Fig. 6: Depdc5-deficient CD8⁺ T cells display enhanced expression of purine catabolic enzyme XO due to hyper-mTORC1-ATF4 activation.

a Volcano plot of differentially expressed genes (DEGs) generated by RNA-seq analysis of Depdc5 WT (Depdc5^WT) and Depdc5 knock-out (Depdc5^KO) CD8⁺ T cells. Red dots represent genes significantly upregulated in Depdc5^KO CD8⁺ T cells (P < 0.05 and log₂ fold change (FC )≥ 0.5), while blue dots represent genes significantly downregulated (P < 0.05 and log₂ FC ≤ 0.5), and grey dots indicate DEGs below the level of significance. b KEGG pathway analysis of upregulated DEGs in a. The top 10 significant KEGG pathways based on upregulated genes are presented by normalized enrichment score (NES) and P value. c Gene set enrichment of “KEGG purine metabolism” pathway in Depdc5^KO relative to Depdc5^WT CD8⁺ T cells. d Heatmap showing normalized expression of purine metabolism-linked genes that were significantly up-regulated in Depdc5^KO CD8⁺ T cells (n = 4). e Heatmap showing normalized expression of genes encoding ROS-generating enzymes in Depdc5^WT and Depdc5^KO CD8⁺ T cells (n = 4). f RT-qPCR analysis of Xdh, Mki67, and related anti-ferroptotic mevalonate pathway genes Hmgcr, Hmgcs1, and Sqle expression in CD8⁺ T cells from Depdc5^ncl and Depdc5^tko mice. Relative mRNA levels were normalized to Gapdh mRNA level (n = 4). g Immunoblotting of XO protein level in Depdc5^WT and Depdc5^KO splenic CD8⁺ T cells. XO molecular weight is 145 kDa (long form) or 125 kDa (short form) while XDH molecular weight is 145 kDa. ERK1/2 served as a loading control. Numbers under the XO immunoblotting bands indicate the density of XO relative to ERK1/2. h Bar graph showing analysis of uric acid levels in Depdc5^WT and Depdc5^KO CD8⁺ T cells (n = 3). i Immunoblotting of ATF4 protein level in Depdc5^WT and Depdc5^KO splenic CD8⁺ T cells. S6K and S6K-pT389 profiles were assessed to determine mTORC1 activity, while p38 served as a loading control. j Heatmap showing normalized expression of ATF4 target genes in Depdc5^WT and Depdc5^KO CD8⁺ T cells (n = 3). k Cellular ROS levels in Depdc5^WT and Depdc5^KO splenic CD8⁺ T cells treated for 4 h in vitro with either vehicle alone or XO inhibitor allopurinol. l Relative lipid peroxidation levels in Depdc5^WT and Depdc5^KO splenic CD8⁺ T cells treated as in k. Error bars indicate means ± SEM (NS, not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001 by unpaired Student’s t-test).