A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation (2022)
Figure 5

Figure 5: Ciliary GPCR stimulation phenocopies ciliary bPAC-driven cyst growth.

(A) Ciliary localization of the EP4 receptor in mIMCD-3 cells. Immunocytochemical labeling of mIMCD-3 cells with an anti-EP4 antibody (pink) and an ARL13B antibody (green) to label cilia. DAPI (blue) was used to label the DNA. The pink arrow indicates the direction and the length of the shift of the channel showing the EP4 labeling. The box indicates the position of the magnified view shown on the bottom left. The scale bars are indicated.

(B) Ciliary cAMP dynamics in mIMCD-3 wild-type (WT) cells measured using 5-HT6-mCherry-cADDis. After baseline measurements, cells were stimulated with 50 nM of Prostaglandin E2 (PGE2). The ratio of ciliary mCherry/cpEGFP fluorescence, normalized to the first 30 s of the recording, is shown as mean (points, solid line) ± S.D. (dotted lines) of 25 cilia (biological replicates) from n = 6 independent experiments.

(C) Ciliary cAMP increase after PGE2 stimulation. Each data-point represents one cilium, data is shown as mean normalized ratio ± S.D. of the time points indicated in light green in (B); p value was calculated using a one-sample t-test compared to unity.

(D) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2. Exemplary images are shown (n = 3 biological replicates).

(E) Quantification of images exemplified in (D). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual biological replicates.

(F) Quantification of the cyst area in PGE2-stimulated mIMCD-3 WT cells in the presence of the PDE4 long isoform-specific activator MR-L8. Data were normalized to PGE2- and control-treated cells and are shown as mean ± S.D. p values calculated using a one-sample t-test compared to 100% are indicated, n = 4 biological replicates.

(G) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2, and DMSO (control) or 10 µM of the EP4 inhibitor L161,982. PGE2 treatment started 2 d after seeding. Exemplary images are shown (n = 3). Scale bars are indicated.

(H) Quantification for images exemplified in (G), including addition of 3 µM AH6809, an EP2 inhibitor (images see Fig. EV3C). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated (p value of n/a indicates that the test is not applicable because both conditions showed no variance). Data points show individual experiments and have been normalized to addition of 100 nM PGE2 (set to 100%) within each experiment.

(I) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or the EP4-inhibitor L161,982 in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or L161,982). Exemplary images are shown (n = 3).

(J) Quantification of images exemplified in (I). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments and have been normalized to the light/DMSO condition (Set to 100%) within each experiment.