Figure 6.

(a) A375 cells stably transfected with shRNA CD271 were subcutaneously inoculated in the left dorsal side of nude mice (n=6). For induction of shRNA, 1 mg/ml doxycycline was added to the drinking water. Tumor growth was monitored for 25 days and the tumor volume was calculated as described in the Materials and Methods section. At the end of the study, tumors were excised, digested and viable cells obtained by Ficoll gradient centrifugation were then analyzed either by flow cytometry (b) or by western blot (c). (d) A375 cells stably transfected with shRNA CD271 were subcutaneously inoculated in the left dorsal side of nude mice (n=6). As previously, shRNA was induced by doxycycline and mice were treated with 45 mg/kg daily of vemurafenib. Tumor growth was monitored for 45 days and the final tumor volume was calculated as described in the Materials and Methods section. At the end of the study, tumors were excised, digested and viable cells obtained by Ficoll gradient centrifugation were then analyzed either by flow cytometry (e) or by western blot (f). (g) Immunofluorescent staining with CD271 (red) and 4',6-diamidino-2-phenylindole (DAPI; blue) in matched pairs of melanoma samples before and after relapse under vemurafenib treatment from three different melanoma patients.