|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. Wrch-1 controls focal adhesions by regulating myosin light-chain phosphorylation. (A) Wrch-1 depletion inhibits myosin light-chain phosphorylation. Cells were transfected with siRNA targeting luciferase (control) or Wrch-1 (W-1) as described in Materials and Methods. Ninety hours after transfection, cells were lysed directly into 2x sample buffer and phosphorylated MLC was analyzed by western blotting. Subsequently, blots were stripped and re-blotted with anti-MLC antibody. Tubulin blot was used as loading control. Levels of phosphorylated MLC from seven independent experiments were quantified as detailed in Materials and Methods and normalized to levels of either MLC or tubulin. *P=10–4, two-tailed t-test. (B) treatment with ML-7 increases stress fiber formation and the number of focal adhesions. Cells were plated onto laminin-coated coverslips for 1 hour and subsequently incubated in the presence of 5 µM ML-7, or DMSO as control. After 48 hours, cells were fixed and processed as described in Fig. 2A. Bars, 10 µm. (C) Quantification of focal adhesions was performed as in Fig. 2D. Data shown are representative of two independent experiments. *P<0.0005, two-tailed t-test. (D) ML-7 inhibits cell migration. HelaS3 cells were transfected with siRNA targeting luciferase (control) or Wrch-1 (W-1) for 72 hours, plated as a confluent monolayer for several hours and subsequently pretreated overnight with either ML-7 (5 µM) or DMSO, and treated with ML-7 or DMSO upon wounding. Migration was quantified 7 hours after wounding, as described in Fig. 4A. Shown is the average ± s.e.m. of eight sites. *P<10–5, two-tailed t-test. Data shown are representative of four independent experiments.
|
