Fig 2: S587R-SASH1 enhanced cellular migration and adhesion abilities, suppressed melanin biosynthesis in PIG1 cells. (A) Wound-healing assays demonstrated 2.04 fold and 1.77 fold increase in S587R-SASH1 transfected PIG1 cells, as compared to WT-SASH1 and S507A-SASH1 cells. (B) Transwell migration assay revealed 2.05 fold and 1.67 fold increase in the number of S587R-SASH1 PIG1 cells that migrated after a 24 h period, as compared to WT-SASH1 and S507A-SASH1 cells. (C) SASH1 mutants obviously reduced the melanin content to 53.1% and 54.6%, as compared to that in WT-SASH1 and S507A-SASH1.

Fig 3: IHC analysis of SASH1 expression in the DUH affected and unaffected individuals. (A) SASH1 protein expression in multiple epithelial layers of a DUH lesion. (B) The normal and homogeneous levels of SASH1 were mainly distributed in the basal and nearby layers of the epidermis of unaffected individuals.

Fig 4: HMGB1 contributes to SASH1 gene methylation.A The Western blotting results showed that, a HMGB1 protein was expressed at a high level in C6 cell total protein compared to cultured astrocyte total protein (upper panel, representative blots of duplicate samples; lower panel, statistical results; n = 4, P = 0.0124). The results of Western blotting after nuclear and cytoplasmic extract isolation showed that, b HMGB1 protein levels in astrocyte nuclei are significantly lower than those in C6 nuclei. (Upper panel, representative blots of duplicate samples. GAPDH was used as the internal control for cytosolic protein, and LaminB1 was used as the internal control for nuclear protein. Lower panel, the statistical results. n = 4, P = 0.0178.). B Schematic diagram of the CpG sites in the 5′ UTR of the SASH1 gene and the DNA sequences occupied by the PCR primers. C Representative results of ChIP analysis. The anti-HMGB1 antibody immunoprecipitated a HMGB1-DNA complex. The DNA region bound by HMGB1 was identified by PCR using SASH1-specific primers (the sequences of primers No. 4 and No. 5 are listed in Table 1), the sites of which are shown in B. The input lanes show the PCR products obtained directly from gDNA of astrocytes and C6 cells, the IgG lanes show mouse IgG-immunoprecipitated controls, and the ddH2O lanes show the blank PCR controls. D HMGB1 overexpression increased the methylation of SASH1 in astrocytes. a Representative images of astrocytes transfected with mCherry-HMGB1 plasmids under bright-field (a1 and a3) and fluorescence (a2 and a4) inverted microscopes and of HMGB1 protein accumulation in nuclei (a4). b Representative PCR results for the SASH1 promoter region using primers (shown in Table 1) specific for methylated (M) and unmethylated (U) gDNA in astrocytes transfected with mCherry or mCherry-HMGB1 constructs. The bar graph (lower panel) shows the M-to-U product ratios. The data are presented as the mean ± SE, n = 3, pmCherry-HMGB1 vs. pmCherry, P = 0.0285. c qRT-PCR results showing that HMGB1 overexpression in astrocytes could significantly decrease SASH1 mRNA levels. P = 0.029

Fig 5: High methylation levels of the SASH1 gene in C6 glioma cells.A Schematic of a CpG island showing the locations of the 17 CpG sites (shown in red text) in the 5′ UTR of the SASH1 gene promoter. B Representative PCR results for the SASH1 promoter region using primers specific for methylated (M) and unmethylated (U) gDNA in astrocytes (AS) and C6 cells. The bar graph (lower panel) shows the M-to-U product ratios. C6 vs. AS, n = 3, P = 0.0194. C Each square indicates a clone of AS or C6 cells. Six clones were subjected to bisulfite sequencing. The clones methylated on individual CpG sites are labeled in black. D A significant increase in the percentage of methylated CpG sites was observed in C6 cells compared to AS cells. P = 0.0002