SI Materials and Methods Materials and Chemicals the DEP and PM Used in This Work Are the Standard Reference Material 2975 and S
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SI Materials and Methods Materials and chemicals The DEP and PM used in this work are the Standard Reference Material 2975 and Standard Reference Material 1648a, respectively, from the National Institute of Standards and Technology (NIST, USA). 1-nitropyrene, 1-nitrofluoranthene (BCR305), naphthalene (91489), fluoranthene (F807) and phenanthrene (P11409) were purchased from Sigma-Aldrich Co. LLC (USA). 6-hydroxynitropyrene (6-OHNP) and 8- hydroxynitropyrene (8-OHNP) were synthesized as previously described (1). Subject characterization We recruited 600 healthy volunteers from 3 cities with discrepant outdoor air pollution levels, including 100 male (mean age 65 years, range: 57-72 years) and 100 female (mean age 66 years, range: 56-72 years) in Zhangjiakou; 100 male (mean age 64 years, range: 57-71 years) and 100 female (mean age 64 years, range: 55-72 years) in Nanjing; as well as 100 male (mean age 66 years, range: from 60-73 years) and 100 female (mean age 64, range: 58-73) in Shijiazhuang. All volunteers were healthy non-smokers, free of any diagnosis of respiratory diseases, and lived in urban communities. Blood and mid-stream urine samples were collected between Dec.10th to Dec. 20th, 2014. For the panel study, we recruited 30 male (mean age 64 years, range: 58-71 years) and 30 female (mean age 65 years, range: 57-73 years) retired COPD patients in Nanjing; 30 male (mean age 65 years, range: 59-72 years) and 30 female (mean age 64 years, range: 57-71 years) retired COPD patients in Shijiazhuang. All subjects were living in urban communities and had been diagnosed with mild-to-moderate COPD in Nanjing Chest Hospital or the Second Hospital of Hebei Medical University according to the classification of Global Initiative for Chronic Obstructive Lung Disease (GOLD). We included the stable COPD patients and excluded those who were current active smokers, www.pnas.org/cgi/doi/10.1073/pnas.2019025117 passive smokers (living with a current smoker), or had severe comorbidities or inflammatory diseases. In addition, 60 healthy volunteers were recruited in each city among the first batch of healthy volunteers. Blood and mid-stream urine samples of COPD patients and healthy volunteers were collected in Jan. 20th and Jan. 28th, 2015 in Nanjing or in Jan. 8th and Jan. 16th, 2015 in Shijiazhuang. The Institutional Review Board in the School of Public Health at Southeast University approved the study protocol. Written consent forms were obtained from all subjects. Spirometry Post-bronchodilator (BD) spirometry testing was performed immediately after blood sampling with the EasyOne Spirometer Model 2001-2S (NDD Medical Technologies, Switzerland). Data of forced expiratory volume in 1 second (FEV1) and Forced vital capacity (FVC) were collected. Collection of particulate matter The 120 COPD patients and 120 healthy individuals wore sampling equipment for 10 h /per day (from 9:00 am to 7:00 pm), from Jan. 21th to 27th, 2015 in Nanjing and from Jan. 9th to 15th in Shijiazhuang. The sampling equipment consisted of AirChek 2000 pumps (SKC Inc, USA) with Triplex PM2.5 cyclones, which sampled at 1.5L/min through 37 mm Teflon filers. Filters were frozen until analysis. Measurement of 1-NP and 1-NP metabolites Half of PM filter were recovered in 10 ml acetone/hexane mixture (1:1) by microwave- assisted extraction. Then the extracts were analyzed using an Agilent 00A GC/MS Triple Quadrupole System (7890A- 7000 series MS, Agilent Technologies Inc, USA) as described(2). The quantitative determination of 1-NP was performed in Multiple Reaction Monitoring (MRM) mode. Metabolites of 1-NP, 6-OHNP, and 8-OHNP in urine samples were measured by HPLC tandem quadrupole MS/MS as described (1). Lentivirus transduction C/EBPα, NDUFA1, NDUFA2, NDUFC2, NDUFS4, and ATP5H overexpression lentiviruses, as well as C/EBPα shRNA, were generated by co-transfection with packaging plasmids, pSPAX2 and pMD2G. The shRNA lentivirus harbored a short- hairpin RNA sequence to target genes. The overexpression lentivirus harbored a target gene coding sequence, which was tagged with c-Myc. Lentivirus (MOI = 30) was added to the HBE cells, and subsequently treated with Blasticidin S for two weeks to obtain stable transduction HBE cells. For experiments, HBE cells were thawed and allowed to grow for three passages before use. The wild-type (WT) HBE cells and lentivirus stable transduction (LST) HBE cells were then treated with 50 μg/ml DEP for 24 h RNA and protein were collected for further analysis. RNA microarray and gene expression analysis HBE cells were seeded in 10 cm culture dishes and exposed to 50 μg/ml DEPs with three biological replicates. Complete medium was removed