Fig. 2

SOX10 activates the transcription of FOXD3 by direct binding to FOXD3 promoter. a HEK293T cells were co-transfected with 500 ng pGL3-FOXD3 (or pGL3-Basic as negative control), 50 ng pRL-TK and an increasing amount of pLentipuro/TO/HA-SOX10 plasmids. After 48 h, cells were lysed and dual-lucfiferase assays were performed. Average relative luciferase activities from three experiments are shown. The expression of SOX10 was verified by western blot. Error bars represent standard deviation. Significance was determined by ANOVA one-way test, ***p < 0.001. b A schematic illustration of FOXD3 promoter region. The positions and sequences of three putative SOX10 binding sites were highlighted. The +1 arrow designated the transcription initiation site. The mutated SOX10 binding sites and the consensus motif are shown. Mutated nucleotides were underlined. c HEK293T cells were co-transfected with 500 ng pGL3-FOXD3 promoter constructs carrying either WT sequence or mutations in either of the three putative SOX10 binding sites, 50 ng pRL-TK and 500 ng pLentipuro/TO/HA-SOX10 for 48 h. Cells were then lysed for dual-luciferase assay. Average relative luciferase activities from three experiments are shown. Error bars represent standard deviation. Significance was determined by ANOVA one-way test, ***p < 0.001. d Sequence alignment of SOX10 binding site #3 in FOXD3 promoters from different species. The SOX10 binding sites were in bold. e A375-TR HA-SOX10 and 1205Lu-TR HA-SOX10 cells were treated with 2 μM Vemurafenib for 6 h. Occupancy of SOX10 (HA) on a region surrounding site #3 in FOXD3 promoter and a region between the GAPDH and CNAP1 genes (negative control) was evaluated by ChIP analysis. Average results from three independent experiments are shown. Error bars represent standard deviation. Significance was determined by ANOVA one-way test, *p < 0.05; **p < 0.01; ***p < 0.001. f Oligo pull-down assays were performed using nuclear extracts from A375 cells treated with or without 2 μM Vemurafenib for 6 h and biotin-labeled FOXD3 promoter fragments containing WT or mutated SOX10 binding site #3. Non-biotinylated DNA fragments (NC) were used as a negative control. The nucleotide sequences of promoter fragments are shown on the right. SOX10 binding sites were underlined and mutated nucleotides were highlighted in bold. Uncropped images are shown in Supplementary Fig. 10