A549 NSCLC cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD), and NCI-H460 NSCLC cell line was purchased from Chinese Academy of Sciences Cell Bank of Type Culture Collection (Shanghai, China). A549 and NCI-H460 cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2.
A549 nsclc cell line
Manufactured by American Type Culture Collection
Sourced in United States
The A549 NSCLC cell line is a well-characterized human lung adenocarcinoma cell line. It is a widely used in vitro model for non-small cell lung cancer research.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
L 798106, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Hyclone, by Cytiva (1 mentions)
Luciferase shrna shluc, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Pft α, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
9 protocols using a549 nsclc cell line
1
NSCLC Cell Line Culture Protocol
2
A549 NSCLC Cell Culture and DMXAA Treatment
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The A549 NSCLC cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS. The cells were maintained at 37°C in a 5% CO2/95% air humidified incubator. DMXAA was dissolved in dimethyl sulfoxide at a stock concentration of 20 mM and stored at −20°C. It was freshly diluted to predetermined concentrations with culture medium. The final concentration of dimethyl sulfoxide was 0.05% (v/v). The control cells received the vehicle only.
3
Culturing A549 and HPAEpiC cells with Ep3 antagonist
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The A549 NSCLC cell line and HPAEpiC human alveolar epithelial cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and maintained at 37°C in a humidified atmosphere with 5% CO2. A549 cells were treated with 1 µM Ep3 antagonist L-798106 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 48 h in the following experiments.
4
Stable NRF2 Knockdown in A549 Cells
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A549 NSCLC cell line was from the American Type Culture Collection (ATCC, Beijing, China). A549-derived stable siRNA knockdown for NRF2 (siNrf2-C27) and, control cell line (siGFP-C5) were generated as described previously 24 (link). The cells were maintained in a growth medium containing Dulbecco's MEM with Glutamax supplemented with 10% fetal bovine serum (FBS) and antibiotics. All cells were cultured at 37oC, in 95% air and 5% CO2, and passaged every 3 to 4 days. All medium supplements for cell culture were from Invitrogen (Shanghai, China).
5
NSCLC Cell Lines and Luciferase Reporters
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The human H322, H358 and A549 NSCLC cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). H358 (H358-Luc) and A549 (A549-Luc) cells stably expressing firefly luciferase were obtained from Optimal (Grenoble, France).
6
Establishment of PTX-Resistant A549 NSCLC Cell Line
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Human A549 NSCLC cell lines were obtained from American Type Culture Collection. The PTX-resistant A549 NSCLC (A549-PTX) cells were established by gradual exposure of A549 cells to increasing concentrations of PTX, as previously described (27 (link)). In order to maintain the PTX-resistant phenotype of A549-PTX cells, 0.1 µM PTX was added into the culture medium. The A549 cells used in the present study were cultured in parallel during the establishment of A549-PTX cells. All cells were cultured in DMEM (Corning, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1X penicillin/streptomycin (HyClone; Cytiva). All cultures were maintained in a humidified tissue culture incubator at 37°C with 5% CO2.
7
Knockdown of CNTN1 in A549 NSCLC Cells
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The NSCLC A549 cell line was purchased from the American Type Culture Collection, and was cultured in high-glucose DMEM (Hyclone; Cytiva), supplemented with 10% FBS (Natocor-Industria Biológica) and 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA). The cell line was thawed every month. Short hairpin (sh)RNA-CNTN1 and control shRNA [Luciferase shRNA (shLuc)] was purchased from Sigma-Aldrich (cat. no. SHC007; Merck KGaA). shRNA-CNTN1 was ligated into a lentiviral plasmid (Takara Bio Inc.) and the shRNA had the following sequences: shCNTN1-1, 5′-CCGGGCCGTGGTTCAGACAATCATACTCGAGTATGATTGTCTGAACCACGGCTTTTTG-3′; and shCNTN1-2, 5′-CCGGCCAAGGATCATCAGTTCAGTACTCGAGTACTGAACTGATGATCCTTGGTTTTTG-3′.
8
NSCLC A549 Cell Hypoxia and PFTα Treatment
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The NSCLC A549 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) in 95% air and 5% CO2 at 37°C.
For hypoxia exposure, cells were incubated and treated in an InVivo2 Hypoxia workstation 400 (Ruskinn Technology Ltd., Bridgend, UK) and flushed with 1% O2, 5% CO2 and 94% N2, which is referred to as hypoxia. Prior to specific treatments, cells were pre-incubated for 2, 4, 6 or 12 h under conditions of normoxia or hypoxia.
For PFTα treatment, 30 µM PFTα (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to cells prior to normoxic or hypoxic exposure for 4 h at 37°C. Then cells were exposed to normoxic or hypoxic condition in RPMI-1640 medium supplemented with 30 µM PFTα.
For hypoxia exposure, cells were incubated and treated in an InVivo2 Hypoxia workstation 400 (Ruskinn Technology Ltd., Bridgend, UK) and flushed with 1% O2, 5% CO2 and 94% N2, which is referred to as hypoxia. Prior to specific treatments, cells were pre-incubated for 2, 4, 6 or 12 h under conditions of normoxia or hypoxia.
For PFTα treatment, 30 µM PFTα (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to cells prior to normoxic or hypoxic exposure for 4 h at 37°C. Then cells were exposed to normoxic or hypoxic condition in RPMI-1640 medium supplemented with 30 µM PFTα.
9
Generation and Validation of Nrf2 Manipulation Cell Lines
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Unless otherwise stated, all chemicals were from Sigma–Aldrich Co., Ltd. (Shanghai, China). The NSCLC A549 cell line was from the American Type Culture Collection. The A549-derived Nrf2-knockdown cell line, siNrf2-C27, and its control cell line siGFP-C5, were described previously [63 (link)]. To generate stable lines overexpressing Keap1, A549 cells were transfected with an mKeap1-pEGFP plasmid expressing mouse Keap-1. After selection in culture medium containing 0.8 mg/ml G418, one clone named mKeap1-C1, which maintained stable overexpression of Keap1 after multiple passages, was chosen for this study. Similarly, a cell line named GFP-c1 was generated after A549 cells were stably transfected with empty pEGFP vector, and served as a negative control.
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