Preparation and treatment of PM2.5
The A549 cell line, was maintained in complete DMEM medium (Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). A549 cells were cultured at 37℃ in a humidified 5% CO2 atmosphere and passaged at confluent condition. The powder of PM2.5 was prepared from standard reference material 1649b (SRM 1649b) which were composed of urban dust purchased from National Institute of Standards and Technology (NIST, Gaithersburg, USA). A total of 50 mg PMs were dispersed in 1 mL dimethyl sulfoxide (DMSO, purity ≥ 99.0%, Sangon Biotech, Shanghai, China), shaken, and ultra-sonicated for 24 h in an ultrasonic bath. Afterwards, 2 μL stock solutions were dispersed in 1 mL ultrapure water for particle-size analysis using a Zetasizer Nano ZS90 zeta Potentiometer (Malvern Panalytical, Malvern, UK), and a stock solution at a concentration of 50 mg/mL were stored at 25°C in the dark. Before the preparation of working solution, ultrasonic vibration was performed for 2 h to ensure that it is a uniformly dispersed suspension. Working solutions at graded concentrations (25, 50 and 100 μg/mL) were prepared by diluting the stock solution with complete DMEM medium. Then, A549 cells were treated with working solution at graded concentrations (25, 50 and 100 μg/mL) and vehicle (0.2% DMSO) for 24 and 48 h. A549 cells without any treatment were used as the control. After treatment, A549 cells were adopted for further studies.
Cell Viability Assay
The cytotoxic effect of PM2.5 on A549 cells was evaluated by MTT assay. Briefly, A549 cells were seeded at a density of 1 × 104 cells per well into a 96-well microtiter plate and cultured for 24 h, allowing cells to adhere to the bottom of 96-well microtiter plate. After A549 cells were treated with 25-100 μg/mL PM2.5 and 0.2% DMSO for 24 and 48 h, 1 g/L 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sangon Biotech, Shanghai, China) working solution was added to replace the growth medium, and the cultures were further incubated in dark at 37℃ for 4 h. Afterwards, the supernatant was removed and 200 μL DMSO was added to dissolve the formed formazan crystals. The optical density (OD) was recorded at an absorbance wavelength of 490 nm using a SpectraMax M5 fluorescence reader (Molecular Devices, Sunnyvale, USA).
Cell Cycle Analysis
Cell cycle was detected by flow cytometry using Cell Cycle Analysis Kit (Beyotime, Shanghai, China) as manufacturer’s protocol. Briefly, after exposure to 25-100 μg/mL PM2.5 and 0.2% DMSO for 24 and 48 h, A549 cells were trypsinized and washed twice with PBS. Afterwards, the treated cells were fixed by 1 mL 70% pre-cooled ethanol overnight at 4°C and subsequently incubated with 500 μL propidium iodide (PI) working solution (20 μg/mL PI and 10 μg/mL RNase) for 30 min at 37°C in the dark. Finally, the distribution in the cell cycle was detected using CytoFLEX flow cytometer (Beckman Coulter, Brea, USA) and analyzed by FlowJo software. Cell cycle was evaluated according to the distribution of DNA content and divided into sub-G1, G1, S and G2/M phases.
Senescence-associated β-galactosidase optical staining
Senescence-associated β-galactosidase (SA-β-gal) activity was detected using Senescence β-Galactosidase Staining Kit (Beyotime, Shanghai, China) as manufacturer’s protocol. Briefly, A549 cells were seeded at a density of 1 × 105 cells per well in a 12-well microtiter plate and exposed to 25-100 μg/mL PM2.5 and 0.2% DMSO for 24 and 48 h. Then, the treated cells were incubated with fixative working solution for 15 min at 25°C. Afterwards, A549 cells were stained with β-galactosidase staining solution overnight at 37°C under the condition of CO2-free. Images were taken in five random fields using optical microscope and analyzed with Image J software.
Senescence-associated β-galactosidase fluorescent staining
Senescence-associated β-galactosidase (SA-β-gal) activity was detected using CellEventTM Senescence Green Detection Kit (ThermoFisher Scientific, Waltham, USA) as manufacturer’s protocol. Briefly, A549 cells were cultured on microscope cover glass in a 6-well microtiter plate for 24 h and treated with 25-100 μg/mL PM2.5 and 0.2% DMSO for 24 and 48 h. Then, the treated cells were fixed with 4% paraformaldehyde for 30 min at 25°C. After rinsed three times with 1% bovine serum albumin (BSA) in PBS, cells were incubated in dark with β-galactosidase staining solution under the condition of CO2-free. Afterwards, cells were incubated with 1 μg/mL Hoechst 33342 nuclear stain (Molecular Probes, Waltham, USA) for 8 min. Finally, the microscope cover glass were mounted at glass slides and analyzed by LSM710 laser scanning confocal microscope (Carl Zeiss, Oberkohen, Germany). At least 1000 cells were scored, and the fluorescence intensity in A549 cells with β-galactosidase foci per 1000 cells were quantitatively analyzed based on each independent experiment.
