene ontology enrichment analysis showing that cluster 3 genes are highly enriched for genes related to cell cycle, mitosis, and chromosome segregation. Box plots follow standard Tukey representation. Statistics are derived using Krushkal–Wallis hypothesis testing with post hoc–paired testing using Benjamini–Hochberg probability adjustments.