Fig 1: Establishment and characterization of MM9H-1 cell line. (A) Clinical imaging of the patient. The patient was a 67-year-old woman who was diagnosed with mucosal melanoma at T4aN1M0 stage. CT axial view reveals a low-density mass around the lower anterior teeth, and the boundary display is not clear. The arrows indicate the primary foci (red), the location of the cryobiopsy specimens, and the metastatic lymph node (yellow). (B) Histopathology of the patient. HE staining of the biopsy shows abundance of tumor cells with pigmentation. Immunohistochemistry analysis shows positive staining for HMB45 and Melan-A and negative for AE1/AE3. (C) Phase contrast image of cultured mucosal melanoma MM9H-1 cells. The cells exhibit a spindle cell-type appearance, a typical fusiform morphology (the 5th passage). (D) MM9H-1 cells are positive for staining of melanoma-specific biomarkers Melan-A, S100B HMB45, S100A6. (E) Cell suspension from the 10th-passage cells formed a black clump at the bottom of the centrifuge tube after centrifugation. (F) Flow cytometry for the purity of MM9H-1. MM9H-1 cells were filtered with or without anti-melanoma (MCSP) microbeads and then stained with or without MCSP antibody. The purity of MCSP-positive cells is >97%. (G-J) Establishment of PDX model. (G) Growth curves of different PDX passages, which start to increase in size at around 10 days after transplantation (P1, P2, P3 = PDX tumors in first, second and third passages). Tumor volume was analyzed with Student's t test. (H) Representative image of PDX-P1 tumors subcutaneously grow in nude mice. (I) H&E staining of the subcutaneous PDX-P3 tumor, with vessel formation indicated by the yellow box. (J) Immunohistochemistry analysis of PDX-P3 shows positive staining for Melan-A, HMB45, S100 and negative for AE1/AE3. * P < 0.05. Abbreviations: CT, computed tomography; HE, hematoxylin and eosin; HMB45, human melanoma black 45; MCSP, melanoma-associated chondroitin sulfate proteoglycan; PDX, patient-derived tumor xenografts
Fig 2: S100A6 is directly transcriptionally regulated by c-Myc (A, B) The mRNA (A) and protein (B) expressions of c-Myc and S100A6 were determined in HaCaT cells with c-Myc overexpression or knockdown. (C, D) The protein expression levels of c-Myc and S100A6 were determined in HaCaT cells pre-treated with 10058-F4 (20 µM). (E) The predicted binding site of c-Myc in the S100A6 promoter region on the Jaspar database (http://jaspar.genereg.net/)(relative profile score threshold 90%) and scheme of mutation strategies in the S100A6 promoter. WT, wild-type; Mut, mutant. (F) ChIP-qPCR products were visualized by agarose gel electrophoresis. (G) The efficiency of ChIP was calculated as percent input = 2% × 2(CT 2%Input Sample–CT IP Sample). (H) 500ng c-Myc overexpression plasmid or control plasmid was co-transfected with 500 ng plasmids containing S100A6 gene promoter/Firefly luciferase construct and 50 ng Renilla into 293T cells in the NG condition. Firefly and Renilla luciferase activities were measured 36 h after transfection. (I) 293T cells were cultured in the NG or HG condition for 5d and then co-transfected with plasmids containing S100A6 gene promoter/Firefly luciferase construct and 50 ng Renilla. Firefly and Renilla luciferase activities were measured 36 h after transfection. OE-NC, the negative control of c-Myc overexpression. shMyc-NC, the negative control of c-Myc knockdown. Data were shown as the mean ± SEM measured in triplicate from three independent experiments. ANOVA followed by Tukey’s multiple comparisons test were performed to analyze the differences. *P < 0.05, **P < 0.01.
Fig 3: High glucose condition can increase the expression of c-Myc by activating the WNT/ß-catenin pathway, which in turn initiates the transcription of c-Myc target gene S100A6, causing keratinocyte differentiation dysfunction.
Fig 4: S100A6 inhibits HaCaT differentiation (A–C) The expression of S100A6 was determined by immunofluorescence assay in HaCaT cells (A) or wound margin tissues of rats (B) and humans (C). The nuclei were stained blue using DAPI. Magnification: ×200, Scale bar =50 µm. The white dotted line represents the epidermis-dermis dividing line. (D) Morphology of undifferentiated and differentiated HaCaT cells was recorded. Magnification: ×100, Scale bar =25 µm. (E–G) The mRNA and protein expressions of c-Myc and S100A6 and protein expressions of TGM1, LOR and K1 were detected after the differentiation of HaCaT cells. (H) HaCaT cells was pre-treated with S100A6 recombinant protein (1 µM) for 24h and then TGM1, LOR and K1 protein levels were measured. (I, J) The mRNA and protein levels of TGM1, LOR, and K1 were determined after knocking down S100A6 in HaCaT cells. (K) TGM1, LOR, and K1 expression levels were determined in S100A6-knockdown HaCaT cells cultured with high glucose. (L) TGM1, LOR, and K1 expression levels were determined in HaCaT cells overexpressing c-Myc or meanwhile knocking down S100A6. shS100A6-NC, the negative control of S100A6 knockdown. NC, negative control. Data were shown as the mean ± SEM measured in triplicate from three independent experiments. ANOVA followed by Tukey’s multiple comparisons test were performed to analyze the differences. *P < 0.05, **P < 0.01.
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