Fig. 6: Matrix organisation changes result in Rho-ROCK engagement and uncoupling of Myosin II from vinculin. | Nature Communications

Fig. 6: Matrix organisation changes result in Rho-ROCK engagement and uncoupling of Myosin II from vinculin.

From: Matrix mechano-sensing at the invasive front induces a cytoskeletal and transcriptional memory supporting metastasis

Fig. 6

a Confocal SoRa images of HT1080 Fibrosarcoma cells showing nuclei (blue), F-actin (magenta), pMLC2 (cyan), and Vinculin (green) at increasing collagen concentrations (1.7-6.6 mg/ml). Scale bars: 10 µm, insets 2 µm. b Quantification of Vinculin/cell area (%) and percentage of pMLC2/Vinculin levels at the indicated collagen I concentrations. Left, n = 35, 28 and 28 cells and right, n = 23, 25 and 22 cells, three independent experiments. c Western blot analysis of Vinculin levels in HT1080 cells grown in different collagen I concentration gels. n = 3 independent experiments. Vinculin and GAPDH were run in the same gel. d Confocal SoRa images of HT1080 Fibrosarcoma cells showing nuclei (blue), F-actin (magenta), pMLC2 (cyan), and Vinculin (green) ± protease inhibitors (PI) at 4 h and 24 h. Scale bars: 10 µm, insets 2 µm. e Quantification of Vinculin/cell area (%) and percentage of pMLC2/Vinculin levels ± PI. Left, n = 23, 18 and 21 cells and right, n = 30, 27 and 25 cells, three independent experiments. f Western blot analysis of Vinculin levels ±PI. n = 3 independent experiments. Vinculin and GAPDH were run in the same gel. g RhoA and RhoC activation by pull-down assays in HT1080 cells ±PI after 2 h treatment, with quantification of GTP-bound and total protein levels. n = 3 independent experiments. h Immunofluorescence images showing F-actin and pMLC2 in HT1080 cells ±PI and ROCK inhibitor (H1152) at 4 h. Scale bar: 20 µm. i Quantification of cell rounding index and pMLC2/cell area from conditions in (h). n = 61, 66 and 59 cells, three independent experiments. Unpaired t-test for pull-down assays (g) and One-way ANOVA with Tukey post-hoc test for all other comparisons (b left, c, e left, f, i). Two-way ANOVA with Tukey post-hoc (b right and e right). Data displayed as violin plots showing individual data points and medians (b left, e left, i), bar plot with mean and SD (b, right, c, e right, f, g). Cartoons in Fig. 6 (a, d, g, h) created in BioRender. Maiques, O. (2025) https://BioRender.com/c83e894.

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