To investigate how itraconazole affects the Hedgehog (Hh) signaling pathway in melanoma cells, we analyzed key proteins involved in this pathway, including SHH, PTCH1, SMO, and the transcription factors Gli1, Gli2, and Gli3 (Moscat et al., 2016) in A375 and A2058 cell lines which were selected due to their robust response to Itraconazole and their relevance to the in vivo studies. This evidence concerns the gene GLI3 and melanoma.