Penicillin and streptomycin

C2C12 cells (mouse skeletal myoblasts) and 293T cells (Human Embryonic Kidney) were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM, Corning, NYC, USA, 10‐013‐CV) with 10% fetal bovine serum (Biological Industries, Beit HaEmek, Israel) and 1% penicillin–streptomycin (KeyGEN, Nanjing, China, KGY0023) at 37 °C supplemented with 5% CO₂.
C2C12 can be differentiated into myotubes by culturing in differentiation medium (DMEM containing 2% horse serum and 1% penicillin and streptomycin), the whole process is about 4 days. Myotube transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The transfection concentration of siRNA was 100 × 10⁻⁹m. The transfection was performed after myotubes formed, and 24 h later, the muscle‐atrophy models were performed. Dexamethasone (Dex, Sigma, MO, USA, D4902), TNF‐α (PeproTech, NJ, USA, 315‐01A), and Ang II (Sigma, Sigma, MO, USA, A9525) were used to induce muscle atrophy according to previous detailed methods.^[44] In short, the myotube cell were incubated with Dex (50 × 10⁻⁶m) for 24 h, TNF‐α (100 ng mL⁻¹) for 24 h, and Ang II (500 × 10⁻⁹m) for 48 h. After incubation, cells were harvested or used for morphological analysis. The sequences for siRNA, short hairpin RNA (shRNA), and plasmid were listed in Table S6, Supporting Information.

The human embryonic heart fibroblast cell line was obtained from the Kunming Cell Bank of Chinese Academy of Sciences. The cardiac fibroblasts were grown in DMEM/F12 (Corning) supplemented with 10% fetal bovine serum (Corning) and 1% penicillin and streptomycin (Key Gen). The cardiac fibroblasts were incubated at 37°C in 5% CO₂ in a humidified environment. The cardiac fibroblasts were seeded on 6 well culture plates and grown to 70%‐80% density over 24 hours. Myofibroblast differentiation was induced using 10 µmol/L Angiotensin II (Sigma). In all experiments, FBS was reduced to 1% 12 hours during treatment with AngII. After cells were differentiated for 12 hours, cells were exposed to PM_2.5 and/or melatonin for another 24 hours.

Human LUAD cell lines, including H1650, H358, HCC827, H23, A549, H1693, H1299 were purchased from American Type Culture Collection (ATCC). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (KeyGen Biotech Co. Ltd., Nanjing, China) with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, U.S.A.) and 1% penicillin and streptomycin (KeyGen Biotech Co. Ltd., Nanjing, China) in a humidified incubator at 37°C containing 5% CO₂. Real-time quantitative PCR primers were provided by Bioengineering. In addition, FastQuant RT Kit (WithgDNase) (Cat:#KR106-02), RNA Extraction Kit (Cat:#RK123), and SuperReal PreMix Plus (SYBR Green) Kit (Cat:#FP205-02) were purchased from TIANGEN. Lipofectamine 2000 transfection reagents were purchased from Invitrogen (Cat:#1854323). Antibodies against GAPDH (Cat:#60004-1-Ig) was purchased from Proteintech and STEAP1 antibody (Cat#88677) was purchased from Cell Signaling Technology. EdU cell proliferation test kit was bought from Solarbio (Cat:#1170).

To analyze apoptosis, the K562 cells were cultured in 6-well plates (cat no. 3516; Corning Incorporated) with at least 3.0×10⁵ cells/well in medium containing RPMI-1640 (Gibco-BRL, Carslbad, CA, USA), 10% FBS (Sijiqing Biological Engineering Materials, Hangzhou, China) and 100 U/ml penicillin and streptomycin (KeyGen Biotech Co., Ltd.) for 24 h. The cells were then treated with different concentrations of virosecurinine (6.25, 25 and 50 μmol/l) for 48 h. The cells were washed twice with cold PBS (cat no. KGB500; KeyGen Biotech Co., Ltd.), followed by the addition of 5 μl annexin V-fluorescein isothiocyanate (FITC; cat no. KGA105; KeyGen Biotech Co., Ltd.) and propidium iodide (PI; cat no. KGA511; KeyGen Biotech Co., Ltd). After 15 min incubation at room temperature in the dark, the cells were analyzed using flow cytometry. Fluorescence was measured using a FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) equipped with an argon laser (488 nm). The cell apoptotic rate was calculated using the internal software system of the FACScan (Becton-Dickinson).