Fig. 5

Coal dust exposure-induced pulmonary apoptosis and necrosis. A The MLE-12 cells, a commonly used murine cell line expressing some type II alveolar epithelial cell markers, were either treated or not with coal dust (0 μg/mL, 200 μg/mL, 400 μg/mL, and 800 μg/mL) for 24 h. Microscopic fluorescent images were used to detect cell apoptosis or necrosis after annexin V/PI staining. Cells with the dual stain of annexin V/PI appear bright green in early apoptosis and red in late apoptosis, while the living and untreated (control) cells appear in a rare green color under a fluorescent microscope. B The counts of annexin V/PI positive cells (under a 400× magnification) at each dose are shown in the graph.  C Representative image of cleaved caspase 3 (CCP3) after immunofluorescent staining revealed apoptotic green cells after coal dust exposure. The untreated cells showed a dark field with negative CCP3 staining. DAPI was used as a counterstain for the nucleus. Apoptosis induced by coal dust in MLE-12 cells presented nuclear degeneration with condensed and damaged chromatin indicated by the DAPI stain. Coal dust was colocalized with cells under bright fields. The data values are expressed as Mean ± SEM. The data were derived from triplicate assays and two independent experiments. *p < 0.05 or **p < 0.05 compared with the untreated cells. D Heatmap of the apoptosis and necrosis expression of type-II alveolar epithelial cells in Gene Ontology (GO) terms. (E) GSEA plots of the top two differentially expressed regulons between coal dust (n = 963 cells) and PBS lungs (n = 1900 cells). The gene set enrichment analysis (GSEA) revealed that coal dust-exposed lungs highly enriched the ATP synthesis coupled proton of the collagen fibril organization and response to mechanical stimulus modules. The p-value was calculated using a permutation test (one-sided) based on phenotype, showing the statistical significance of the enrichment score