after 24 h treatment. Adherent cells were then collected. The total RNA was extracted using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. An Agilent Array platform (Agilent Technologies, Santa Clara, CA, USA) was employed for microarray analysis of total RNA extracted from HBE cells as described (3). An absolute fold change of 2 or more and 0.05 adjusted P-value were set as cut-off to evaluate the significance of gene expression differences of raw data. Database for Annotation, Visualization, and Integrated Discovery (DAVID 6.7) functional annotation tool was used to analyze differentially expressed genes. The P-value was set to 0.05 to denote the significance of GO enrichment in the differentially expressed mRNA list. The pathway analysis for differentially expressed mRNAs was performed on the KEGG database (P-value was set as 0.05). Cell growth assays Cellular growth was evaluated using the Cell Counting Kit-8 (Nanjing Jiancheng Bioengineering Institute, China). HBE cells were plated at a density of 1 × 104 per well in a 96-well plate and treated with 0, 0.1, 1, 10 μg/ml 1-NP coupled with or without C/EBPα lentivirus infection with 8 biological replicates for each concentration. Accordingly, 10 μl of CCK-8 was added to each well. The cells were incubated for 4 h at 37°C. Absorbance was determined at 450 nm. Cell viability affected by 1-NP was monitored every 24 h up to 3 days. Transmission electron microscopic observation HBE cells were treated with 0 or 10 μg/1-N ml P for 24 h, then collected and fixed. Ultra-thin sections were stained with uranyl acetate and lead citrate, and then observed in a transmission electron microscope (JEOL-1010, Japan). Metabolomics analysis GC/TOFMS analysis was performed using an Agilent 7890 gas chromatography system coupled with a Pegasus 4D time-of-flight mass spectrometer. HBE cells were exposed to DEPs (0, 10, 20, and 50 μg/ml) for 24 h with 9 biological replicates. The cell lysate sample and post-exposure blood samples from 120 COPD patients and 120 healthy volunteers were then prepared and analyzed as previously described(4). Animal experiments Male C57BL/6 mice (23–25 g) were purchased from Vital River Laboratory Animal Technology (China). Homozygous Atg7fl/fl mice on a C57BL/6 background were generated by insertion of a loxP sequences within introns 13 and 14 of Atg7 gene. The conditional lung Atg7-/- were modeled as previously described(5). Briefly, 6-week-old mice were anesthetized and 50 μL Lenti-Cre virus (105 infectious particles) was administered dropwise into one nostril until the virus was completely inhaled. The intranasal delivery method was completed in 3-5 min per mouse. The first batch of animal experiments included three groups (with ten C57BL/6 mice in each group): control with a sham treatment of 10 μL PBS; mice treated with a single exposure of 5 μg DEPs (10 μL DEPs at a concentration of 0.5 μg/μL); or mice exposed to 50 μg DEPs (10 μL DEPs with a concentration of 5 μg/μL) through intratracheal instilation. Mice were sacrificed on the 29th day of DEP administration. A second batch of mice were divided into three groups (with ten C57BL/6 mice in each group): control with a sham treatment of 10 μL PBS; mice treated with a single exposure of 1 μg 1-NP (10 μL 1-NP with a concentration of 0.1 μg/μL); or mice exposed to 5 μg 1-NPs (10 μL 1-NPs with a concentration of 0.5 μg/μL) through intratracheal instilation. Mice were sacrificed on the 29th day of 1-NP administration. A third batch of mice was divided into four groups (with ten C57BL/6 mice in each group): control mice treated with control shRNA lentivirus in PBS; mice treated with single C/EBPα shRNA lentivirus in PBS; mice treated with single 5 μg/mouse 1-NP treatment; and mice treated with 5 μg/mouse 1-NP coupled with C/EBPα shRNA lentivirus. Mice received a single intranasal instilation with 1 × 108 TU/mouse one week before DEP exposure. A single dose of 5 μg 1-NPs was administered through intratracheal instillation. Mice were sacrificed on the 29th day of 1-NP administration. A fourth batch of mice was divided into four groups (with ten C57BL/6 mice in each group): control mice treated with control vector lentivirus in PBS; mice treated with single C/EBPα vector lentivirus in PBS; mice treated with single 5 μg/mouse 1-NP treatment; and mice treated with 5 μg/mouse 1-NP coupled with C/EBPα vector lentivirus. Mice received a single intranasal instillation with 1 × 108 TU/mouse one week before 1-NP exposure. A single dose of 5 μg 1-NP was administered through intratracheal instillation. Mice were sacrificed on the 29th day of 1-NP administration. A fifth batch of mice included four groups (with ten mice in each group): control wild type (WT) C57BL/6 mice (sham); 5 μg/mouse 1-NP treatment of WT mice; control ATG7-knock out (KO) mice (sham); 5 μg/mouse 1-NP treatment of ATG7-KO mouse.