Table 1.
Candidate qPCR reference gene names and primer sequences.
Table 2.
Desired cell line characteristics for modeling Hh signaling.
Fig 1.
Ciliation time course and cilia length for immortal cell lines.
A) Proportion of ciliated cells at 0, 8, 16, 24, 48, 72, and 96 hours post serum starvation. We performed three replicates for each time point, with error bars denoting the range for each time point across three batches. The numbers of cells counted at each time point and in each batch are listed in the table below the graph. B1 = batch 1, B2 = batch 2, and B3 = batch 3. B) Cilium length across time points. Black bars in each column represent median length. Significant p-values are denoted as follows: 0.01 to 0.05 (*), 0.001 to 0.01 (**), and <0.001 (***). No *’s are shown for differences that are not statistically significant. C) Proportion of ciliated cells without and with stimulation. Each symbol represents the proportion of ciliated cells in one batch. No differences between untreated and treated cells within each cell line were statistically significant using the Mann-Whitney test. D) Cilium length without stimulation and with stimulation. Large symbols = median cilium length within a batch. Small symbols = cilium length of one cilium. One coverslip was imaged for each cell line and time point. The total number of cells counted across three batches are listed below each column of the graphs.
Fig 2.
SMO and GPR161 localization in response to Hh pathway stimulation.
Normalized fluorescence intensity (au) of A) SMO and B) GPR161 with and without pathway stimulation. Large symbol = median normalized fluorescence intensity for individual coverslip data. Small symbol = normalized fluorescence intensity of one cilium. Measurement from three separate batches are indicated by a unique symbol shape and color. Total number of cilia measured listed in the second row below the graph. Representative images are shown below graphs for unstimulated (-SAG) and stimulated (+SAG) cells. Two-tailed Student’s t-test was performed. P-values represented significant differences if they ranged from 0.01 to 0.05 (*), 0.001 to 0.01 (**), and <0.001 (***).
Fig 3.
GLI1 and PTCH1 expression in response to Hh pathway stimulation.
A) GLI1 and B) PTCH1 target gene induction. Each data point represents the expression fold change of stimulated cells from the same batch. Expression fold change was normalized to unstimulated values, which were set to 1. Two-tailed Student’s t-test was performed. P-values represented significant differences if they ranged from 0.01 to 0.05 (*), 0.001 to 0.01 (**), and <0.001 (***).
Table 3.
Overview of results.