Fig 2: The effects of AT1 receptor inhibitor candesartan on the expression and activation of Rho GTPases and Rho-associated kinase (ROCK). Cells were pretreated with candesartan (5 µM) for 6 h and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) Detection of Rho GTPases activity by pulldown assay. Histograms show the ratios of Rho GTPases GTP-bound to total Rho GTPases. * p < 0.05, vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3). (B) Western blot analysis of the expression of ROCK1, ROCK2, MYPT1, and phospho-MYPT1 (Thr696). Histograms show the ratios of ROCK1 or ROCK2 to ß-tubulin or phospho-MYPT1 to total MYPT1. * p < 0.05, vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3).

Fig 3: MYPT1 suppressed MAPK8/N-cadherin expression to influence the migration and invasion of ccRCC cells. (A, D) Western blotting of the indicated proteins in ccRCC cells. The bar plots show the relative band intensities (mean ± SEM) of three independent experiments. (B, E) Cell migration as determined by wound healing assay. Red lines denote the margins of the wound. (C, F) Cell invasion as determined by Transwell assay. Matrigel was added to the upper chamber. Scale bars: 200 µm. Statistical analysis was performed with a two-tailed unpaired Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). ccRCC, renal clear cell carcinoma; MAPK, mitogen-activated protein kinase; MYPT1, myosin phosphatase target subunit 1.

Fig 4: RLC phosphorylation in MYPT1-deficient dorsal arteries in response to PE stimulation.Dorsal arteries of MYPT1 KO (Mypt1?SM/?SM) and control (Mypt1+/+) mice were collected and stimulated with 10 µM PE. The stimulated arteries were sampled for RLC phosphorylation via urea/glycerol PAGE. a Representation of typical RLC phosphorylation assay results after stimulation. b Quantification of RLC phosphorylation. The RLCp level was expressed as the percentage of the total RLC level. The bars indicate the mean values ± SEM; n = 5; *p < 0.05; t-test.

Fig 5: MYPT1 suppressed ccRCC metastasis in vivo. (A) The lung metastasis model showed that MYPT1 overexpression significantly inhibited ccRCC lung metastasis. Representative gross images of the lungs and bioluminescence images of lung metastases are shown. Arrows indicate pulmonary metastatic nodules. Scale bars: 500 µm. (B, C) Nude mice injected with MYPT1-overexpressing Caki1 cells had fewer and smaller lung metastases. Representative images of HE staining of the lungs and statistical analysis results are shown. Scale bars: 200 or 50 µm. The pulmonary metastatic nodules were counted under a microscope, and the results were summarized. Data presented as mean ± SEM (n = 5). Statistical analysis was performed with a two-tailed unpaired Student's t test (*P < 0.05). ccRCC, renal clear cell carcinoma; HE, haematoxylin–eosin; MYPT1, myosin phosphatase target subunit 1.