Fig 2: Identification of the signal adaptor CRKL as an EMT-related interaction partner of SASH1. (A) Immunoblot analysis of parental HCT116 cells transiently transfected with control vector or a vector to express SASH1 with a C-terminal V5 tag. Cells were stimulated with 20 ng/mL TNF or vehicle control for 48 hours. Immunoblot quantifications also are shown (unpaired t test; n = 3 independent experiments; P = .0094). (B) Immunofluorescence microscopy (scale bar: 20 μm) of control or SASH1-V5–transfected cells, either untreated or stimulated with 20 ng/mL TNF for 48 hours. Fixed cells were stained with an anti-V5 antibody (red), anti–E-cadherin antibody (green), and 4′,6-diamidino-2-phenylindole for nuclei (DAPI; blue). (C) To screen for potential interaction partners of SASH1, a yeast 2-hybrid screen was performed with SASH1 as bait protein and a human placental cDNA bank. Shown are protein coding hits of good and high confidence (significance A–C), and the corresponding number of articles from “ and EMT” searches in the PubMed database. In addition, SASH1 with an N-terminal GFP tag was transiently expressed in HEK293 cells and precipitated to analyze co-precipitating proteins via mass spectrometry. Yeast 2-hybrid hits that also were found to interact with SASH1 via co-precipitation/mass spectrometry are indicated. (D) Endogenous CRKL co-precipitates endogenous SASH1 in HCT116 and HEK293 cell lysates, while the isotype control antibody shows no signal. (E) HEK293 cells transiently expressing SASH1 with a C-terminal V5 epitope were stained for immunofluorescence microscopy (scale bar: 10 μm) with an anti-V5 and anti-CRKL antibody. SASH1-V5 and CRKL showed a pronounced co-localization at phase-dense structures in peripheral regions of the cell (arrowheads). (F) Immunofluorescence microscopy (scale bar: 20 μm) of HeLa cells expressing SASH1-V5 deletion constructs with wild-type (WT) or mutated (mut) nuclear localization signals. Cells were fixed and stained with an anti-V5 antibody (red) and DAPI for nuclei (blue). COIP, co-immunoprecipitation; IP, immunoprecipitation; ISO, isotype antibody; Mr (K), molecular range (kilodalton); PARP, Poly(ADP-Ribose)-Polymerase 1; SIGN, significance.

Fig 3: Melanogenic components were induced by SASH1 mutations. (A) IHC analysis demonstrated that in the affected epithelial tissues with Y551D mutation,heterogeneous expression of SASH1 was exhibited. Heterogeneous expression of Pmel17, tyrosinase, Rab27a and TYRP1 in affected epithelial layers was induced by SASH1 Y551D mutation, which caused heterogeneous distribution and more synthesized melanin in different affected layers. Original magnification: ×400 and scale bar: 10 μm. Red arrows denote the representative positive cells of SASH1, Pmel17, TYRP1, tyrosinase and melanin. (B) 5–6 visual fields in each section of affected and normal control individuals were photographed. The staining intensity and percentage of SASH1‐, Pmel17‐, TYRP1‐, tyrosinase‐ and melanin‐positive cells were calculated and scored. Statistical significance of the scores was determined using one‐way anova with LSD correction and represented as the mean ± S.D. Cartograms were plotted using GraphPad Prism 5. ※ denotes P < 0.01. SASH1: SAM and SH3 domain containing 1.

Fig 4: Elevated melanin secretion and increased tyrosinase activity were induced by SASH1 mutations, however, eliminated by silencing MAP2K2 and CREB. (A) Mutated exogenous SASH1 was more induced than wild‐type of exogenous SASH1 by p53 in HEK‐293T cells. 1 μg of HA‐p53 plasmid and 1 μg of GFP‐SASH1 (wild‐type and mutated) were cotransfected into HEK‐293T cells pre‐transfected with HA‐MC1R plasmid. Cells were lysed and subjected into Western blot analysis along with GAPDH as loading control. Relative protein levels of endogenous SASH1 (bottom panels) were determined by Western blot (top panels) in three independent experiments. ※ P < 0.01 compared to 0 μg groups. (B) and (C) SASH1 mutations increased the activity of tyrosinase (B) and resulted in elevated melanin secretion (C) in NHEMs. NHEMs were infected with wild‐type and mutant SASH1 in 10‐cm dish and cultured in FBS‐free M2 medium. At 48 hrs after infection, cells were washed gently and lysed with IP‐WB lysis buffer. The tyrosinase activity analysis and melanin content assay were performed as per the instruction of manufacturer and determined using one‐way anova with LSD correction on SPSS version 16.0 and showed as the mean ± S.D. Cartograms were plotted using GraphPad Prism 5. ※ denotes P < 0.01. # denotes P < 0.05. (D) MAP2K2 was silenced by two specific MAP2K2 siRNAs efficiently as indicated by Western blot of NHEMs. (E) MAP2K2 silencing by its specific siRNAs induced decreased tyrosinase activity in NHEMs transiently transfected with wild‐type and mutant SASH1 genes as well as empty pEGFP‐C3 vector. The tyrosinase activity was determined using one‐way anova with LSD correction on SPSS version 16.0 and showed as the mean ± S.D. ※ denotes P < 0.01. # denotes P < 0.05. (F) MAP2K2 silencing by its specific siRNAs decreased melanin secretion in NHEMs transiently transfected with wild‐type and mutant SASH1 genes as well as empty pEGFP‐C3 vector. Melanin content was determined using one‐way anova with LSD correction and showed as the mean ± S.D. (G) Two specific CREB siRNAs could efficiently down‐regulate CREB expression which was silenced as indicated by Western blot of NHEMs. (H) CREB silencing induced decrease in tyrosinase activity in NHEMs. (I) CREB deletion by its specific siRNAs caused decreased melanin secretion in NHEMs. Tyrosinase activity and melanin content were determined using one‐way anova with LSD correction and showed as the mean ± S.D. ※ denotes P < 0.01. NHEMs: normal human epithelial melanocytes; SASH1: SAM and SH3 domain containing 1.

Fig 5: Mutations in SASH1 increase SASH1 expression in vitro and in vivo. (A) Mutation sites in the SASH1 gene in three families with DUH. (B) Western blotting demonstrated the differential and increased expression of mutant SASH1 proteins compared to that of wild‐type SASH1 in different A375 cells. (C) HE staining, SASH1 and Mitf immunohistochemical analysis of the epidermal tissues from the Y551D‐mutation DUH‐affected individuals and normal controls. Heterogeneous expression of the SASH1 protein was observed in all of the epithelial layers in the epidermal tissues from the Y551D‐mutation DUH‐affected individuals as compared with that of normal controls (NC). Heterogeneous distribution of melanocytes was detected in the epithelial layers of DUH‐affected individuals using the melanocyte marker Mitf as compared with that of normal controls. 40× magnification. Scale bar = 20 μm. Red arrows denote the representative positive cells of SASH1 and Mitf.