Adrenocortical cells in vitro and in vivo lack ARL13B-positive primary cilia. (A) Immunofluorescence of NCI-H295R cells (top) and NIH3T3/Smo-mEos2 cells (bottom) for acetylated tubulin (AcTUB; cyan), ARL13B (magenta), and nuclear DAPI (blue). The cyan arrowhead denotes an AcTUB-positive cilium, while the magenta arrowhead denotes a cilium where ARL13B colocalizes with AcTUB. Scale bar, 10 µm. (B) Quantification of the percentage of ciliated NCI-H295R and NIH3T3/Smo-mEos2 cells, counted as cells positive for AcTUB or ARL13B. The number of counted cells is given under the graph. Data are presented as mean ± SD, n = 6–8 replicates, pooled from three experiments. ***, P < 0.001. (C) Mouse adrenal immunofluorescence of AcTUB (top) and ARL13B (bottom), costained with SF1, a nuclear steroidogenic marker (magenta), and nuclear DAPI (blue). The dashed line represents the approximate border between the adrenal capsule and cortex. Cilia are indicated by an asterisk. Scale bar, 10 µm. (D) Quantification of the percentage of ciliated cells within the adrenal cortex and capsule, counted as cells positive for AcTUB or ARL13B. Each data point represents the quantification for one adrenal gland, presented as mean ± SD. **, P < 0.01. (E) Model representing the SHH-producing cortical and SHH-responding capsule cells in the adrenal gland. Subset of subcapsular cortical cells produce SHH (magenta) and signal to the overlying capsule cells (green), which possess ARL13B-positive primary cilia and respond by Gli1 expression. The cortical cells themselves (magenta and blue) do not respond to autocrine SHH signaling, possibly because of a lack of ARL13B-positive cilia.