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. Author manuscript; available in PMC: 2020 Jul 22.
Published in final edited form as: Curr Biol. 2019 Jul 11;29(14):2339–2350.e5. doi: 10.1016/j.cub.2019.06.032

Figure 4. EPAC2 regulates GDMT nucleation downstream of cAMP in β-cells.

Figure 4.

A-L) MIN6 cells (white dotted line) following nocodazole washout. A-B) DMSO control. C-D) EPAC activator (8-pCPT-2O-Me-cAMP). E-F) EPAC inhibitor HJC0197. G-H) EPAC inhibitor ESI-09. I-J) 8-br-cAMP and EIS-09. K-L) KCl and ESI-09. Golgi (Giantin, red) and emerging MTs (EB1, cyan). Centrosome (left inset, yellow arrowhead) and GDMT nucleation (right inset, white arrow). M) GDMT nucleation in each condition for low (left) and high glucose (right). p < 0.001, one-way ANOVA and Tukey’s multiple comparison test; n = 51-86 cells. N) GDMT nucleation in DMSO (grey), EPAC activator (red) and ESI-09 (blue) in low and high glucose in β-cells of intact islets. 5-95% boxplots, p < 0.001, one-way ANOVA and Tukey’s multiple comparison test; n = 40 cells from 4 islets for each condition. O) Top: western blots for EPAC1 and EPAC2 in MIN6 cells. GAPDH, loading control. Bottom: western blots from MIN6 cells transduced with shRNA as indicated probed for EPAC2 and GAPDH (loading control). P-S) A single β-cell (white dotted line) in an islet following nocodazole washout. EPAC activator (P-O). ESI-09 (R-S). Low glucose (P, R). High glucose 5 minutes (Q, S). Golgi (Giantin, red) and emerging MTs (EB3, cyan). Centrosome (left inset, yellow arrowhead) and an example of GDMT nucleation (right inset, white arrow). T-W) MIN6 cells (white dotted line) following nocodazole washout. T-U) Scrambled shRNA. V-W) EPAC2 shRNA#1. Golgi (Giantin, red) and emerging MTs (EB1, cyan). Centrosome (lower, left inset, yellow arrowhead), GFP signal (upper right inset) and GDMT nucleation (lower, right inset, white arrow). All images are maximum intensity projections of three confocal z-planes. See also Figures S3, S4 and S5.