A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation (2022)
Figure 4

Figure 4: Ciliary cAMP signaling is maintained by isoform-specific PDE4 activity.

(A) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark or during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubated with DMSO (control), 250 µM IBMX, a ubiquitous PDE inhibitor, or 10 µM Rolipram, a PDE4-specific inhibitor. Exemplary images are shown (n = 3). Scale bars are indicated.

(B) Quantification of data exemplified in (A). 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 experiments.

(C) mIMCD-3 cells stably expressing cyto-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO or 10 µM rolipram. Exemplary images are shown (n = 4-5). Scale bars are indicated. A matching 3D culture, incubated with 250 µM IBMX, is shown in Fig. EV2B.

(D) Quantification of data exemplified in (C) and Fig. EV2B (250 µM IBMX). Data are shown as mean ± S.D., p values calculated using an unpaired Student's t-test are indicated. Data points show individual experiments.

(E) Measurements of ciliary cAMP levels using cilia-Pink Flamindo. mIMCD-3 cells expressing cyto-bPAC have been pre-treated with 10 μM rolipram or DMSO (ctrl) for 10 min before the measurement. Ciliary cAMP levels have been determined as the mean ciliary fluorescence intensity during the first 60 s after light stimulation (measurement interval 10 s); p value for a Kolmogorov-Smirnov test is indicated. Each data point represents an individual cilium (31 cilia (biological replicates) from n = 12 independent experiments (DMSO) and 32 cilia (biological replicates) from n = 6 independent experiments (rolipram)). Bars display median and interquartile range.

(F) Analysis of Pde4 isoform expression (long and short isoforms) in wild-type mIMCD-3 cells, revealed by immunoblotting against PDE4A, PDE4B, PDE4D, and PDE4D. The original immunoblots are shown in Appendix Fig. S2.

(G) Immunocytochemical labeling of mIMCD-3 cells with antibodies against PDE4A-D (pink) and an ARL13B antibody (green) to label cilia. DAPI (blue) was used to label the DNA. Pink arrow indicates the direction and the length of the shift of the channel showing the PDE4 labeling. The box indicates the position of the magnified view shown on the bottom of each panel. The image in the box shows only one confocal plane, whereas the whole image is shown as maximal projection. The control shows cells only stained with the secondary antibody.

(H) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO (0 µM), 3 µM, or 10 µM of the Pde4-long-isoform activator MR-L8. Exemplary images are shown (n = 3). Scale bars are indicated. The respective dark controls are shown in Fig. EV2E-F.

(I) Quantification of data exemplified in (H). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data have been normalized to the Light, 0 µM MR-L8 condition (Set to 100%) within each experiment. Data points show individual experiments.