Fig. 1.

AMPK regulates atypical protein complex Cζ (aPKCζ) localization during junction assembly. A: confluent Madin-Darby canine kidney (MDCK) cells were stably transfected with empty pSUPER vector [AMPK wild-type (WT)] or pSUPER containing shRNA directed against AMPKα1 [AMPKα1 knockdown (KD)] and were incubated in media containing 5 μM Ca²⁺ [low-Ca²⁺ media (LCM)] for 16 h. Fresh LCM or media containing 1.8 mM Ca²⁺ (Ca²⁺ switch) were introduced for 2 h. Cells were fixed and immunostained for aPKCζ. Scale bar, 30 μm. B: quantification of aPKCζ 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 atypical protein complex Cζ (aPKCζ) per cell within each of the four selected fields of view. P values were calculated by two-way ANOVA with Sidak’s multiple-comparison test. ***Significant difference due to the Ca²⁺ switch (P < 0.001) compared with LCM conditions or AMPKα1 KD. C: confluent WT MDCK cells were incubated in media containing 5 μM Ca²⁺ (low-Ca²⁺ media, LCM) for 16 h. Fresh LCM with or without 2 mM AICAR was introduced for 1 h. Cells were fixed and immunostained for aPKCζ. Scale bar, 30 μm. Arrowheads indicate sites of junction assembly. D: quantification of aPKCζ 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 aPKCζ per cell within each of the four selected fields of view. **Significant difference due to the presence of AICAR (P < 0.0001) compared with LCM conditions based on the Student’s t-test.