Quantification of ACE

Materials and Methods
Western blot
To prepare whole-cell extracts, cells were washed for three times and lysed by RIPA lysis buffer containing protease inhibitor for 30min on ice. Next, the cells were harvested by scratching and were sonicated. The protein  concentration  was determined with Bradford assay (BioRad). Equal amounts of protein(20-30g) were resolved by SDS-polyacrylamide gels and were transferred to nitrocellulose （NC） membranes(GE  Healthcare  Life  science,  Lot.G9597136).Then the non-specific binding sites were blocked in 5% (w/v)non-fat dry milk at room temperature  for 1h.The membranes were incubated overnight at 4℃ with primary antibodies in TBS-Tween (0.2% v/v) containing 5% BSA. Antibodies against ALDH2 (abcam, ab108306), vinculin, γH2AX and actin (MBL, PM053-7) were used. The secondary antibody  was Goat anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated IgG. The antibodies were diluted according to manufacturers’ instructions.
 

Cell lines and growth conditions
Human embryonic kidney cells(HEK293T) and Human non-small cell lung  carcinoma cells were obtained from American Type Culture Collection (ATCC) and were tested and authenticated by DNA typing at Shanghai Jiao Tong University Analysis Core.
The HEK293T cells were cultured in DMEM essential medium with 10% fetal calf serum. 16HBE (Human bronchial epithelial cells) cells were as described previously (Oner et al., 2018). HBEC cells were cultured in serum-free medium, and all other cells were cultured in RPMI-1640 essential medium with 10% fetal calf serum, at 37℃ in a humidified incubator in an atmosphere of 95% air and 5% CO₂.
 

Transfections and viral infections
HEK293T cells were transfected with lenti-shRNA(or nonspecific sequence)-ALDH2 plasmid or lenti-ALDH2(or GFP) plasmid used for virus production for ALDH2 knock down or overexpression.Lipofectamine 2000(plasmid:lipo2000 1:4) was used as transfection reagent.The medium was replaced after 4-6 hours.Then the lentivirus medium was collected to infect NSCLC cells after 48 hours. The stably transfected cells were selected by puromycin (2 μg/ml).Then the cells were expanded and collected for westernblot analyses.
 

Quantitative real-time (Q)-PCR
Human lung cancer tissues samples were obtained from Shanghai Chest Hospital, Shanghai Jiao Tong University (Shanghai, CHINA). Tissues were firstly lysed using trizol, then total RNA were extracted with RNA Extract Kit and cDNA were prepared from 1.5ug of total RNA using Fast Quant Kit. The Q-PCR analysis was performed on ABI 7300 real-time PCR machine. All Ct values were standardized by actin’s Ct value. And the following primers were used：
ALDH2 F: 5’-CCTCACCGCCCTCTATGTG-3’
R:5’-CGGCCAATCTCAGTGGAGC-3’
β -actin  F: 5’- GCTCTTTTCCAGCCTTCCTT-3’ 
R: 5’-CTTCTGCATCCTGTCAGCAA-3’;
 

Colony formation assay
500 cells were seeded in 6cm-dish.After cultured in 37℃ for 15 days，the cells were stained with 0.1% crystal violet at room temperature for 1h.Then the dishes were photographed and the cells were counted.
 

Sphere culture assay
The NSCLC cells were cultured in 1640 medium contain with 10% and were plated in low-adhered plates in order to simulate a 3-D culture room. Every well contains 10000 cells in 24-well plate. The medium was changed every 3 days. Take care the spheroids size to aviod necrosis in the center of the spheres. The number of spheroids was counted and photographed under microscope after 2 weeks.

Tumorigenicity in nude mice
Eight-week-old nude mice were injected subcutaneously with A549-sh-NS/ALDH2 (1.0x106 cells) combined with matrigel. Three weeks later, the volume of tumors were measured and further analyzed. The nude mice were obtained from Shanghai Jiao Tong University, School of Medicine Animal Care [experimental animal use permission No: SYXK (Shanghai) 2008-0050] and were maintained in the specific pathogen-free animal facility in the university.
 

Side population assay
The cultured cells were detached with trypsin, resuspended in RPMI-1640 culture medium supplemented with 37℃ 2% fetal bovine serum , and the cell suspension was adjusted to a density of about 1×10⁶ /ml. Samples were separated to the control group and the experimental group. All samples were stained with Hoechst-33342 (5μg/ml). The control group was also added verapamil (50μmol/L) or ko143 (5μM) to inhibit the efflux of the dye by blocking ABCG2 transporters or ABCB1 transporters for gating of SP cells. All samples incubated in water at 37℃ for 120 min . In this period, shake the incubation tubes several times. The samples were immediately centrifuged at 4 ℃after the incubation, resuspended in ice-cold Hank's Buffer. Propidium iodide (PI, 2μg/ml) was added to gate out the dead cells. Then the cell suspension was filtered through a 40 μm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) to obtain a single suspension of cells. Flow cytometry analysis were conducted on Beckman Coulter CytoFLEX S. Hoechst-33342 was excited by ultraviolet laser, 450 nm filter received Hoechst blue light, and 675 ALP received Hoechst red light. Finally, the data was analyzed in the CytExpert software.

