Membrane-associated SHH on adrenocortical carcinoma cells signals to adjacent fibroblasts. (A) Schematic representation of the experiment. Mouse fibroblasts were co-cultured adjacent to NCI-H295R cells. In B and C, Shh-LIGHT2 were used for measuring luciferase activity as a readout of Gli1 transcription, while in D and E, NIH3T3/Smo-mEos2 were used for direct visualization of SMO enriched in primary cilia. (B) Shh-LIGHT2 cells were co-cultured in direct contact with NCI-H295R, in the presence or absence of lipoproteins, and treated with 10 µg/ml 5E1, 10 µM cyclopamine, or the appropriate controls. Data are presented as mean ± SD, n = 12–24 replicates, pooled from 3–6 experiments. *, P < 0.05; ****, P < 0.0001. (C) Shh-LIGHT2 and NCI-H295R cells were co-cultured either in direct contact or in wall-separated culture inserts, where they share the same culture medium. SAG treatment was used as a positive control for Shh-LIGHT2 activity. Data are presented as mean ± SD, n = 5–7 replicates, pooled from three experiments. **, P < 0.01. (D) Immunofluorescence of 40,000 NIH3T3/Smo-mEos2 cells, treated either with 200 nM SAG or co-cultured with 40,000 NCI-H295R cells, labeled for ARL13B–cilia (magenta), Smo-mEos2 (green), StAR, a steroidogenic marker present in mitochondria of NCI-H295R cells (red), and nuclear DAPI (blue). ARL13B-positive cilia are denoted with magenta arrowheads. Ciliary SMO enrichment is indicated with an asterisk. Scale bar, 10 µm. (E) Quantification of the percentage of NIH3T3/Smo-mEos2 cells with ciliary SMO enrichment. The number of cells and cilia counted is given under each experimental condition. Data are presented as mean ± SD, n = 4–6 replicates, pooled from two experiments. *, P < 0.05; **, P < 0.01.