Fig. 6
AKT phosphorylation as a mediator of Kcnj13 function in tracheal SM cells. a Immunostaining for αSMA (red) and p-AKTser473 (green) and DAPI staining (blue) of transverse sections of E14.5 WT (n = 6) and Kcnj13T38C/T38C (n = 6) tracheas. b Quantification of mean p-AKTser473 fluorescence intensity in E14.5 WT (n = 6) and Kcnj13T38C/T38C (n = 6) tracheal SM cells. c Western blotting for phosphorylated AKT, total AKT and GAPDH in P0 WT (n = 12) and Kcnj13T38C/T38C (n = 12) tracheas. d Quantification of relative p-AKTser473 levels in P0 WT (n = 12) and Kcnj13T38C/T38C (n = 12) tracheas. e Western blotting for phosphorylated AKT, total AKT and GAPDH in P0 tracheas after DMSO (n = 16) or 50 μM VU590 (n = 16) treatment. f Quantification of relative p-AKTser473 levels in P0 tracheas after DMSO (n = 16) or 50 μM VU590 (n = 16) treatment. g Immunostaining for αSMA (red) and DAPI staining (blue) in dorsal views of E12.5 WT (n = 16) and Kcnj13T38C/T38C (n = 8) tracheas after a 48 h DMSO treatment, and Kcnj13T38C/T38C (n = 8) tracheas after a 48 h 0.5 μM A443654 treatment. Quantification of SM cell orientation (h) and NAR (i) of E12.5 WT (n = 8) and Kcnj13T38C/T38C (n = 8) tracheas after a 48 h DMSO treatment, and Kcnj13T38C/T38C (n = 8) tracheas after a 48 h 0.5 μM A443654 treatment. j Phalloidin (green) and DAPI staining (blue) in dorsal views of E12.5 WT (n = 8) and Kcnj13T38C/T38C (n = 8) tracheal SM cells after a 48 h DMSO treatment, and Kcnj13T38C/T38C (n = 8) tracheal SM cells after a 48 h 0.5 μM A443654 treatment. k Quantification of mean phalloidin fluorescence intensity in SM cells (as in j). l A working model for the KCNJ13/AKT pathway in tracheal SM cells. KCNJ13 is expressed in tracheal SM cells to maintain intracellular positive charges at physiological levels, which leads to AKT phosphorylation and actin polymerization, which are essential for SM cell alignment and polarity. When KCNJ13 is inactivated, intracellular positive charge levels increase, leading to AKT hypophosphorylation and actin depolymerization, ultimately resulting in altered SM cell alignment and polarity during tubulogenesis. Scale bars: 50 μm (a), 20 μm (g, j). *P < 0.05; **P < 0.01; ***P < 0.001; unpaired Student’s t-test, mean ± s.d.
