1.1 Preparation and characterization of black carbon and cadmium
The carbon black powders (Printex U and SB4A) are purchased from Degussa Inc. Corp. The carbon black was adjusted to 2 mg/ mL and ultrasonically with 100 Hz, 200 W water bath for 2 h. The morphology of the CB samples was tested with The transmission electron microscope (TEM). To test the hydrated particle size of the CB samples, Zeta-sizer was carried out. Afterwards, X-ray photoelectron spectroscopy (XPS) was tested to identify the surface functional groups.
1.2 Cell line and cell culture
The human lung epithelial cell line (BEAS-2B) was purchased from the American Type Culture Collection (Manassas, VA, USA) and cells were cultured in phenol red-free RPMI-1640 medium (Gibco BRL Life Technologies Inc, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen) in the humidified incubator with 5% CO2 at 37 ˚C, as reported (Li et al. 2019).
1.3 Cytotoxicity assay
Cytotoxicity of CBs (SB4A/ Printex U) was tested according to the manufacturer’s protocol of Cell Counting Kit-8 (CCK-8) (Solarbio Science & Technolo Co., Ltd., Beijing, China). 2,000 BEAS-2B cells were inoculated into 96-well plates, and lung epithelial cells were subjected to different concentration of CBs（10, 20, 20, 100, 200 μg/ml）and Cadmiun (0.1, 0.5, 1, 2, 5 μM) at different concentration for 24 h. The cell viability of cells was determined following the provided reagents as previous reported (Zhu et al. 2020).
1.3 Confocal assay
BEAS-2B cells were seeded into 6-well plates, exposed with CBs at concentration of 20μg/ml, following with/without Cd treatment for 24 h. BEAS-2B cells were then incubated with PI staining for 20 min at room temperature. PI fluorescence of PI was tested with TCS SP5 microscope (excited at 488 nm and visualized at 630 nm). The photographs of lung cells were taken at random fields with (n=4).
1.4 Flow cytometric apoptotic cell death analysis by annexin V FITC- PI staining
BEAS-2B cells (1×105) were stained with anti-annexin V and propidium iodide (PI) antibody, according to the manufacturer’s protocol (KeyGen Biotech, Nanjing, People’s Republic of China). Afterwards, apoptosis ability of BEAS-2B cells were analyzed in the fluorescence-activated cell sorting (BD, Franklin Lakes, USA). Percentage of death cells were corresponded to annexin V+–PI+, and the percentage of apoptotic death cells was shown with annexin V+–PI−.
1.5 Western blot analysis
Various proteins in cells protein concentrations were performed with Western blot analysis as previously described (Wang et al. 2019). Cells were harvested, and then lysed in RIPA lysis buffer for 30 min. After measured with BCA protein assay kit, equal amounts of protein were subjected to SDS-PAGE electrophoresis, followed by Western blotting. Afterwards, proteins were transferred to PVDF membranes (Millipore). The primary antibodies (Abs) and the secondary antibodies were shown in Fig. S1. β-actin was used as a loading control for normalization with software Image J.
1.6 RNA expression analysis by real-time quantitative PCR (RT-qPCR)
BEAS-2B cells were treated with CBs (0, 20, 50 μg/ml) at different concentrations for 24 h. Expression of RNA was tested with Real-time PCR as previously described (Gao et al. 2020). β-actin was used as the invariant control. Results were from experiments in triplicate. All of the primer sequences for PCR analysis are presented in Table S1.
1.7 Animal experiment
BALB/C male mice (6–8 weeks old) were purchased from the Beijing Vital River Laboratory Animal Technology, and housed in aseptic animal facility under specific pathogen-free (SPF grade). BALB/C male mice were divided into 6 groups (5 mice in each group). CBs (2.5 mg/kg) and Cd (10mg/L) were intratracheally instilled into mice with lung exposure after intraperitoneal anesthesia (pentobarbital sodium). For the combination of CBs and Cd, mice were treated with CBs following with Cd after 24 h. Mice were sacrificed 24 h post administration for 4 weeks, and mice were sacrificed 24 h post the last administration. As positive control, lipopolysaccharide (LPS) was intratracheally instilled into the lungs with 2.5 mg/kg body weight. All animal experimental protocols were approved by the Animal Ethics Committee at the Research Center for Eco Environmental Sciences, Chinese Academy of Sciences.
1.8 Histological analysis and immunohistochemistry analysis
Lung tissues were fixed with 10% formaldehyde solution in PBS after sacrificed. After embedded in paraffin, the lung tissue specimens were sliced into sections at thicken of 4μm，following with hematoxylin-eosin (H&E) staining with standard protocols. Briefly, slices were pictured with optical microscope (Axio Scope A1, Carl Zeiss, Inc., Germany). The photographs of fields on each slide were taken at random fields with (n=10).
1.9 Statistical analysis
All data were represented in terms of the mean ± standard deviation (SD). Statistical analysis was performed with independent t-test or One-Way ANOVA test. Values were considered to be statistically significant with *: P < 0.05 and #: P < 0.001.