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. 2021 Apr 30;9:634649. doi: 10.3389/fcell.2021.634649

FIGURE 6.

FIGURE 6

pAKT(S473) localizes in close proximity to the PIs at the transition zone. (A) Ciliated hTERT-RPE1 cells were immunostained with pAKT(S473) (green), CEP164 or TCTN1 (red) and ARL13B (grayscale) antibodies and imaged by STED microscopy (confocal resolution image of the ARL13B stained axoneme is shown). Right panels show merged image at lower magnification. Arrows indicate transition zone pAKT(S473) signal, arrow heads indicate CEP164 or TCTN1, bar indicates 1 μm. (B) Graph shows the lateral diameter between the highest intensity points of the pAKT(S473), CEP164 or TCTN1 puncta perpendicular to the plane of the axoneme. Bars represent mean ± SEM, n = 3 independent experiments, ≥30 cilia imaged per experiment and all cilia with two distinct pAKT(S473), CEP164 or TCTN1 puncta measured, statistical significance was determined using one-way ANOVA (p < 0.0001) followed by Tukey’s post hoc test, ***p < 0.001, ****p < 0.0001. (C) Graph shows the axial distance between the highest intensity point of the pAKT(S473) signal and CEP164 or TCTN1 parallel to the plane of the axoneme. Bars represent mean ± SEM, n = 3 independent experiments, ≥ 30 cilia imaged per experiment and all cilia with distinct pAKT(S473), CEP164 or TCTN1 puncta measured, statistical significance was determined using Student’s t-test (p = 0.7284). (D) Representative image showing the method used for the lateral diameter and axial distance measurements, bar indicates 1 μm. (E) Ciliated hTERT-RPE1 cells were immunostained with pAKT(S473) (green), TCTN1 (red) and ARL13B (grayscale) antibodies and imaged by STED microscopy (confocal resolution image of the ARL13B stained axoneme is shown). Arrow indicates ring shaped transition zone pAKT(S473) morphology, bar indicates 1 μm.