Journal: The American journal of pathology

Article Title: The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critical for Corneal Endothelial Cell Protection and Graft Survival after Transplantation.

doi: 10.1016/j.ajpath.2021.10.016

Figure Lengend Snippet: Figure 1 Corneal endothelial cells (CEnC) express high levels of the receptor for a-MSH. A: Immunostaining for a-MSH receptor, melanocortin receptor 1 (MC1R), on murine (mCEnC) and human (hCEnC) CEnC lines and primary CEnCs. Blue: DAPI, purple: MC1R. B and C: Representative Western blot bands of MC1R in murine and human CEnC lines (B) and in primary murine corneal endothelium lysates (C). SIT lysate: small intestine tissue lysate used as positive control for mouse MC1R (mMC1R). A-375: human skin epithelial cell lysate used as positive control for human MC1R (hMC1R). Scale bars Z 50 mm.

Article Snippet: The mouse small intestine tissue (SIT) lysate (No. PK-AB718-1408; PromoCell, Heidelberg, Germany) and A375 human skin epithelial cell lysate (No. sc-3811; Santa Cruz Biotechnology, Dallas, TX) were used as positive controls for mouse and human MC1R, respectively.

Techniques: Immunostaining, Western Blot, Positive Control

Journal: Biosensors

Article Title: Malignant Melanoma-Derived Exosomes Induce Endothelial Damage and Glial Activation on a Human BBB Chip Model

doi: 10.3390/bios12020089

Figure Lengend Snippet: Exosomes derived from A375 cells were more easily absorbed by human brain microvascular endothelial cells. ( A ) Schematic diagram of exosomes purification from tumor cells by differential centrifugation. ( B ) Western blotting showing protein level of exosomal markers (CD63, CD81) in 4 types of tumor cell-derived exosomes ( n = 3). ( C ) Representative TEM images of 4 types of tumor cell-derived exosomes ( n = 2). ( D ) Representative confocal images of hCMEC/D3 cells 24 h after incubation with exosomes (2 μg/mL and 10 μg/mL) derived from 4 different types of tumor cells ( n = 4). Exosomes were labeled by PKH67 (green) fluorescent dye. ( E ) Quantification of PKH67 fluorescence intensity for each group based on ( D ) ( n = 4). Data are presented as mean ± SEM, and are analyzed using a one-way ANOVA with Bonferroni post-test (***: p < 0.001).

Article Snippet: A375 cells (iCell, #A375) were cultured in DMEM medium (Gibco, #C11995500BT) supplemented with 10% FBS and 1% P/S.

Techniques: Derivative Assay, Purification, Centrifugation, Western Blot, Incubation, Labeling, Fluorescence

Journal: Biosensors

Article Title: Malignant Melanoma-Derived Exosomes Induce Endothelial Damage and Glial Activation on a Human BBB Chip Model

doi: 10.3390/bios12020089

Figure Lengend Snippet: A375 cell-derived exosomes caused BBB damage and glial activation. ( A ) Side views of the BBB chip 24 h later following exposure of A375 cell-derived exosomes (green) to the vascular side ( n = 3). ( B ) Representative confocal images of glial cells 24 h later following exposure of A375 cell-derived exosomes labeled by PKH67 dye to the vascular side ( n = 3). ( C ) BBB permeability assay of 10 kDa FITC-dextran following exposure of A375 cell-derived exosomes to the vascular side ( n = 4). Data are presented as mean ± SEM, and are analyzed using unpaired Student’s t -test (*: p < 0.05). ( D ) Representative confocal images of brain endothelial cells (VE-cadherin) 72 h later following treatment of A375 cell-derived exosomes ( n = 3). The areas indicated by the white boxes were enlarged on the right. ( E ) Western blotting showing the protein level of VE-cadherin and ZO-1 in brain endothelial cells 72 h later following treatment of A375 cell-derived exosomes ( n = 3). GAPDH was used as an internal control. ( F , G ) Representative confocal images of astrocytes (GFAP; F ) and microglia (IBA1; G ) 72 h later following treatment of A375 cell-derived exosomes ( n = 3). ( H ) Bar graph showing IL-6 concentration in culture supernatant of vascular channel 72 h later following exposure of A375 or A549 cell-derived exosomes to the vascular side detected by ELISA kit ( n = 3). ( I ) Bar graph showing IL-6 concentration in culture supernatant of glial channel 72 h later following exposure of A375 or A549 cell-derived exosomes to the vascular side detected by Elisa kit ( n = 3). ( H , I ) Data are presented as mean ± SEM, and are analyzed using a one-way ANOVA with Bonferroni post-test (**: p < 0.01; ***: p < 0.001). ( J ) Representative confocal images of brain endothelial cells and glial cells stained with PI dye 72 h later following treatment of A375 cell-derived exosomes. Cells positive for PI were indicated by white arrowheads ( n = 4). ( K ) Bar graph showing the percentage of PI + cells for each group based on ( J ) ( n = 4). Data are presented as mean ± SEM, and are analyzed using unpaired Student’s t -test (***: p < 0.001).

Article Snippet: A375 cells (iCell, #A375) were cultured in DMEM medium (Gibco, #C11995500BT) supplemented with 10% FBS and 1% P/S.

Techniques: Derivative Assay, Activation Assay, Labeling, Permeability, Western Blot, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Biosensors

Article Title: Malignant Melanoma-Derived Exosomes Induce Endothelial Damage and Glial Activation on a Human BBB Chip Model

doi: 10.3390/bios12020089

Figure Lengend Snippet: RNA-seq analysis of brain endothelial cells and glial cells following treatment of A375 cell-derived exosomes on the BBB chip. ( A ) Heat-map showing the transcriptional changes of brain endothelial cells and glial cells on BBB chip 3 days after treatment of A375 cell-derived exosomes ( n = 3). ( B , C ) Volcano plots showed the modulated genes in brain endothelial cells ( B ) or glial cells ( C ) 3 days after treatment of A375 cell-derived exosomes. Genes differentially expressed with fold-change >1.5 and p < 0.05 were marked in color. p -values were calculated using a two-sided, unpaired Student’s t-test with equal variance assumed. ( D ) Venn diagrams depicted the down-regulated DEGs shared or unique between brain endothelial cells and glial cells. ( E ) Venn diagrams depicted the up-regulated DEGs shared or unique between brain endothelial cells and glial cells. ( F , G ) Dotplot showed the enriched GO terms based on up-regulated ( F ) or down-regulated ( G ) genes in brain endothelial cells following treatment of A375 cell-derived exosomes. ( H , I ) Dotplot showed the enriched GO terms based on up-regulated ( H ) or down-regulated ( I ) genes in glial cells following treatment of A375 cell-derived exosomes. ( F – I ) The color of the dots represents the rich factor, and the size represents the input number for each GO term.

Article Snippet: A375 cells (iCell, #A375) were cultured in DMEM medium (Gibco, #C11995500BT) supplemented with 10% FBS and 1% P/S.

Techniques: RNA Sequencing, Derivative Assay