MIIB enhances MIIA-initiated actomyosin bundling in CHO.K1 cells. (A) Cells were transfected with control, MHCIIA, or MHCIIB shRNA-containing plasmids to inhibit expression of the indicated isoform. The cells were plated on 2 µg/ml fibronectin for 60 min, then fixed and stained with rhodamine-conjugated phalloidin to visualize actin (left) or antibodies against the indicated MII isoform (middle). Representative cells are shown. The right column shows inverse colored, higher magnification of the boxed regions in left column. In the MIIA-depleted cells, there are few organized actomyosin bundles; in the MIIB-depleted cells, the bundles are small and thin. Images were captured using a confocal microscope (FV300; Olympus). Bar, 10 µm. (B–D). Quantification of the actin bundling phenotypes illustrated in A. Parameters evaluated are: (B) number of bundles per cell, divided into “short” (<10 µm) and “long” (>10 µm), n ≥ 35 cells/condition; (C) bundle length, and (D) thickness (n ≥ 150 bundles from >30 cells/condition). (B–D) P is the significance using the nonparametric Mann-Whitney U test (in C and D; asterisks refer to P vs. control cells). Error bars indicate ±SD.