Fibroblasts from Rothmund–Thomson syndrome patients have abnormal spindle axis and more micronuclei. Fibroblasts from Rothmund–Thomson syndrome patients.

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Fibroblasts from Rothmund–Thomson syndrome patients have abnormal spindle axis and more micronuclei. Fibroblasts from Rothmund–Thomson syndrome patients have abnormal spindle axis and more micronuclei. (A) Expression of RECQL4 in HeLa, HEK293T, and U2OS immortalized cell lines is compared with the expression in healthy (GM00323, GM01864) and Rothmund–Thomson syndrome patient (AG05013, AG18371) fibroblasts, using human RECQL4 antibody. (B) Rothmund–Thomson syndrome fibroblasts (AG05013, AG18371) show increased amount of micronuclei as compared with healthy fibroblasts (GM00323, GM01864). More than 1,000 interphase cells per cell line were analyzed for the presence of micronuclei (DAPI stained dots) in the cytoplasm, which was identified by the α-tubulin staining. The pictures show a Rothmund–Thomson syndrome fibroblast (AG05013) with a micronucleus (arrow). Scale bars: 5 μm. (C) Fibroblasts were stained with α-tubulin (green) and γ-tubulin (magenta) antibodies and chromatin with DAPI (blue). The tilting of the spindle axis with respect to the culture plate was quantitated based on γ-tubulin staining (centrosomes) as in the scheme. The pictures show examples of spindle axis lateral views from a healthy (GM00323) or Rothmund–Thomson syndrome (AG18371) fibroblast. The plot shows the angle of the mitotic spindle axis with respect to the culture plate. The difference between the two control fibroblast cell lines GM00323 and GM01864 and one patient cell line (AG05013) has P values of 0.02 and 0.01, respectively, the P values of the control and the second patient cell line (AG18371) are 0.06 each. Scale bars: 1.5 μm. Hideki Yokoyama et al. LSA 2019;2:e201800120 © 2019 Yokoyama et al.