Flow Cytometry analysis of CD44 positive and CD24 negative population
The NSCLC cells cultured in 6cm-dish were collected and washed two times using PBS. Then the cells were incubated in 400μl 1640 contained with 1% CD44-APC, CD24-PE(BD Pharm ingen) at 4℃ for 30minutes,whereas the control were incubated with IgG. The cells were suspended in 400μl PBS for Flow Cytometry analysis after incubation. The gates were established using the negative controls cells stained with IgG. Finally, the data was analyzed in the Flowjo 7.6.1software.
 

Wound healing test
The same amounts of cells were seed into a 6-well plate. When the cells were grown to 90%, scratched a wound at the bottom of the wells using a pipette tip. Changed the medium and took pictures by an inverted microscope. Then the wounds were photographed every 6 hours.
 

Transwell assay
The A549-GFP and A549-ALDH2 cells were seeded in the top well of the transwell chambers (Costar) at a density of 2 × 104/well. A total of 1ml of the conditional medium (CM) was added in the bottom chamber. Following cultivation for 24 h, the top well surface of the filter was wiped with a cotton swab to remove the cells. Then the migrating cells were fixed with 4% paraformaldehyde for 20mins.Lastly, cells were stained with 0.1% crystal violet for 1 h after washing. Migrating cells were observed and captured by an inverted microscope (Nikon ECLIPSE).
 

Quantification of Acetaldehyde
800μl 80% acetonitrile and 200μl Dinitrophenylhydrazine was added to lung tissue samples. Then the tissues were homogenized three times at 5500rpm for 20s using Bertin Precellys 24 Dual Multifunctional sample homogenizer. After placed in -80℃ for 1h  and  room  temperature  for 4h,the tissue homogenate was derivatization.  Then centrifugated at 20000 g for 10 min, the supernatant was took for vacuum drying. Finally, 200μl acetonitrile was added to reconstitution the samples for LC-MS（AB SCIEX 4000) analysis.  For the cell samples detection, 80% methanol was used as extraction reagent. The other steps are the same as the tissue extraction method.

Comet Assay
The A549-GFP and A549-ALDH2 cells were treated with 4mM ACE for two days, cells were harvested and mixed with low-melt agrose (0.5%) at a ratio of 1:10(v/v) and pipetted onto glass slides. After placed at 4℃ in the dark for 30min,the slides were immersed into lysis buffer(100mM EDTA,2.5M NaCl,10mM Tris,1% Triton X-100,10% DMSO) at 4℃ for 1h.Put the slides into alkaline buffer solution(300mM NaOH,1mM EDTA, pH＞13) for 1h to unwind DNA and then electrophoresed for 30min in 25V.Gently immersed slides into neutralizing buffer (0.4M Tris, pH 7.5) for 5min and then immersed into 70% ethanol for 5min.Dried samples completely at room temperature overnight. Placed SYBR Green onto each sample for 30min.The slides were observed and photographed by epifluorescence microscopy. The comet tail counted by CASPLab software.
 

HE staining
Dewaxed the paraffin slices using xylene and gradient alcohol. Then immersed the slices into hematoxylin for 3-8min to stain the Nucleus and washed with water. Following back to blue using 0.6% ammonia water, rinsed with running water. Next, put the slices into Eosin staining solution for 1-3min to stain cytoplasm. At last, dehydrated using gradient alcohol and xylene and sealed with neutral gum. Acquiesced images and analyzed by an inverted microscope.
 

Soft Agarose
2% Agarose combined with 1640 containing 10% Fetal bovine serum(FBS) (1:3 v/v; final concentration, 0.7%) was added to 24-well (0.5ml per well), then the agarose solidified for 10 minutes at 4℃. NSCLC cells (500 cells in 50 ul medium) were mixed in the 1640 medial contained 0nM, 100nM, 350nM or 1μM Alda-1(MCE,HY-18936 ) and putted on the solidified agarose. On the top layer of the wells, 0.5ml mixture of 2% Agarose combined with 1640+10%FBS (1:6 v/v; final concentration, 0.35%) along with matrigel (1:30 v/v) was added. The plate was incubated in 37℃/5% CO₂ incubater. After 2 weeks, established colonies were counted and photographed.

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