Fig. 4: TTC7A deficiency modifies the structural organization of chromatin.

a Left: confocal images of DAPI nuclear staining in control and patient purified naïve CD4+ T cells; scale bar: 5 µm. Right: corresponding interactive 3D surface plots illustrating the spatial intensity of DAPI. b DAPI intensity variation in control and patient CD4 T cells. Cells were isolated from peripheral blood, activated for 7 days in the culture, and analyzed for chromatin compaction. DAPI nuclear mask was applied and each nucleus was subdivided into squares of 12 pixels size in order to calculate the DAPI intensity variance. Mean of three independent experiments ± SEM. Total number of resting cells from controls (n = 100) and patients (n = 93); Total number of activated cells from controls (n = 109) and patients (n = 60). Turkey’s multiple comparisons test multiples p-value < 0.05. c Fluorescence lifetime maps in control and patient nuclei. Fluorescence excitation decay of YoYo-1 bound to DNA was measured by symphotime software. All four images are 9.59 µm width. d Mean fluorescence lifetime ± SEM of the whole nucleus was calculated in resting and activated CD4+ T cells. Mean of three independent experiments ±SEM. Total number of resting cells from controls (n = 88) and patients (n = 85). Total number of activated cells from controls (n = 77) and patients (n = 79). Turkey’s multiple comparisons test multiples; ****p-value < 0.0001; ***p-value < 0.001. e The nuclear area of the focal plane of control and patient B lymphoblastoid cell lines. Cells were stained with DAPI and imaged on the ImageStream system, then analyzed with IDEAS software. Area was measured using the feature mask-morphology (µm²). Graph is a mean ± SEM of three independent experiments with five independent controls and four patients. Total number of cells in controls = 27,191 and patients = 21,186. Unpaired t test p-value < 0.0077. f Confocal microscopy immunostainings of H3k27me3 (green fire blue). We quantified the intensity ratio (log10) of H3k27me3 between the center and the peripheral zone of activated T lymphocytes nuclei. The peripheral zone is set at 10 pixels from the outer layer of the nucleus; the rest is considered to be the center. Scale bar: 5 µm and 1 µm. Mean ± SEM of five independent experiments. Total number of cells in controls = 413 and patients = 500; Paired t test on means; *p-value < 0.0364