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. 2015 Jul 6;112(29):9034–9039. doi: 10.1073/pnas.1501032112

Fig. S3.

Fig. S3.

Neurofibromin depletion with siRNA (siNF1_8; siNF1_15) in mel-WT cells. (A) qRT-PCR analysis of NF1 transcript expression in mel-WT-1 and mel-WT-2 cells transfected with either siRNA targeting different domains of NF1 (siNF1_8 and siNF1_15) or siCtrl. qRT-PCR results are presented as fold change relative to siCtrl-mel-WT cells after normalization against housekeeping gene 18S. Results are expressed as mean ± SD. (B) Western blot analysis of neurofibromin expression in mel-WT-1 and mel-WT-2 cells transfected with siNF1_8, siNF1_15 or siCtrl. α-actinin served as a loading control. (C) Melanin content in siCtrl-mel-WT-1 and WT-2 and mel-WT-1 and WT-2 cells transfected with siNF1_8 and siNF1_15. Measurements were performed using 105 cells of each cell type. Data are presented as mean ± SD normalized to the expression in siCtrl-mel-WT-1 cells. (D) Western blot analysis of tyrosinase expression in mel-WT-1 and WT-2 cells transfected with in siNF1_8, siNF1_15 and siCtrl-mel-WT-1 and WT-2 cells. β-actin served as a loading control. (E) Representative EM images of melanosome maturation in mel-WT cells transfected with siNF1 (siNF1_7) and siCtrl-mel-WT. Characteristic immature (stage I/II) and mature (stage III/IV) melanosomes are observed in the soma of melanocytes. (Scale bar: 1 µm.) (F) Quantification of melanosome maturation in siNF1-mel-WT and siCtrl-mel-WT cell lines. Data are presented as mean ± SD (n = 100 stages of melanosomes). (G) Western blot analysis of Phospho-p44/42 ERK1/2 in siCtrl-mel-WT-1 and WT-2 and mel-WT-1 and WT-2 cells transfected with siNF1_8 and siNF1_15. Total ERK served as a control. (H) Direct ELISA analysis of cAMP content in siCtrl-mel-WT-1 and Wt-2 and siNF1_8 and siNF1_15-mel-WT-1 and WT-2 cells. Measurements were performed using 105 cells of each cell type. Results were normalized to siCtrl-mel-WT-1 and are expressed as mean ± SD.