NMIIB is critical for traction force generation in fully spread, nonmigrating cells. (A) NMIIA and NMIIB protein expression levels in MDA-MB231 cells stably infected with lentiviral shRNA constructs to deplete NMIIA or NMIIB. (B) Diagram showing measurement of traction stress, a measure of force generation for cells attached to 30-µm2 patterned squares of fibronectin. Cells were plated and allowed to adhere for 1 or 16 h, and bead positions were imaged before and after trypsin treatment to determine contractile stresses. (C and D) Traction stress measurements were collected on NT shRNA control, NMIIA-shRNA, or NMIIB-shRNA 4T1 cells (C) or MDA-MB-231 cells (D) at 1 h (open bars) and 16 h (closed bars). *, P < 0.01; ***, P < 0.0001; n = 30 cells per condition; error bars represent SEM. (E) MDA-MB 231 were transiently transfected with either a NMIIB-GFP fusion construct or a control-free GFP construct and expression was verified via Western blot. (F and G) MDA-MB 231 NMIIB-shRNA cells were transfected with a GFP-NMIIB fusion construct and traction stress was measured on patterned fibronectin at 1 h (F) or 16 h (G) after plating. GFP intensity of individual cells was quantified and correlated with levels of traction stress. Dashed lines represent 95% confidence interval; n = 21 and 33 for 1 and 16 h time points, respectively. (H and I) MDA-MB 231 cells were plated on fibronectin-coated coverglass for 1 or 16 h before fixation and DAPI staining. They were then analyzed by spinning-disk confocal microscopy to generate x–z projections for nuclear height quantification. (H) Representative x–z projection DAPI images of MDA-MB 231 control, NMIIA shRNA, or NMIIB shRNA at 16-h time point. Vertical bar, 5 µm. (I) Nuclear height among conditions is unchanged at 1 h (open bars). NMIIB knockdown significantly increased nuclear height, whereas NMIIA knockdown had no effect compared with control at 16 h (shaded bars). **, P < 0.01; ***, P < 0.0001; n = 60 cells per condition.
