A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation (2022)
Figure 3

Figure 3: Ciliary cAMP signaling controls cell proliferation via mTOR signaling.

(A-C) Analysis of Ki67 expression in wild-type (WT) mIMCD-3 and cilia-bPAC cells, kept in the dark or stimulated with light (1 h light/1 h dark, 72 h, 465 nm, 38.8 µW/cm²). (A) Cells were labeled with DAPI (blue) to label the DNA and a Ki-67 antibody (look-up table indicated). The DAPI channel was used as a mask for the Ki67 signal. Arrows indicate cells considered as high-Ki67 cells in the quantification shown in (B). Scale bars are indicated. (B) Quantification of high Ki67 expressing cells. The fraction of Ki67 high cells was calculated in wild-type (WT), cilia-bPAC, and cyto-bPAC mIMCD-3 cells, kept in the dark or after light stimulation and the fold change compared to WT dark was calculated. Data are shown as mean ± S.D., n = 9, 134 to 5509 (median: 702) cells were analyzed per condition and experiment, each datapoint corresponds to an individual experiment; p values were calculated using an unpaired, two-sided Student's t-test. (C) Immunocytochemistry of mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during continuous exposure to light for 3 days (top) or 9 days (bottom) (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, with an ARL13B antibody (magenta) to label cilia, and a Ki-67 antibody (shown on right, look-up-table indicated). Arrows indicate Ki67-high cells.

(D) Immunoblotting of lysates from mIMCD-3 wild-type (WT) cells and mIMCD-3 cells stably expressing cilia-bPAC for S6 ribosomal protein (S6RP) phosphorylation at Ser235/236 (pS6RP). Levels of total S6RP protein (S6RP) and beta-Tubulin have been determined as controls. Cilia-bPAC cells were treated with DMSO (control) or with 10 nM of Rapamycin. All cells were stimulated with light (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²).

(E) Quantification of the pS6RP/S6RP ratio. Data are shown as mean ± S.D., n > 3; p values were calculated using a paired, two-sided Student's t-test. Each data point shows an independent experiment.

(F) Quantification of high Ki67 expressing cells. The fraction of Ki67 high cells was calculated in wild-type (WT), cilia-bPAC, and cyto-bPAC mIMCD-3 cells after light stimulation and the presence or absence of 10 nM rapamycin, and the fold change compared to cilia-bPAC dark was calculated. Data are shown as mean ± S.D., n = 9, 134 to 16781 (median: 1309) cells were analyzed per condition and experiment, each datapoint corresponds to an individual experiment; p values were calculated using an unpaired, two-sided Student's t-test. Data for cilia-bPAC in the dark and after light stimulation (both without rapamycin) has been taken from (B) but normalized as indicated here.

(G) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubation with 10 nM rapamycin. Light exposure started 2 d later than incubation with 10 nM rapamycin. Exemplary images are shown (n = 3).

(H) Quantification of the data exemplified in (G). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using a paired, two-sided Student's t-test are indicated.