lung tissues  (StatLab Medical Products Inc)

Journal: ACS Nano

Article Title: Toward a ToxAtlas of Carbon-Based Nanomaterials: Single-Cell RNA Sequencing Reveals Initiating Cell Circuits in Pulmonary Inflammation

doi: 10.1021/acsnano.5c12054

Figure Lengend Snippet: Cell mapping of the CBN-specific course of pro-inflammatory cytokines. The scoring of the “pro-inflammatory response” pathway exhibited CBN-specific patterns in alveolar epithelial cell types (a), airway epithelial cells (b), macrophage cell types (c) and also mesenchymal cell types (d). (e). Heatmap of BAL cytokine protein level compared to relative mRNA level in defined “inflammatory niche” including alveolar and airway epithelial cells, macrophages, and mesenchymal cells. (f). The UMAPs of CBN-specific cytokines, two identified cytokines were shown for each CBN, the localization of each cytokine in cell types is circled and labeled on the plots. (g). The dotplot of specific cytokine gene induction caused by different CBN in different cell types. (h). UMAP of Cxcl2 and Cxcl5 . Experiments were performed as n = 3 for BAL cytokine measurements, n = 4 for all transcriptomics analysis. One-way ANOVA (nonparametric analysis; Kruskal–Wallis Test) followed by Dunn’s multiple comparisons test was used for statistical analysis.

Article Snippet: Murine epithelial lung tissue cell lines (LA-4; cat. no. ATCC CCL-196; MLE-12, cat no. CRL-2110), murine fibroblast cell line (CCL-206; cat. no. Mlg 2908), and murine macrophage cell line (J774.1; cat. no. TIB-67) were purchased from American Type Culture Collection (ATCC) instructions.

Techniques: Labeling

Journal: ACS Nano

Article Title: Toward a ToxAtlas of Carbon-Based Nanomaterials: Single-Cell RNA Sequencing Reveals Initiating Cell Circuits in Pulmonary Inflammation

doi: 10.1021/acsnano.5c12054

Figure Lengend Snippet: CNP exposure caused neutrophil attraction and formed cellular communications within the local alveolar environment (a). UMAP embedding illustrates cell types and states in the epithelial niche. (b). Treatment-dependent alteration of cell relative frequencies with activation of AT2 (AT2 activated) for CNP and DWCNT and alveolar differentiation intermediates upon MWCNT exposure. (c). Dotplots displaying the top 5 marker genes for each annotated cell type. (d). Heatmap of gene set variation analysis of hallmark signaling pathways on AT2-activated cells. (e). Matrixplot of pro-inflammatory cytokine gene expression caused by different CBN in AT2-activated cells. (f). Csf2 induction by CNP exposure in MLE12 cells after 6 and 24 h measured by qPCR. (g). Cxcl1 induction by CNP exposure in MLE12 cells after 6 and 24 h measured by qPCR. In (f, g), data are shown as mean ± SEM ( n = 3). For each time point, a Student t test was performed between two groups. P value was shown and P value <0.05 was considered statistically significant. (h). BAL CXCL1 protein level measured by ELISA. (i). The CXCL1 release into MLE-12 cell supernatant exposed to different CBN after 24 h. (j). Mouse primary epithelial cells (EpCAM+ cells) isolation and CXCL1 release into supernatants induced by different CBN. (k). Connectomes based on induced differential gene expression (DGE) analysis (treatment vs sham) show computationally inferred cellular communication strength in response to CNP. For each circle plot, edge weight and color represent the number of ligand–receptor pairs between interacting cell types. (l). The visualization of NicheNet analysis by circosplot illustrates the connectome of the situation before in (j) described prevailing interaction between different epithelial cells upon CNP exposure. The lower part of the circosplot is identified as “sender” cell types whereas the upper part draws the “receiver” cell types, signaling interaction by different genes were shown. For (h–j), data are shown as the mean ± SEM ( n = 3), one-way ANOVA followed by Dunn’s multiple comparisons test was used for statistical analysis.

Article Snippet: Murine epithelial lung tissue cell lines (LA-4; cat. no. ATCC CCL-196; MLE-12, cat no. CRL-2110), murine fibroblast cell line (CCL-206; cat. no. Mlg 2908), and murine macrophage cell line (J774.1; cat. no. TIB-67) were purchased from American Type Culture Collection (ATCC) instructions.

Techniques: Activation Assay, Marker, Protein-Protein interactions, Gene Expression, Enzyme-linked Immunosorbent Assay, Isolation

Journal: ACS Nano

Article Title: Toward a ToxAtlas of Carbon-Based Nanomaterials: Single-Cell RNA Sequencing Reveals Initiating Cell Circuits in Pulmonary Inflammation

doi: 10.1021/acsnano.5c12054

Figure Lengend Snippet: CNT exposure causes acute epithelial damage and DAMP release (a). BAL total protein levels detected after CBN treatment as a measurement for epithelial damage. (b). TUNEL assay of the DNA damage caused by different CBN. DAPI: blue, Phalloidin 488: green, TUNEL: red. Scale bar: 20 μm. (c). The quantification of TUNEL-positive events in mouse lung tissue at 12 h ( n = 3). (d). Double staining of TUNEL-positive cells in AT1 and AT2 cells marked by AQP5 and pro-SPC. DAPI: blue, AQP5 or pro-SPC: green, TUNEL: red. Scale bar: 20 μm. The quantification of TUNEL & APQ5 double positive events (e) and TUNEL & APQ5 double positive events (f) in mouse lung tissue at 12 h. (g). UMAP displays density and distribution of cells showing a high DAMP score in epithelial niche. (h). A matrixplot of DAMP-related gene expression caused by different CBN in AT2-activated cells. (i). IL-33 release caused by CBN into BAL fluid measured by ELISA. (j). IL-33 release into the supernatant of mouse AT2-like cells LA4 caused by CBN measured by ELISA. (k). Double staining of TUNEL-positive cells in AMs marked by CD11c. DAPI: blue, CD11c: green, TUNEL: red. Scale bar: 20 μm. (l). The quantification of TUNEL & CD11c double positive events in mouse lung tissue at 12 h. (m). IL1α release caused by CBN into BAL fluid measured by ELISA. (n). The visualization of Il1a expression and localization by UMAP. (o). IL1α release upon CBN exposure in isolated mouse primary AMs. The expression of Ccl2 (p) and Ccl3 (q) induced by CBN exposure in AM-like J774.1 cells. CNP (50 μg/mL), DWCNT (50 μg/mL), and MWCNT (30 μg/mL) were used in the in vitro study. Data were shown as the mean ± SEM ( n = 3 or 4), one-way ANOVA followed by Dunn’s multiple comparisons test was used for statistical analysis.

Article Snippet: Murine epithelial lung tissue cell lines (LA-4; cat. no. ATCC CCL-196; MLE-12, cat no. CRL-2110), murine fibroblast cell line (CCL-206; cat. no. Mlg 2908), and murine macrophage cell line (J774.1; cat. no. TIB-67) were purchased from American Type Culture Collection (ATCC) instructions.

Techniques: TUNEL Assay, Double Staining, Gene Expression, Enzyme-linked Immunosorbent Assay, Expressing, Isolation, In Vitro