Fig. 2: Cytogenetic analysis of SSc skin fibroblasts.
From: Centromere defects, chromosome instability, and cGAS-STING activation in systemic sclerosis
Fibroblasts from skin biopsies of healthy individuals (a) and SSc patients (b–g) were arrested in metaphase with colchicine. Chromosomal spreads were stained with anti-CENPA (red) or anti-CENPB (green) antibodies. (In b, c, and f only anti-CENPB). The nuclei/chromosomes were counterstained with DAPI. Shown are representative pictures of at least 10 micrographs. a Healthy fibroblasts showing normal ploidy, b Aneuploidy in dcSSc fibroblasts showing 52 chromosomes, c Micronuclei in SSc fibroblasts (arrows). d Nuclear defects and micronuclei (arrows) in dcSSc and lcSSc. e Loss of centromere identity in micronuclei from SSc fibroblasts. The arrow indicates a micronucleus stained with CENPB but not CENPA antibodies. f Cytoplasmic centromere proteins (arrows) in fibroblasts from ACA-positive lcSSc patients. Yellow indicates colocalization of CENPA and CENPB. g Western blotting analysis of CENPA, GAPDH and H3K9Me3 in cytoplasmic and chromatin fractions from SSc fibroblasts grown in the presence of colchicine. GAPDH (cytoplasmic) and H3K9Me3 (chromatin) confirmed the specificity and purity of the fractions. CENPA was detected mostly in nuclear chromatin fractions as expected but was found leaked into the cytoplasmic fraction in lcSSc patient 025, who has ACAs. Patients 043 and 116 with dcSSc are seronegative for ACAs. A faint band is seen in the cytoplasmic fraction in patient 043. A more quantitative description of the data is found in Supplementary Table 1.
