Table 1.
Sequences of primers used in quantitative RT-PCR.
Fig 1.
The effect of PM2.5 concentrations on cell viability in 16HBE cells.
Cells were exposed to various concentrations of PM2.5 (1.56, 7.81, 15.63, 31.25 and 62.50 μg/cm2 (5, 25, 50, 100 and 200 μg/mL) for 24 hours. Cell viability was expressed as a percentage of unexposed control (mean ± S.D.). (*) indicates significant difference (p < 0.05) when compared to control, (**) represents significant difference (p < 0.01) compared to control.
Fig 2.
PM2.5 induces ROS generation in 16HBE cells.
The cells were incubated with 5 μM DCFH-DA and then treated with PM2.5 at the concentrations of 25 μg/cm2. ROS production was monitored with the fluorescence intensity by microscopic analysis. A. Changes in intracellular ROS levels before and after PM2.5 exposure. Time 0 means the start of a measurement. The arrow indicates the time at which PM2.5 at the concentration of 25 μg/cm2 was added to culture media. The three lines show the DCF fluorescence intensity obtained from three arbitrary single cells in the same field of the microscope. B. Fluorescent localization of ROS. The time labeled on the time series photographs corresponds to the time point in Fig 2A. While the first image was recorded just before the start of PM2.5 exposure (as control), the second and third ones were recorded at 10 and 60 minutes after exposure. Scale bar in graphs, 20 μm.
Fig 3.
Analysis of differentially expressed genes between PM2.5 treated and untreated samples.
A. Gene expression level of control versus PM2.5 treated. FDR<0.001 and the absolute value of log2 fold change ≥ 1 were used as the threshold to judge the significance of gene expression difference. B. Summary of the numbers of differentially expressed genes in the PM2.5 treated sample versus untreated control.
Table 2.
Top 10 up-regulated genes in PM2.5 treated 16HBE cells.
Table 3.
Top 10 down-regulated genes in PM2.5 treated 16HBE cells.
Fig 4.
Gene Ontology (GO) classification of genes differentially expressed between PM2.5 treated and untreated samples.
The functions of genes identified cover three main categories: biological process, cellular component, and molecular function. The left and right y axes indicate the percentage and the number of genes in a category respectively.
Fig 5.
The top 10 biological processes identified by GO analysis of differentially expressed genes.
Bars represent-log(P-value). P-value was calculated by hypergeometric test.
Table 4.
The significant pathways for the DEGs.
The arrows ↑ and ↓ indicate up- or down-regulation.
Fig 6.
Correlation between RNA-seq data and qRT-PCR data.
The fold changes were calculated using the 2-△△Ct method comparing PM2.5 treated cell to control untreated samples. All Ct values were normalized to GAPDH.
Fig 7.
The fold changes in CYP1A1, IL-8 and IL-6 expression between control and PM2.5 treated 16HBE cells confirmed by western blot analysis.
A-C. The expression of CYP1A1, IL-8 and IL-6, respectively, detected by western blot analysis of cell lysates. In each gel image the left lane (1) contained untreated control cells and the right lane (2) contained cells treated with 25 μg/cm2 PM2.5. GAPDH was used as a loading control. D. Quantitation of western blot band intensity (n = 3). Data is expressed as mean ± SD. (*) indicates significant difference (p < 0.05) when compared to control, (**) represents significant difference (p < 0.01) compared to control.
Fig 8.
Cytokine release in 16HBE cells exposed to various concentrations of PM2.5.
16HBE cells were exposed to PM2.5 for 5 hours and 24 hours, whereafter the culture supernatants were collected and assessed for (A) IL-6, (B) IL-8 and (C) TNF-α. The data are mean ± SD of independent experiments (n = 3). (*) indicates significant difference (p < 0.05) when compared to control cells (exposed to 0 μg/cm2 PM2.5 over the same period), (**) represents significant difference (p < 0.01) compared to control.