Fig 2: The immunoblots of (A) active RhoA, (B) total RhoA, (C) normalized Rho GTP, (D) P-LIMK2, (E) LIMK2 and (F) normalized P-LIMK were analyzed using densitometry. The results are presented in bar graphs as mean ± S.D. *P<0.05. P-LIMK2, phosphorylated LIM domain kinase 2; LIMK2, LIM domain kinase 2.

Fig 3: EGF activates LIMK2 and Cofilin expression in MG63 cells through ROCK. (A) Phosphorylation of LIMK2 and Cofilin in MG63 cells treated with 10 ng/mL EGF for 2, 6 and 12 h, as detected by western blotting. Quantification of LIMK2 (B) and Cofilin (C) phosphorylation in MG63 cells treated with 10 ng/mL EGF for 2, 6 and 12 h; (D) Phosphorylation of LIMK2 and Cofilin pretreated with Y27632 for 2 h followed by the addition of EGF, as detected by western blotting. Quantification of Cofilin (E) and LIMK2 (F) phosphorylation in MG63 cells pretreated with Y27632 for 2 h followed by the addition of EGF; (G) Expression of LIMK2 and Cofilin in MG63 cells treated with LIMK2 siRNA and Cofilin1 siRNA, as detected by western blotting. siR-Ribo™ served as the negative control (NC); (H) Phalloidin staining of stress fibers and DAPI staining of MG63 cells treated with LIMK2 and Cofilin1 siRNAs. siR-Ribo™ served as the negative control, scale bar = 50 μm. * p < 0.05, ** p < 0.01.

Fig 4: CLU interacts with ROCK1 to suppress metastatic traits. (A,B) IP–MS analysis was performed using 293FT cells with ectopic expression of CLU-Flag. Metastasis-related regulator ROCK1 was identified as a CLU binding protein. (C) Co-IP assay was performed in 293FT cells to confirm the interaction between precursor CLU and ROCK1. (D) Representative images of immunofluorescent co-staining of intracellular CLU and ROCK1 in CLU-overexpressing and vector-control cells. (E) PLA assay was performed to confirm the interaction of CLU and ROCK1 in H460 and A549 cells. (F,G) The effect of Y27632 treatment in CLU-silenced H460 cells and vector-control cells on their migration and invasion abilities. (H) Cell morphology had no difference in CLU-overexpressing H1299 cells and CLU-silenced H460 cells. F-actin was used to detect the cytoskeleton in those cells. (I) The effect of CLU on ROCK1, p-MLC2, Cofilin, p-LIMK1 and LIMK2 in CLU-overexpressing H1299 and H1975 cells and CLU-silenced H460 cells. GAPDH was used as a loading control. Results are presented as mean ± SD, *** p < 0.001, ns means no significance.

Fig 5: Low dose X-ray irradiation-induces LIMK2 and Cofilin phosphorylation. The ROCK inhibitor blocked 0.5-Gy X-ray irradiation-induced actin reorganization and LIMK2 phosphorylation. Cells were pretreated (+Y-27632) or not (-Y-27632) with 10 µmol/l Y-27632, and subsequently with 0 or 0.5-Gy X-ray irradiation for the indicated time points. Western blotting was performed to detect the levels of P-LIMK2 and P-Cofilin in each group (A). The immunoblots were analyzed by densitometry, and the intensity of the GTP-RhoA bands was normalized to the intensity of the corresponding total Rho band. The ratios presented in bar graphs are shown as mean ± SD of P-LIMK2 and P-Cofilin (B-G) (*P<0.05). ROCK, Rho-associated protein kinase; LIMK2, LIM domain kinase 2; P, phosphorylated.