Fig. 1. Comparison of the effects of aPKC kn and of myosin-II inhibitors on the junction development of MTD1-A cells. (A) Myosin inhibitors and aPKC kn, a dominant-negative mutant of aPKC, block junction development similarly but affect actin reorganization differently. Confluent monolayers of MTD1-A cells transformed by adenovirus vectors encoding β-galactosidase (LacZ) or aPKC kn were cultured in LC medium for 40 hours and then subjected to a Ca²⁺ switch in the presence or absence of the myosin inhibitors blebbistatin (100 µM) or Y27632 (20 µM). The cells were fixed at 6 hours after the Ca²⁺ switch and stained with an anti-ZO-1 antibody (magenta in merged images) and phalloidin (green in merged images). The images shown are single confocal sections that were selected to demonstrate the ZO-1 staining most clearly. Enlargements of the boxed regions in the merged views are shown at the bottom. For cells overexpressing aPKC kn, two typical images are presented, in which the left and right panels show cells exhibiting tightly and loosely bundled circumferential actin cables, respectively (each represents 40% and 60% of aPKC-kn-overexpressing cells, respectively). White arrowheads, circumferential actin cables; arrows, punctate staining of F-actin on spot-like AJs; yellow arrowheads, radial actin fibers. (B) Schematic presentation of the intermediate state of F-actin organization observed in aPKC-kn-overexpressing cells. (C) RNAi knockdown of aPKC isoforms in MTD1-A cells. Subconfluent MTD1-A cells were transfected with the indicated siRNA oligonucleotide duplexes (NS, non-silencing siRNA; 3, aPKC siRNA; 1, aPKC siRNA). Top panels: western blot analyses of total extracts of cells subjected to the indicated RNAi. aPKC was specifically detected by an anti-aPKC (human aPKC) antibody, whereas both aPKC isoforms (aPKC and aPKC) were simultaneously detected by an anti-aPKC antibody (C20) that reacts with both isoforms. GAPDH was used as a loading control. The data for E-cadherin, -catenin and β-catenin indicated no significant change in the expression levels of these AJ proteins. Images shown underneath: the indicated cells were immunostained with an anti-aPKC antibody (C20). (D) aPKC knockdown suppresses junction development after a Ca²⁺ switch, in a similar manner to aPKC-kn overexpression. The indicated cells were subjected to a Ca²⁺ switch as described in A and then immunostained with an anti-ZO-1 antibody (magenta in merged images) and phalloidin (green in merged images). Scale bars: 10 µm.