Rpmi 1640

The human LUAD cell lines (H322 and A427) were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium high glucose (KeyGEN BioTECH)/ RPMI 1640 (KeyGEN BioTECH) with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified chamber with 5% CO₂.

BF211 was synthesized in our laboratory as previously described.4 (link) Distearoyl phosphoethanolamine-PEG²⁰⁰⁰ (²⁰⁰⁰PEG-DSPE), cholesterol (CH), hydrogenated soybean phospholipids (HSPC) were obtained from Lipoid GmbH (Ludwigshafen, Germany). Ammonium sulfate was purchased from Nanjing Chemical Reagent Company (Nanjing, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 4% paraformaldehyde (PFA), 4´,6-diamidino-2-phenylindole (DAPI), DiD, DiR, DiI, PKH-26, trypsin-EDTA solution, DMEM and RPMI 1640 were supplied by KeyGEN BioTECH Co. Ltd (Nanjing, China). Creatine Kinase (CK) kit and lactate dehydrogenase (LDH) kit, Doxorubicin (Dox) were purchased from Nanjing JinYibai Biological Technology Co. Ltd. (Nanjing, China). Cinobufagin was supplied by Sigma Corporation (St. Louis, MO, USA). Poloxamer 188 (P188) (PEO/PPO/PEO ratio is 80/27/80, MW 8600) and Tween 80 for injection were purchased from Feiyu BioTECH Co. Ltd (Nanjing, China). HPLC-grade methanol (MeOH), acetonitrile (ACN) and formic acid were obtained from Merck (Darmstadt, Germany). Deionized (DI) water produced by a Milli-Q^® Plus System (Billerica, MA, USA) with a resistivity of 18.2 mΩ·cm-1 was used in all the experiments. All other chemicals and reagents were obtained from Sigma Aldrich (Shanghai, China), and used without further purification.

HCT116 and HT29 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Corning) and RPMI1640 (KeyGEN BioTECH), respectively, supplemented with 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (FBS, HyClone) at 37 ^oC with 5% CO₂. To investigate the functional roles of miR-4260 in colorectal cancer cells in vitro, HCT116 and HT29 cell lines were starved for 6 hrs and then transfected with miR-4260 mimic (50 nM, RiboBio, Guangzhou, China), inhibitor (100 nM, RiboBio, Guangzhou, China), or negative controls for 48 hrs. To investigate whether miR-4260 contributes to the regulation of colorectal cancer via targeting MCC and SMAD4, siRNA for these two genes (75 nM RiboBio, Guangzhou, China) was transfected in HCT116 and HT29 cells in combination with miR-4260 inhibitor for 48 hrs.

Liver epithelial cell-line THLE-2 and HCC cell lines (HuH7, Hep3B, MHCC97-H, LM3) were commercially bought from BioVector NTCC Inc. (Beijing, China). Cell lines were incubated with Roswell Park Memorial Institute-1640 (RPMI-1640; KeyGen, Nanjing, China) containing 10% new-born bovine serum and 1% penicillin-streptomycin. Cells growth was performed in a 5% CO₂ incubator at 37°C.

A purchased human airway epithelial cell line transfected with human papillomavirus type 16 (HPV16) E6/E7 oncogenes (16HBE; American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI 1640 (KeyGen Biotech, Nanjing, China) supplemented with 10% heat-inactivated FBS (Scientan, Beijing, China) and 1% penicillin–streptomycin (Scientan). The cells were incubated at 37°C in 5% CO₂ with humidification, then exposed (or not) to PM_2.5 (100 μg/mL) or 10% CSE. To investigate effects of Wnt5a on inflammation induced by PM_2.5 and CSE, 16HBE cells were pretreated with the Wnt5a antagonist BOX5 (200 μM; Merck Millipore, Burlington, MA, USA) for 1 hour before adding 100 μg/mL PM_2.5 or 10% CSE.

PC12 cells were purchased from Beina Biology (BNCC337644, Beina Biotech, Beijing, China) and cultured in RPMI1640 (KeyGENBioTECH, Nanjing, China) with 10% fetal bovine serum (Biological Industries, Beijing, China) in 5% CO₂ at 37°C, as previously described (Wang et al., 2011). Cells were grown to 60% confluence for used in experiments.