To study the molecular mechanism by which nonmuscle myosin II (MII) regulates protrusion and adhesion dynamics in migrating cells, NIH3T3 cells were treated with 50 µm blebbistatin (BBS) for 15 min. and stained for βPIX (green) and myosin IIB (red). βPIX staining is transformed into numerous small puncta along the membrane after BBS treatment, and that myosin staining is excluded from the leading edge. These findings help elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells. This image is the original data file from Fig. S4, “Potential involvement of βPIX in blebbistatin-induced morphological changes in MII-regulated cell protrusion and adhesion.” J. Cell Biol. 2010. Vol. 190(4):663–674.
Cells were cultured in DME (Invitrogen) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen) at 37°C in a humidified 5% CO2 incubator. Cells were treated with 50 µM blebbistatin, 15min, fixed using 3.7% paraformaldehyde in PBS for 15 min, permeabilized using 0.2% Triton X-100 in PBS for 2 min, blocked with 2% BSA in PBS, and stained with the indicated primary antibodies for at 4°C overnight, followed by incubation with a secondary Alexa Fluor 488–, 546– or 594–conjugated antibody. F-actin was visualized with TRITC or Alexa Fluor 350–conjugated phalloidin. Images were captured by Leica TCS SP2 confocal microscope with HCX PL APO 63X objective.
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