Western blot assay
The protein expression of γ-H2AX, cGAS, STING, phospho-NF-κB (p-p65), TNF-α, IL-6, IL-8, p16 and p21 was determined by Western blot. Briefly, after A549 cells were treated with PM2.5 in 60-mm dishes, total proteins were harvested by lysing in ice-cold RIPA lysis buffer containing 1 mM PMSF (Beyotime, Shanghai, China). The concentration of total proteins was measured by Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, USA). Equal amounts of proteins were separated by 10% SDS-PAGE gel and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat milk or BSA in TBST for 90 min at 25°C, the PVDF membrane was overnight incubated by employing primary antibodies of γ-H2AX (1:2000, Millipore, Billerica, USA), cGAS (1:1000, Cell Signaling Technology, Danvers, USA), STING (1:1000, Cell Signaling Technology, Danvers, USA), p-p65 (1:5000, Cell Signaling Technology, Danvers, USA), TNF-α (1:2000, Abcam, Cambridge, UK), IL-6 (1:1000, Abcam, Cambridge, UK), IL-8 (1:1000, Abcam, Cambridge, UK), p16 (1:2000, Cell Signaling Technology, Danvers, USA), p21 (1:2000, Cell Signaling Technology, Danvers, USA) and β-actin (1:2000, TransGen, Beijing, China) at 4°C. Afterwards, the PVDF membrane was subsequently incubated with HRP-conjugated secondary antibodies (1:5000, TransGen, Beijing, China) for 1 h at 25°C. β-actin was used as the internal control. The images of protein bands were visualized using an enhanced chemiluminescence (ECL) detection system. Densitometric analysis of the images was performed with Image J software.
The activation of cGAS was assessed by immunofluorescent assay. Briefly, A549 cells were cultured on microscope cover glass in a 6-well microtiter plate for 24 h and treated with 100 μg/mL PM2.5 and 0.2% DMSO for 48 h. After fixing with 4% paraformaldehyde for 30 min at 25°C and permeabilizing with 0.5% Triton X-100 in PBS for 30 min at 25°C, the treated cells were blocked with 5% BSA in 0.1% Triton X-100 for another 1 h at 25°C, followed by employing primary antibody of γ-H2AX (1:1000, Novus Biologicals, Littleton, New Zealand) for 2 h at 25°C. Afterwards, cells were blocked with 5% non-fat milk in 0.1% Triton X-100 for 1 h at 25°C before incubating by employing primary antibody of cGAS (1:1000, Cell Signaling Technology, Danvers, USA) overnight at 4℃. Finally, after incubating by employing secondary antibody of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:2000, TransGen, Beijing, China) and Alexa Fluor 555-conjugated donkey anti-mouse IgG (1:2000, TransGen, Beijing, China) for 2 h at 25°C, nuclei were counterstained with 1 μg/mL DAPI for 10 min at 25°C. Images were captured using a LSM710 laser scanning confocal microscope (Carl Zeiss, Oberkohen, Germany) and processed with a LSM image browser.
Treatment with cGAS and NF-κB inhibitor, and pretreatment with Se-Met
A549 cells were exposed to 100 μg/mL PM2.5 with 10 μM PF-06928215 (a inhibitor of cGAS, MedChemExpress, New Jersey, USA) or 5 μM BAY 11-7082 (a inhibitor of NF-κB, MedChemExpress, New Jersey, USA) for 48 h. In addition, 20 μM selenomethionine (Se-Met, Macklin, Shanghai, China) was prepared to pretreat exponentially growing A549 cells for 12 h. Afterwards, treated cells were used to determine the expression level of cGAS/STING/NF-κB pathway- and senescence-related proteins, as well as the percentage of SA-β-gal-positive cells and fluorescence intensity of SA-β-gal foci as described above.
Data from at least three independent experiments were performed for statistical analysis and presented as mean ± S.D. Statistical significance between different groups was assessed using one-way analysis of variance (ANOVA) with Fisher’s least significant difference test. The value of p < 0.05 was considered to indicate a statistically significant difference (*).