MIIA and MIIB control different stages of adhesion maturation. (A) Cells were transfected with control, MHCIIA, or MHCIIB shRNA-containing plasmids to inhibit expression of the indicated isoform. The cells were plated on coverslips coated with 2 µg/ml fibronectin for 60 min, fixed, and stained with antibodies against the indicated MII isoform (left) or endogenous vinculin (middle). Images were captured using a confocal microscope (FV300; Olympus). Representative cells are shown. The right column shows inverse colored, higher magnification of the boxed regions in the middle column. In the MIIA-depleted cells, there are no elongated adhesions; in the MIIB-depleted cells, some adhesions around the periphery are somewhat elongated. Bar, 10 µm. (B and C) Quantification of the adhesion phenotypes including adhesion elongation represented as the axial ratio (B) and area (C). MIIA-depleted cells were not included in the analysis as they only display small, nonelongated, nascent adhesions. n is indicated, and P represents significance using the nonparametric Mann-Whitney U test.