Fig 2: ROCK inhibitors such as DJ4 may reduce the phosphorylation of downstream targets such as MLC2 and MYPT1. Blocking ROCK-related pathways can potentially have therapeutic benefits by restoring proliferation and apoptotic cascades that may be improperly signaling due to malignant transformations arising from cancer.

Fig 3: Pendrin gene ablation does not change the percentage of myosin light chain phosphorylation in the mouse aorta.Aortas were isometrically mounted and phosphorylation was determined under either baseline conditions or following stimulation with phenylephrine at the ED50 concentration (0.3 µM) or at a concentration giving maximal stimulation (10 µM). The percentage of MLC20 phosphorylation was determined using 2-D electrophoresis. U represents the unphosphorylated light chain spot, whereas P1 & P2 are, respectively, the singly and doubly phosphorylated light chains. N = 4 in each group.

Fig 4: PP1Ba and PP1Bb dephosphorylate the regulatory myosin light chain.HeLa cells were transfected with zebrafish Mypt1 and GFP-PP1Ba (A, B) or zebrafish Mypt1 and GFP-PP1Bb (C, D). The HeLa cells were immunostained using an anti-phospho myosin light chain 2 antibody, co-stained with DAPI and imaged using confocal microscopy. Panels A and C show anti-phospho MLC2 staining, while B and D show a merge of GFP (green), phospho-MLC2 (red) and DAPI (blue). (E) Purified GST-MLC2 was run either as an untreated control (MLC2) or phosphorylated using ZIPK (Phosph-MLC2). The prephosphorylated MLC2 was dephosphorylated by Mypt1 (1-300)-PP1Ba or PP1Bb complexes immunoprecipitated from HEK293T cell lysates or using controls of treatment with beads from a myc-IP from untransfected cells (myc IP) or cell expressing only Mypt1 (1-300) and no additional catalytic subunit (Mypt1). Dephosphorylation (0), mono (1) and di-phosphorylation (2) was detected by band shift using a phos-tag SDS-PAGE gel as described in the materials and methods. HeLa cells were grown on fibronectin coated coverslips and treated with media containing either 0.1% DMSO (F) or 50 µM blebbistatin (G) for 4 hours. After treatment the HeLa cells were stained with DAPI (blue) and Alexa 568-phalloidin (red) and imaged with confocal microscopy and a color merged image is shown.

Fig 5: FZD7-DAAM1-RhoA-ROCK1/2 supports tumour initiation and metastasis in vivo.a Schematic of experiment (left) and tumour weight (right) of ROCKi (H1152 or GSK269962A) pre-treated A375M2 cells 18 days post-subcutaneous injection into NSG mice (n = 5 mice for control and H1152, n = 4 for GSK269962A). b–d Quantification of b melanoma cell shape score, c H-score of p-MLC2 staining and d ki-67 positive cells in A375M2 tumours from (a). e Limiting dilution assay estimating TIF (top) and tumour volume (bottom) of shControl and shFZD7 WM1361 cells when injected at different dilutions (500,000, 50,000 and 5,000 cells) into NSG mice (Number of tumours per condition indicated in table). TIF was determined using ELDA. f–h Representative images (top) and quantification (bottom) of f melanoma cell shape score, g H-score of p-MLC2 staining and h ki-67 positive cells in TB and IF of shControl (n = 16) and shFZD7 (n = 9) derived tumours from 50,000 cells’ condition from (e). Scale bar, 100 µm; inset, 25 µm. i Limiting dilution assay estimating TIF (top) and tumour volume (bottom) of shControl and shDAAM1 WM1361 cells when injected at different dilutions (500,000, 50,000 and 5,000 cells) into NSG mice (Number of tumours per condition indicated in table). TIF was determined using ELDA. j–l Representative images (top) and quantification (bottom) of j melanoma cell shape score, k H-score of p-MLC2 staining and l ki-67 positive cells in TB and IF of shControl (n = 10) and shDAAM1 (n = 5) derived tumours from 50,000 cells’ condition from (i). Scale bar, 100 µm; inset, 25 µm. m Representative confocal images (left) and percentage of fluorescence area (right) of mouse lungs 24 h (n = 6 mice) and 3 weeks (n = 5 mice) after tail vein injection into NSG mice of WM1361 cells expressing shControl and shDAAM1. Scale bar, 50 µm. a, m Graphs show mean ± s.e.m. b–l Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. a–d One-way ANOVA with Dunnett post-hoc test. e, i Two-tailed Mann–Whitney test. f–h, j–l One-way ANOVA with Tukey post-hoc test. m Two-tailed t-test. For all graphs, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The exact significant p values for *p, **p and ***p are provided in Supplementary Table 1. Schematics in this figure were created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).