Cell lines and cell culture
HUVECs were obtained from the American Type Cell Culture Collection (ATCC, lot no.: CRL-1730) and cultured in RPMI-1640 medium (Gibco Laboratories, Grand Island, N.Y.) supplemented with 10% fetal calf serum (Gibco) in a humidified, 5% CO2 atmosphere at 37°C. Cells were passaged at 80% confluence and grown to full confluence for the experiments. Each experiment was performed in triplicate and repeated independently at least three times.
Preparation of CSE.
CSE was prepared as previously described . Briefly, one nonfiltered Fu-Rong cigarette (tar: 12 mg/cigarette; nicotine: 1.1 mg; and carbon monoxide: 14 mg; China Tobacco Hunan Industrial., Changsha, China) was burned, and the smoke was passed through 25 ml of phosphate-buffered saline via a vacuum pump. This 100% CSE solution was adjusted to 7.2~7.4 and filtered through a 0.22-m membrane filter to remove large particles and bacteria before use. CSE was freshly prepared within the 30 min preceding each experiment.
After serum starvation for 24 h, HUVEC medium was supplemented with 2.5% CSE for 24 h as described previously . To investigate the role of RESV in CSE induced apoptosis, after serum starvation, HUVECs were pretreated with 40 μM RESV (R5010, Sigma-Aldrich Co., St Louis, MO, USA) for 2 h, followed by cotreatment with 2.5% CSE for 24 h. To examine the effects of autophagy on HUVEC apoptosis, the cells were cultured for 2 h in the presence or absence of the potential 5 mM of the autophagy inhibitor 3-methyladenine (3-MA) (A8353, APExBIO, Houston, TX, USA) or 50 nM of the autophagy inducer rapamycin (Rapa) (A8167, APExBIO, Houston, TX, USA) prior to CSE intervention. For Notch1 inhibition, HUVECs were treated with γ-secretase inhibitor DAPT (D5942, Sigma-Aldrich Co., St Louis, MO, USA) for 24 h.
Flow cytometry analysis
Apoptosis was assessed using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA). After treatment, cells were harvested and washed twice with ice-cold PBS and binding buffer and incubated with AnnexinV-FITC in dark for at least 15 min. Propidium iodide (PI) was added in the dark for 10 min at room temperature. Apoptosis was determined by flow cytometry (A00-1-1102, Beckman, USA). The degree of early apoptosis was determined as the percentage of cells positive for Annexin V and negative for PI by flow cytometry. Cells staining negative for Annexin V and PI were considered to be normal. Cells staining positive for Annexin V and negative for PI represented early apoptosis. Finally, cells staining positive for both Annexin V and PI were classified as late apoptosis.
Notch1 lentivirus and siRNA Transfection
cDNA encoding a constitutively active form of Notch1 consisting of the intracellular domain (N1ICD, base pairs: 5,308-7,665; amino acids: 1,770-2,555) was synthesized and subcloned into the multicloning site of the lentiviral vector pNL-IRES2-EGFP to generate pNL-N1ICD-EGFP by the GeneChem Corporation (Shanghai, China). siRNA duplexes were produced by RiboBio Corporation (Guangzhou, China) against human Notch1. Scrambled control siRNA, which was used as a negative control (NC), was also designed and obtained from RiboBio Corporation. Lentivirus and siRNA transfections were performed using Lipofectamine 3000 (ThermoFisher Scientific, USA) following the manufacturer’s instructions. Cells of each group were collected 48‐72 hours after transfection. The transfection efficiency of N1ICD was determined by counting the percentage of EGFP positivity cells. In our experimental conditions, the transfection efficiency average reached 75.37%±3.21.
Quantitative Real-time polymerase chain reaction (qPCR)
After extraction, RNA was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Subsequently, qPCR was performed using a StepOne Plus real-time PCR system (Life Technologies, Carlsbad, CA, USA). All of the primers were obtained from Sangon Biotech (Shanghai, China). All mRNA expression values were presented relative to β-actin and analyzed by the 2-ΔΔCt method. The primers used for qPCR is listed in Table 1.
Western blot analysis
Cells were harvested and digested in a lysis buffer. Total protein concentration was determined by the BCA protein assay kit. Proteins were separated by 10% SDS-PAGE and were transferred onto PVDF membranes, then incubated with antibodies to Notch1 (#3608, Cell Signaling Technology, Danvers, MA, USA), N1ICD (#4147, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-3 (#9664, Cell Signaling Technology, Danvers, MA, USA), LC3b (14600-1-AP, Proteintech Group, Inc., Rosemont, IL, USA), Beclin1 (11306-1-AP, Proteintech Group, Inc., Rosemont, IL, USA), mammalian target of rapamycin (mTOR) (20657-1-AP, Proteintech Group, Inc., Rosemont, IL, USA), Hes1 (ab108937, Abcam, Cambridge, UK) and P62/SQSTM1 (18420-1-AP, Proteintech Group, Inc., Rosemont, IL, USA). After washing, membranes were incubated with HRP-conjugated secondary antibody for 1 h. The bound complexes were detected using enhanced chemiluminescence detection system (Santa Cruz Biotechnology, Dallas, TX, USA). β-actin (600008–1, Proteintech Group, Inc., Rosemont, IL, USA) was probed to confirm equal protein loading and transfer.
Quantitative data are presented as the means±SD. Statistical analysis was performed using a software package (SPSS 21.0, SPSS Inc., Chicago, IL, USA). One-way ANOVA was used for multiple groups to analyze any differences between two groups. Tukey’s test or Dunnett’s T3 test was used for post hoc multiple comparisons according to the homogeneity test of variance. A value of p < 0.05 was considered to be statistically significant.