Fig. 3

PreMel express HEM-specific proteins. Expression of MITF and TYR was evaluated by Western blot in HEM, A375 cells, ADSC, and PreMel. a In HEM and A375 cells, there is a band at 75 kDa and a doublet at 70 kDa, which represent MITF-A and MITF-M isoforms, respectively. Bands were denominated as I (lower band) or II (upper band) only for quantification. The reported band for N-glycosylated TYR is 80 kDa, but in HEM, multiple bands (*) appear that correspond to less N-glycosylated forms of the enzyme. Vinculin was used as a loading control. b Quantification shows that there is a significant increase in MITF-A levels in PreMel. c–d Each band of the MITF-M doublet was quantified as I and II. e TYR protein levels rise in response to the differentiation induction. f MITF localization in HEM is both nuclear and cytoplasmic. g MITF distributes similarly in PreMel compare to HEM. h ADSC express lower levels of MITF. i Nucleus/cytoplasm ratio of fluorescence intensity of MITF shows that the cellular distribution of MITF is similar between PreMel and HEM. Data are normalized with respect to DAPI intensity. TYR localization is cytoplasmic in j HEM and k PreMel, but not all differentiated cells are positive for TYR. l In ADSC, TYR is not detectable. m Quantification of TYR⁺ HEM, ADSC, and PreMel. A similar pattern is observed for PMEL in n HEM and o PreMel, with a low percentage of positive cells. p There is no expression of PMEL in ADSC. q Quantification of PMEL⁺ HEM, ADSC and PreMel. Scale bar = 100 μm (f–g) and 50 μm (j–p). n = 3, *P < 0.05 one-way ANOVA, compared to ADSC