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. 2020 Sep 29;11:4902. doi: 10.1038/s41467-020-18584-5

Fig. 4. Actin stress fibers dominate cytoskeletal anisotropy-dependent cellular responses.

Fig. 4

a Cell stiffness under different stress modes before adding Latrunculin A (No LatA control), after adding Latrunculin A (1 μM) for 2 min (+LatA 2 min) or 5 min (+LatA 5 min). P = 0.0075 between No LatA and +LatA 2 min under 45° stress mode; P = 0.023 between No LatA and +LatA 2 min under 90° stress mode; P = 0.039 between No LatA and +LatA 2 min and P = 0.0032 between +LatA 2 min and +LatA 5 min under in-plane stress mode; P < 0.001 between other conditions under each stress mode. Mean ± s.e.m.; n = 12 cells; nine independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Normalized mean squared displacement (MSD) of all individual GFP spots when the stress (15 Pa at 0.3 Hz) was applied at 0°, 45°, or 90° out-of-plane mode or in-plane mode before adding LatA (b), after adding LatA for 2 min (c), or for 5 min (d). Mean ± s.e.m.; n = 12 cells, nine independent experiments. e Representative images of RNA FISH at no stress, 0°, 45°, or 90° out-of-plane mode or in-plane mode. Stress (15 Pa at 0.3 Hz) was applied before adding Latrunculin A (No LatA control), after adding Latrunculin A for 2 min (+LatA 2 min), or 5 min (+LatA 5 min). The yellow dashed lines highlighted the area of the nucleus. The GFP chromatin domains (green color) and the DHFR FISH fluorescence (red color) in the white box of each image were enlarged and shown on the right. The brightfield image of each cell was shown in an inset at the left site and the scale bar was 3 μm within each inset. The black dot was the magnetic bead attached to the cell surface. The nucleus was highlighted with a dashed line. The scale bar on the top right panel was 3 μm. f DHFR transcription varies with duration of LatA treatment for both in-plane and out-of-plane modes. P = 0.0023 between No LatA and +LatA 2 min under 0° stress mode; P = 0.0048 between No LatA and +LatA 2 min under in-plane stress mode; P < 0.001 between other conditions under each stress mode. Mean ± s.e.m.; 0°: n = 38, 45, and 47 cells; in-plane: n = 58, 89, and 41 cells; 45°: n = 35, 52, and 40 cells; 90°: n = 28, 31, and 62 cells for No LatA control, +LatA 2 min and +LatA 5 min, respectively; three independent experiments. Control was the cells applied with the same stress but not treated with LatA. **P < 0.01; ***P < 0.001. The dashed line indicated the DHFR gene expression levels without applying the stress. P values were calculated and corrected using two-tailed Student’s t-test and Bonferroni correction. Source data are provided as a Source data file.