Comparative analysis of the nuclear F-actin networks in A3

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Comparative analysis of the nuclear F-actin networks in A3 Comparative analysis of the nuclear F-actin networks in A3.01 T cells and NIH3T3 fibroblasts. Comparative analysis of the nuclear F-actin networks in A3.01 T cells and NIH3T3 fibroblasts. (A) STED microscopy of endogenous nuclear actin filaments with Alexa Fluor 488 and Atto 647N Phalloidin in A3.01 T cells without stimulation (−) or after stimulation (+) with PMA/Iono. (B) Workflow for the quantification of nuclear actin filaments. STED images of endogenous nuclear actin filaments and segmentation based on supervised machine learning for comparative quantification between A3.01 T cells stimulated with PMA/Iono (left) and NIH3T3 (3 T3) cells stimulated by serum (right). (C) Example of NIH3T3 cytoplasmic actin stress fibers. Stimulated A3.01 T cells and NIH3T3 show nuclear filaments that are similar in size (D) and do not differ in the number of junctions per filament (E). ns, not significant. A3.01 show fewer nuclear actin filaments per cell (F) compared with stimulated NIH3T3 cells. The overall size of the nuclear actin filament network (sum of all nuclear actin filaments) is higher in NIH3T3 cells than in A3.01 cells (G), but the number of filaments in the nucleus is comparable (H). When normalized to the nuclear area, the network size between A3.01 and NIH3T3 cells appears to be similar (I). (J and K) Anisotropy of nuclear actin filaments: No orientation is favored by nuclear actin filaments, and all angles in the orientation range are occupied to a similar extent by nuclear actin filaments in stimulated A3.01 (J) and NIH3T3 (K) cells. For comparison, cytoplasmic actin stress fibers (stress) are plotted in (J) and show a characteristic isotropic peak in the range of orientation. Scale bars, 5 μm. Arrowheads indicate examples of nuclear F-actin filaments (B) or stress fibers (C). Statistical significance was determined by unpaired t test (n = 19 A3.01 T and n = 18 NIH3T3 cells quantified from two independent experiments). Only filaments longer than 200 nm are considered in the quantitative analysis. **P ≤ 0.01, ***P ≤ 0.001. N. Tsopoulidis et al. Sci. Immunol. 2019;4:eaav1987 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works