Fig. 5.

Atypical protein complex Cζ (aPKCζ) inhibition decreases both Ca²⁺ switch- and AMP-activated protein kinase (AMPK)-mediated zonula occludens-1 (ZO-1) localization to sites of junction assembly and affects the level of interaction between ZO-1 and afadin during junction assembly. Confluent Madin-Darby canine kidney (MDCK) cells were incubated in media containing 5 μM Ca²⁺ [low-Ca²⁺ media (LCM)] for 16 h. Cells without aPKCζ inhibition (−aPKCζi) were introduced to fresh LCM and switched to media containing 1.8 mM Ca²⁺ (NCM; Ca²⁺ switch) or 1 mM AICAR for the indicated time points (top). Cells subjected to aPKCζ inhibition (+aPKCζi) were pretreated with the indicated concentration of aPKCζ psuedosubstrate inhibitor for 2 h before being introduced to fresh LCM, NCM, or 1 mM AICAR containing aPKCζ inhibitor for the indicated quantity and time points. A: cells were fixed and immunostained for ZO-1. Scale bar, 30 μm. B and C: quantification of ZO-1 localization at sites of junction assembly. Data were analyzed for each experimental condition based on four randomly obtained views from three independent coverslips. Error bars represent the SE of the length of ZO-1 per cell within each of the four selected fields of view. ****Significant difference in ZO-1 length following treatment with aPKCi (both 10 and 25 μM) after 2 h of the Ca²⁺ switch (P < 0.0001) and following aPKCi at 10 μM after 1 and 2 h of AICAR treatment (P < 0.0001). P values were calculated based on two-way ANOVA with Sidek’s multiple-comparison test. D: cell lysates were probed with the indicated antibodies by Western blot analysis. E and F: cell lysates from each condition were obtained and immunoprecipitated using antibody directed against afadin and protein G agarose beads. Equal amounts of immunoprecipitates were then separated on SDS-PAGE and probed with anti-ZO-1 antibody. Total cells lysates were simultaneously subjected to immunoblotting using anti-ZO-1 and afadin antibodies. G: quantification of the immunoreactive signal for ZO-1 in immunoprecipitates was normalized to the level of immunoprecipitated afadin. Data were analyzed for each experimental condition based on five independent experiments and represent mean intensity relative to LCM level ± SE. * or **Significant difference due to the presence of aPKCζ inhibition under LCM (P < 0.05) or LCM + AICAR (P < 0.01) conditions based on the two-way ANOVA with Sidek’s multiple-comparison test.