Organization of stress fibers and distribution of NMIIA and NMIIB in spreading REF-52 fibroblasts with and without depletion of each NMII paralog. (A) Western blotting showing expression of NMIIA (left), NMIIB (middle), and NMIIC (right) in wild-type COS-7 cells and in REF-52 cells expressing nontargeting shRNA (shNt) or shRNAs targeting NMIIA (shA) or NMIIB (shB). (B–D) REF-52 cells expressing nontargeting shRNA (B, shNt) or shRNA targeting NMIIA (C, sh NMIIA) or NMIIB (D, sh NMIIB). Cells were preextracted before fixation and stained with phalloidin (magenta) and NMIIA (cyan) and NMIIB (yellow) antibodies. Examples of stress fiber types are indicated by arrows in B and show radial (r), ventral (v), and transverse arcs (t). Left panels show xy maximum projections, whereas right panels show z slices along yellow lines. Bars: (xy panels) 20 µm; (z panels) 5 µm. (E) REF-52 cells expressing nontargeting shRNA (left) or shRNA targeting NMIIA (middle) or NMIIB (right). Cells were directly fixed and stained with phalloidin (magenta) and vinculin antibody (green). Bar, 20 µm.