br Wound healing assay br A
2.6. Wound healing assay
A549 NCT-501 were seeded in 6-well plates at density of 1 × 106 cells/ well. After reaching nearly confluence, a wound was created by using a p200 pipette tip in the middle area of confluent cells and then FBS free medium was used to remove the detached cells. Then cells were treated with either vehicle or CAA45 at the desired concentrations at 37 °C for 24 h. Images were taken by using phase-contrast microscopy (Olympus, IX51, Tokyo, Japan). The cell migration area was then calculated by ImageJ software (the formerly scratch area minus the final vacant area between cells). The ratio of migration area to the formerly scratched area indicates how many A549 cells migrate.
2.7. Transwell migration assay
Cells were suspended in serum-free DMEM and transferred on the top of transwell chambers (1 × 105 cells per Transwell) (Corning, New York, USA). DMEM with 10% fetal calf serum was placed in the lower chambers in the presence or absence of CAA45. After incubation for 24 h, the migrated cells in the bottom of the chamber were fixed by using 90% alcohol and stained with crystal violet staining solution (Beyotime, Jiangsu, China). Images were taken by using phase-contrast microscopy (Olympus, IX51, Tokyo, Japan). Five random fields per well were counted.
2.8. Apoptosis assay
Cells were seeded in 6-well plates at density of 1 × 105 cells/well and were allowed to grow overnight. Then the cells were treated with either DMSO or CAA45 (0.10, 0.20 and 0.30 μM) at 37 °C for 24 h. The cells were then trypsinized, repeatedly washed with cold PBS for three times, and centrifuged at 1200 rpm for 5 min- to pellet. Then the cells were resuspended to ~ 5 × 105 cells/mL in binding buﬀer (100 μL), stained with annexin-V-fluorescein isothiocyanate (FITC) (Sangon Biotech, Shanghai, China) and DAPI (Beyotime, Jiangsu, China) for 10 min at room temperature. Subsequently, the apoptotic cells were
immediately analyzed on a flow cytometer (Accuri C6, BD Biosciences). Cells undergoing early and late apoptosis were labeled by both annexin-V and DAPI, respectively.
Cells (1 × 105 cells/well) were seeded in 6-well plates and treated with diﬀerent concentrations of CAA45 (0.10, 0.20 and 0.30 μM) or 0.1% DMSO for 24 h at 37 °C. Then cells were harvested, washed with PBS and fixed with 70% precooled ethanol at −20 °C overnight. The fixed cells were washed with PBS and incubated with RNase A (Beyotime, Jiangsu, China) for 30 min at 37 °C. Finally, cells were stained with PI (Beyotime, Jiangsu, China) for 10 min at room tem-perature and analyzed on a flow cytometer (Accuri C6, BD Biosciences).
2.10. Monodansylcadaverine (MDC) incorporation assay
MDC incorporation assay was used to detect the formation of au-tophagic vacuoles. A549 cells were plated on coverslips in 24-well plates (2 × 104 cells/well) and treated with diﬀerent concentrations of CAA45 (0.06, 0.12 and 0.25 μM) or DMSO (0.1%) or rapamycin (0.30 μM) for 12 h. Cells were then washed twice with PBS and in-cubated with fresh medium containing 0.05 mM MDC (Solarbio, Beijing, China) for 30 min at 37 °C. Then cells were washed four times with PBS and the autophagic vacuoles were detected on a confocal laser scanning microscopy (Leica TCS SP8, Solms, Germany).
2.11. Ad-mCherry-GFP-LC3B transfection
Cells (1 × 104 cells/well) were seeded on glass coverslips in 24-well plates and were allowed to grow overnight, then were transfected with Ad-mCherry-GFP-LC3B adenovirus (MOI value was 20) (Beyotime, Jiangsu, China) in 200 μL DMEM medium containing 10% FBS for 6 h. Subsequently cells were washed twice with PBS and treated with CAA45 (0.06, 0.12 and 0.25 μM) or rapamycin (0.30 μM) for 12 h. The treated cells were fixed with 4% paraformaldehyde for 20 min and washed three times with PBS. Finally, autophagy was observed using a confocal laser scanning microscopy (Leica TCS SP8, Solms, Germany).
A549 cells were treated with desired concentrations of CAA45 for
24 h. Cells were then washed twice with ice-cold PBS and the total cellular protein was extracted by using western blot and IP cell lysis buﬀer kit (Beyotime, Jiangsu, China) following manufacturer's in-structions. Protein concentrations were quantified using BCA Protein Assay Kit (Beyotime, Jiangsu, China) and then denaturized with SDS loading buﬀer at 100 °C for 8 min. Equal amounts of total protein (40 μg) was separated by 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE) and then transferred onto poly-vinylidene fluoride (PVDF) membrane, blocked with 5% fat-free dry milk/0.05% Tween 20 at room temperature for 2 h, breezed with de-sired primary monoclonal antibodies overnight, followed with horse-radish peroxidase-conjugated goat anti-rabbit secondary antibody for 1 h. Finally, the immune reactive band was scanned in the enhanced chemiluminescence (ECL)-detecting reagents (Beyotime, Jiangsu, China) and optical density (OD) values were analyzed using Image J software.