Non-transcriptional Hh-responsiveness of the generated iMEFs. A. Example of a migration assay using wild type (Gli3+/+) iMEFs in a Transwell system with varying concentrations of Shh as chemoattractant. Fluorescence was read every two minutes and expressed as relative fluorescence unit (RFU). B. Example of a migration assay using wild type (Gli3+/+), Gli1-/-2-/- and Gli2-/-3-/- iMEFs with 2 μg/ml Shh as chemoattractant. No-attractant condition was subtracted and migration starting points were set to t = 0. Robust migration was observed for each cell line. C. Total migration data from several experiments as performed for B, pooled and expressed as fraction of wild type iMEF migration (Gli3+/+, set to 1, n = 5). To determine whether the migration response was significantly different relative to wild type, a 95% confidence interval was calculated based on the mean and standard deviation of the observations. Reported significant differences thus have a P value of < 0.05.