Fig. 8: Gene expression deficits in OT-I Mrtfab^−/− cells.

See also Supplementary Data 1 and 2. A Purified naive wildtype or Mrtfab-null OT-I CD8⁺ T cells were stimulated as shown⁵³. RNAseq was carried out on naive cells, cells activated by TCR crosslinking for 24 h, or activated cells cultured in IL-12 for 16 h (“activated/rested cells”) and then stimulated with IL-2 for various times. Data show mean values ± SEM of three biological replicates, each representing cells purified from pooled lymph nodes from individual animals. B (i) Normalised z-scored read counts of activated/rested cells stimulated with IL-2, grouped according to gene expression patterns by unsupervised clustering. (ii) Genes showing significant changes upon IL-2 stimulation at any time point in wildtype (left) and Mrtfab-null cells (right). Genes showing an absolute fold-change greater than 2 at p < 0.05 (DESeq2) are color-coded. Fold-change and padj values are reported in Supplementary Data 1. (iii) Genes whose expression is impaired by MRTF inactivation, in activated/rested cells (left) or IL-2 stimulated cells (right), displayed as in (ii). (iv) Genes identified as candidate SRF targets in TPA-stimulated MEFs⁴² or serum-stimulated NIH3T3 fibroblasts⁴¹. C Gene ontology categories significantly overrepresented (p < 0.01) in the clusters identified in (B) are summarised by standard Jaccard index scores. Significance was established by hypergeometric testing using the “phyper” function from the R library “stats” and p values were adjusted for multiple comparisons using the “p.adjust” function with the Benjamini & Hochberg method from the same library. St JI scores and adjusted p values are reported in Supplementary Data 2. Gene ontology categories significantly overrepresented (P < 0.01) in the clusters identified in (B), summarised by Standard Jaccard Index. D Reactome pathway categories (https://reactome.org) significantly overrepresented (P < 0.01) in the clusters identified in (B) are summarised by Standard Jaccard Index scores, with significance established and reported as in (C). E Expression of β- and γ-actin in MACS-purified OT-I WT and OT-I Mrtfab^−/− lymph node cells following TCR activation assessed by qRT-PCR. MACS-purified OT-I WT and OT-I Mrtfab^−/− cells from the spleens of tamoxifen-fed bone marrow-reconstituted mice were activated in vitro with plate-bound anti-CD3/CD28 (5 μg/ml) for 24 h, then transferred with supernatant to uncoated wells and cultured 5 h with or without anti-IL-2 blocking antibody. Data show mean values ± SEM of three independent experiments, normalised to expression in naive OT-I WT cells. F Immunoblot analysis of β-actin, γ-actin, and total actin expression in MACS-purified OT-I WT and OT-I Mrtfab^−/− lymph node cells following TCR activation. Cells were activated in vitro as in (E) and transferred with supernatant to uncoated wells for a further 24 h. Protein levels were normalised to those of naive cells. Source data are provided as a Source Data file.