Fig 2: RhoA mediates the effect of MiR-23a-ATG12 axis on melanoma invasion and metastasis downstream of AMPK signaling. (A, B) Immunoblotting analysis of RhoA, phosphorylated-RhoA and phosphorylated-MYPT1 expression in ATG12-silenced and miR-23a-overexpressed A2058 and A375 cells. Actin was used for normalization. Data represent the mean ± SD of triplicates. (C, D) anti-miR-23a-A2058 cells co-transfected with the siRNA against RhoA or control were subjected to the cell invasion and migration assay. Representative fields of the invaded and migrated cells are shown. Scale bar = 100µm. The invaded and migrated cells were also quantified on the right. Data represent the mean ± SD of triplicates. (E) anti-miR-23a-A2058 cells co-transfected with siRNA against RhoA or control were subjected to wound-healing assay. Experiments were repeated three times with similar results. Scale bar = 100µm. (F, G) ATG12-overexpressed A2058 cells co-transfected with the siRNA against RhoA or control were subjected to the cell invasion and migration assay. Representative fields of the invaded and migrated cells are shown. Scale bar = 100µm. The invaded and migrated cells were also quantified on the right. Data represent the mean ± SD of triplicates. (H) ATG12-overexpressed A2058 cells co-transfected with the siRNA against RhoA or control were subjected to wound-healing assay. Experiments were repeated three times with similar results. Scale bar = 100µm. P value was calculated by two-tailed Student's t-test. *P < 0.05.

Fig 3: Effects of GC combination on phosphorylation of MYPT1, ROCK activity and ratio of active RhoA in colon tissue in NAFLD induced by HFD. (A) p-MYPT1 was detected by western-blot. The relative expression levels of proteins were corrected by MYPT1. The data represents the fold changes relative to that in control group. (B) ROCK activity and ratio of active RhoA in colon tissue. C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate, p-MYPT, phosphorylated myosin phosphatase target subunit, ROCK, Rho-associated kinase, NAFLD, non-alcoholic fatty liver disease.*P < 0.05, **P < 0.01.

Fig 4: PI3K inhibition blocks carbachol-induced MYPT1 and MLC phosphorylation in HASM cells. Time-dependent inhibition of carbachol-induced (10 min, 10 µM) MYPT1 and MLC phosphorylation in HASM cells by CAL-101 (A) and LY294002 (B) (1 µM). Data are representative of five different HASM donors (n = 5 donors, mean ± SEM). *P < 0.05. P values are time points compared with basal. (C) Concentration-dependent inhibition of carbachol-induced MYPT1, Akt and MLC phosphorylation in HASM cells by LY294002 and CAL-101. Data are representative of five different HASM donors. *P < 0.05. P values compared with basal (n = 5, mean ± SEM). (D) CAL-101 (1 µM) prevents carbachol- (10 min, 10 µM) induced MYPT1 (pMYPT1) and MLC (pMLC) phosphorylation, but has little effect on KCl- (10 min, 50 mM) mediated MYPT1 and MLC phosphorylation in HASM cells. Data are representative of five different HASM donors (n = 5, mean + SEM).

Fig 5: Rho kinase activation is greater in HASM cells obtained from subjects with asthma as compared to those of non-asthmatic subjects. HASM cells obtained from fatal asthmatic and non-asthmatic subjects were lysed and subjected to immunoblot for phosphorylated MYPT1 (A), or elisa to measure ROCK activation (B). Data represent mean + SEM fold change of MYPT1 phosphorylation as compared with tubulin control (immunoblotting) and fold compared with basal (for elisa). *P < 0.05. n = 7 asthma and seven non-asthma for (A). n = 5 asthma and seven non-asthma for (B).