Fig 2: 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 3: Vanishing white matter (VWM) human astrocyte subtypes differ in morphology and mRNA expression level from control human astrocyte subtypes. (A, B) Differential expression analysis revealed significant differentially expressed genes (DEGs) between VWM and control in human white matter (WM) astrocytes (A; control, n = 3; VWM, n = 4) and human gray matter (GM) astrocytes (B; control, n = 4; VWM, n = 4). The volcano plots indicate significant DEGs in red, with the 15 most significantly up- or downregulated DEGs labeled. (C) Significantly enriched Gene Ontology (GO) terms classified the DEGs in different categories of biological processes. (D, E) Morphology of control (D) and VWM (E) human WM astrocytes was visualized using CD44 and SOX9 immunostaining. (F–H) Morphological analysis showed differences in roundness (F), area (G), and perimeter corrected for area (H) between VWM and control human WM astrocytes based on the CD44 immunostaining. (I) A quantitative polymerase chain reaction on VWM human WM astrocytes (n = 4) showed differential expression of S100B, SLC25A18, MLC1, SOX9, CD44, NESTIN, ALDH1L1, ALDOC, SLC1A2, AQP4, GFAP, and BLBP relative to control human WM astrocytes (n = 4). *p < 0.05. (J) The cleaved caspase 3 (CC3)-positive percentage of the 4,6-diamidino-2-phenylindole (DAPI)-positive cells was determined in oligodendrocyte cultures containing media conditioned by GM or WM astrocytes derived from control (n = 4) and VWM (n = 4) lines. Bars represent mean ± standard error of the mean.

Fig 4: Effects of 8-day olanzapine and 4-PBA treatment on cumulative food intake, weight gain, hypothalamic S100B, GFAP, and TLR4 signaling pathways. (A) Food intake and (B) cumulative weight gain. (C,E) Representative western blot and densitometry analysis of GFAP, S100B, TLR4, MyD88, phospho-p65, MMP-9, phospho-p38, and phospho-ERK1/2 (44 and 42 kd) in the hypothalamus of the olanzapine-, vehicle-, or 4-PBA-treated rats. (D,F) Western blot analysis of protein expression of GFAP, S100B, TLR4, MyD88, phospho-p65, MMP-9, phospho-p38, and phospho-ERK1/2 (42/44 kd) in the hypothalamus of rats. All data were presented as mean ± SEM. Statistical significance was defined as p < 0.05 ($p < 0.05, $$p < 0.01, $$$p < 0.001 vs. control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. OLZ). Con, control; OLZ, olanzapine; OLZ + 4-PBA H, olanzapine + 4-PBA high dose; OLZ + 4-PBA L, olanzapine + 4-PBA low dose; 4-PBA H, 4-PBA high dose.

Fig 5: Spheroid formation and tumorigenicity of MM9H-1 cells and establishment of mucosal melanoma PDX model. (A) Proliferation of MM9H-1 and A375 cells was monitored by xCELLigence RTCA system. (B-D) Sphere-forming assay. (B) Phase contrast image of a sphere from MM9H-1 cells in 96-well plate. (C) HE staining shows that MM9H-1 cells aggregated into regular spheres after 4 days of suspension culture, and the arrow indicates melanin pigmentation in cells. (D) Immunohistochemistry analysis of the sphere shows positive staining for a triple-antibody cocktail against cytoplasmic HMB45, Melan-A, and Tyrosinase (red). Hochest was used to stain nucleus (blue). (E-H) Tumor formation in immunodeficient mice. (E) Tumor growth curves of the subcutaneous tumors formed by the 10th, 50th and 90th passage MM9H-1 cells (n = 3). Tumor volume was analyzed with Student's t test. Image of tumor formation of MM9H-1 cells in the subcutaneous (F, 10th passage cells) and orthotopic regions (G, 90th passage cells). (H) H&E staining of the MM9H-1-xenografted tumor in subcutaneous regions, with vessel formation indicated by the yellow box. (I) Immunohistochemistry analysis of MM9H-1-xenografted tumor shows positive staining for Melan-A, HMB45, S100 and negative for AE1/AE3. (J) Typical gross appearance of liver metastasis in nude mice injected intrasplenically with MM9H-1 cells. (K) HE staining for the liver metastasis. The black arrows indicate the metastatic foci with extensive melanin deposition. Abbreviations: HE, hematoxylin and eosin; HMB45, human melanoma black 45