EP 4 stabilizes ciliary cAMP in SHH-stimulated cells to maintain anterograde IFT and promote SMO ciliary accumulation. (A) cADDis-expressing IMCD3 cells were pretreated overnight with vehicle or L161,982 (10 µM). The following morning, cADDis activity was monitored by live imaging to track the ciliary cAMP response as cells were treated with forskolin (FSK, 100 μM) for 1.5 min prior to the addition of the SMO agonist SAG (1 μM) or vehicle control. Fluorescence ciliary intensity was recorded over 10 min in live cell imaging mode. An average of ∼six cilia were recorded for each condition and the experiment was performed twice. A representative experiment is shown. (B) Anterograde IFT velocity was calculated in IMCD3 cells by tracking IFT88-GFP movement in the presence and absence of SHH, L161,982 (10 µM), or vehicle control. IFT velocity was calculated for 30 cilia per condition across four experiments. Velocity is shown as a violin plot with SD indicated. (C and D) Average ciliary length and SMO ciliary intensity were quantified in IMCD3-bPAC cells exposed to control or SHH-conditioned media in the absence or presence of 10 µM L161,982 in control or blue light-exposed cells. Significance was determined by one-way ANOVA. For all experiments, a P value of <0.05 was considered statistically significant. Significance is denoted as follows: *<0.05, **<0.01, ***<0.001, ****<0.0001, and ns, P > 0.05. Data are represented as mean ± SD. For all ciliary length and SMO ciliary intensity experiments, 50–100 cells per condition were analyzed over at least three independent experiments.