ROCK1 and ROCK2 differentially regulate front–back polarity through isoform-specific mechanisms. (A) Immunofluorescence of CHO.K1 cells cotransfected with either ROCK isoform–specific shRNA or control empty vector and the indicated GFP construct were plated on 2 µg/ml fibronectin for 2 h (left) or overnight (right), and actin filaments were stained with rhodamine phalloidin (magenta). ROCK1 shRNA cells coexpressing RLC-DD–GFP are outlined in white to indicate cell morphology. (B and C) Quantification of cell perimeter (B) and polarity index as measured by cell length divided by width (C), after 2 h or overnight plating on 2 µg/ml fibronectin; n = 39/106 control cells, 42/64 ROCK1 shRNA cells, and 26/89 ROCK2 shRNA cells (n = 2 h/overnight plating). (D) Quantification of polarity index in control cells (Ctrl; white bars) or ROCK1 shRNA cells coexpressing either GFP alone (gray bars) or RLC-DD–GFP (black bars); n = 39/42/33 cells at 2 h and n = 106/64/42 cells after overnight plating (n = control/ROCK1 shRNA + GFP/ROCK1 shRNA + RLC-DD–GFP). (E) Quantification of polarity index in control cells (white bars) or ROCK2 shRNA cells coexpressing either GFP alone (gray bars) or cofilin-S3D–GFP (black bars) or RLC-AD–GFP (diagonal stripes); n = 39/26/22 cells at 2 h and n = 106/89/12/44 cells after overnight plating (n = control/ROCK2 shRNA + GFP/ROCK2 shRNA + cofilin-S3D GFP/ROCK2 shRNA + RLC-AD GFP). Error bars indicate SEM.