Fig. 7.

Regulation of MSO by Cx43 involves PI3K–pAKT. (A,B) S1 cells were treated with vehicle (control) or with AGA, or with 1 μM of PI3K inhibitor LY294002 (LY), or with AGA+LY294002 pre-lumen assembly and until day 4 in 3D culture. Graphs show mean±s.e.m. percentages of cells with proper MSO (i.e. tangential to the acinus circumference) based on immunostaining for α-tubulin and analysis of at least 60 mitotic cells per group (A), and mean±s.e.m. ratios of expression levels of downstream effector of PI3K, p-AKT, over AKT, normalized to the control group based on quantification of western blot bands (B); n=3, one-way ANOVA with Dunn's comparison. A representative image of western blots for one of the replicates is also shown in B; lamin B is used as loading control. (C) Representative images of western blots for PI3K, AKT and p-AKT expression from 3D cultures of S1 cells transduced with Cx43 shRNA (shRNA), non-specific sequence shRNA (NSS) or empty vector (EV). Graph shows mean±s.e.m. relative levels of p-AKT expression compared to total AKT upon Cx43 silencing, normalized to expression in NSS control cells. (D) S1 cells were treated with 5 μM aPKC pseudosubstrate inhibitor until day 4 of 3D culture. Graph shows mean±s.e.m. percentages of cells with MSO tangential to the acini circumference based on α-tubulin immunostaining and analysis of a minimum of 60 cells; n=3; *P<0.05, ***P<0.001; unpaired t-test.