Fig. 6: Loss of Zfp335 leads to altered nuclear envelope architecture, accumulation of cytosolic dsDNA and promotes cGAS/STING signaling.

a Representative histograms and gating of Baf phosphorylation as measured by proximity ligation assay (PLA). Percent phosphoserine-Baf and geometric MFI in parenthises are shown. Phosphoserine-Lamin B1 PLA was used as positive control. Quantification of Baf phosphorylation based on geometric MFI (b) or percent positive cells (c). n = 5 mice per genotype. d Representative immunofluorescence images of full cell thickness maximum intensity projections (left) and profile plots (right) of nuclear envelope staining in ex vivo DN4 thymocytes. Profile plots are based on white lines shown in merged images. Scale bars represent 10 µm. Quantification of frequency of cells with high nuclear-associated Lamin B1 (e), mean DAPI pixel intensity (f) or mean nucleus sphericity (g) for ex vivo DN4 thymocytes. e, g n = 5 mice per genotype, f n = 124 WT and n = 490 Zfp335cKO cells. Representative images (h) and quantification (i) of cytoplasmic dsDNA in WT (n = 548 cells, 4 mice) or Zfp335cKO (n = 268 cells, 6 mice) thymocytes following 3 days in OP9-DL1 culture. Magenta arrow indicates OP9-DL1 cell. GSEA enrichment plots for T-cell-specific STING signaling gene signature in DP bulk (j) or DN4 scRNA-seq datasets (k). l UMAP projection of IRF3 gene signature in WT or Zfp335 mutant DN4 thymocytes. m Violin plots of proapoptotic Bcl2 gene expression in WT or Zfp335 mutant DN4 thymocytes. n Normalized cGAMP concentration for WT (n = 3 mice) or Zfp335cKO (n = 3 mice) thymocytes. Representative histograms (o) and quantification (p) of phospho-STING, -TBK1, and -IRF3 in WT (n = 4 mice) or Zfp335cKO (n = 6 mice) thymocytes following 3 days in OP9-DL1 culture. WT thymocytes treated with 250 µg/mL cridanimod (WT + CMA) for 2 h were used as a positive control. q Representative images of phospho-IRF3 staining related to o,p. P-values determined by two-tailed Mann–Whitney U-test (b–i), unpaired two-tailed Student’s t-test (n), or two-way ANOVA with post hoc Tukey’s test (m) or Sidak’s test (p). Data shown are compiled from one (j–m), two (h, i, n–p), or three (a–g) independent experiments utilizing male and female mice. Plots show mean ± SEM (b, c, e–g, n, p) or median and interquartile range (f, i). Source data are provided as a Source Data file.