FIGURE 6.

Dynamin‐independent, Ca²⁺‐sensitive regulation of APJ endocytosis by miR‐204 in H9C2 cells. (A) Effects of dynasore (100 μM) and barbadin (100 μM) on APJ expression in the MEF and WCL following stretch (24 h) or miR‐204 overexpression (24 h) in H9C2 cells. Quantification of APJ in the MEF (n = 5). (B) Effects of dynasore on ERK1/2 activation following stretch or miR‐204 overexpression (n = 5). (C) Effects of ionomycin on APJ expression in the MEF in Ca²⁺ supplemented media. (D) Representative images of cardiomyocytes showing the effect of ionomycin on the formation of Rab5a and APJ puncta and their colocalisation in Ca²⁺‐supplemented media. (E) Effects of BAPTA‐AM (B, 2 μM, 24 h) on the miR‐204‐induced decrease in APJ expression in the MEF. (F) Representative images of cardiomyocytes showing the effect of miR‐204 overexpression and BAPTA‐AM (2 μM, 24 h) on the formation of Rab5a and APJ puncta and their colocalisation. In (D) and (F), the pixel colocalisation of APJ and Rab5a was determined using the RG2B plugin of ImageJ, and images were captured at ×100 magnification. (G and H) Effects of miR‐204 overexpression on the levels of Rab5a, Rab7 and Rab11 in H9C2 cells (G) and quantification (H). *p < .05, **p < .01, ***p < .001 versus indicated group. Data are shown as mean, and error bars represent SEM. B, BAPTA‐AM; SC, scrambled control; miR‐204 M, miR‐204 mimic; SFG, stain‐free gel