Figure 1.
Analysis of MLCK expression in normal and cancer tissues.
Quantitative PCR (A) and western blot (B) analyses of normal and cancer tissues. RNA was isolated from various normal and cancer tissues and total MLCK (A), including all splice variants of nmMLCK and smMLCK, was detected using primers targeting exons 26 and 27. The gene for H3F3A was used as an internal control in all experiments. The data in Panel A depict the averages of qPCR analyses performed in triplicate and the error bars show the standard deviation. Panel B shows a western blot analysis using affinity purified antibodies to MLCK. GAPDH was used as a loading control.
Figure 2.
Analysis of MLCK expression in normal and cancer cells in culture.
Quantitative PCR to detect total MLCK (A) and western blot (B) analyses of normal and cancer cells in culture. RNA and protein were isolated from various cells and analyzed as described in Figure 1. The data in panel A depict the averages of qPCR analyses performed in triplicate.
Figure 3.
Contraction of 3D collagen gels.
HeLa, LNCaP, MCF7, MCF10A, HUF and primary prostate cells were seeded in 24 well dishes in liquid collagen. After the collagen hardened, the gels were released from the sides of the wells and allowed to contract for 24 hrs. The wells were photographed from above at the times shown. Note that the HUF and primary prostate cells contract the gels to a greater extent than HeLa and LNCaP cells and that MCF 10A cells contract more than MCF-7 cells. Scale bar = 2 mm.
Figure 4.
Effect of inhibitors on 3D gel contractions.
HUF (left) and HeLa (right) cells were grown in 3D cultures and treated with inhibitors as described. The gels were photographed 24 hrs later and the surface area of the individual gels was quantified. The data represent the mean +SEM. One way ANOVA * = p value < 0.05, *** = p value < 0.001.
Figure 5.
Quantification of MLC phosphorylation of cells treated with inhibitors.
The phosphorylated and unphosphorylated MLC were separated by urea-glycerol gel electrophoresis, blotted to nitrocellulose and identified using an antibody the 20 kD MLC.
Figure 6.
Micrographs of cells grown in collagen gels.
HeLa cells were transfected with Lifeact mCherry (red) or Lifeact-GFP and grown in collagen gels. These gels were not released from the walls of the wells to prevent motion artifacts. Panel A shows control (untreated) cells extending filapodia that contact neighboring cells (also see movie in Figure S2). Panels B & C show cells that were treated with 20 μM ML-7 (B) or 6 μM cytochalasin D (C). Cells treated with ML-7 continue to actively extend filopodia. Cells treated with cytochalasin D stop extending filopodia and the actin in these cells appears to collect in large aggregates. The insets are blow ups of the boxed areas. Size bar = 10 μm.