Human metastatic NSCLC cell lines H2030‐BrM3 (K‐rasG12C mutation) (P9) and PC9‐BrM3 (EGFRΔexon19 mutation) (P40) were kindly provided by Professor Joan Massagué (Metastasis Research Center, Memorial Sloan Kettering Cancer Center, New York, NY) and authenticated by left ventricle inoculation as previously described.26 , 27 Parental human NSCLC cell line PC9 (P10) was a gift from Dr. Jasmine G. Lee (Department of Internal Medicine, Division of Respiratory Medicine, University of California Davis, Davis) and authenticated by erlotinib treatment in a viability assay.28 Human NSCLC cell line H2030 was obtained from American Type Culture Collections (ATCC) (Manassas, VA) and passaged for fewer than 6 months after receipt from ATCC. Cell lines were maintained in RPMI‐1640 supplemented with 10% heat‐inactivated foetal bovine serum and streptomycin (100 mg/mL), and were cultured at 37°C in a humid incubator with 5% CO2.
H2030
Manufactured by American Type Culture Collection
Sourced in United States
The H2030 is a laboratory incubator designed for the cultivation and maintenance of cell cultures and microbial samples. It provides precise temperature and humidity control to support optimal growth conditions.
Automatically generated - may contain errors
Lab products found in correlation
H1299, by American Type Culture Collection (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
H1975, by American Type Culture Collection (10 mentions)
H1650, by American Type Culture Collection (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
H2122, by American Type Culture Collection (6 mentions)
Hcc827, by American Type Culture Collection (5 mentions)
Calu 1, by American Type Culture Collection (5 mentions)
H2170, by American Type Culture Collection (5 mentions)
Beas 2b, by American Type Culture Collection (5 mentions)
Sk lu 1, by American Type Culture Collection (3 mentions)
H1792, by American Type Culture Collection (3 mentions)
H1435, by American Type Culture Collection (3 mentions)
Sb431542, by Cayman Chemical (2 mentions)
Nih3t3 e1 fibroblasts, by American Type Culture Collection (2 mentions)
Gant61, by Cayman Chemical (2 mentions)
Hpi 1, by Abcam (2 mentions)
H2009, by American Type Culture Collection (2 mentions)
Sw1573, by American Type Culture Collection (2 mentions)
32 protocols using h2030
1
Metastatic NSCLC cell line authentication
2
Cell Culture Protocols for Cancer Research
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NIH3T3-E1 fibroblasts were obtained from ATCC (Manassas, VA, USA) and cultured as previously described [25 (link),26 (link)]. The human lung cancer cell lines A549 and H2030 were obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS and antibiotics as previously described [23 (link)]. CAPAN-1 human pancreatic adenocarcinoma cell line was purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and antibiotics as previously described [23 (link)]. Sufu-/- mouse embryonic fibroblasts were a gift from Dr. Matthew Scott of Stanford University. TGFβRI-deficient mink lung epithelial cells, R1BL17, were maintained in MEM containing 10% FBS and non-essential amino acids as described [27 (link)]. Gant61, and SB431542 were obtained from Cayman Chemical Company (Ann Arbor, MI, USA), HPI-1 was obtained from Abcam (Cambridge, UK), TGFβ1 was obtained from R&D Systems (Minneapolis, MN, USA). Carboplatin was purchased from Cayman Chemical Company (Ann Arbor, MI, USA).
3
Culturing and Validating Lung Cell Lines
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Immortalized bronchial epithelial cells (BEAS-2B) and nine human lung cancer cell lines (A549, H1299, H1650, H1975, H2030, H292, H460, H838, SK-LU-1) were purchased from ATCC (American type culture collection). All cell lines were confirmed to be free of mycoplasma contamination. BEAS-2B and H292 cells were cultured in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (FBS). A549, H1299, H1650, H1975, H2030, H460, H838 and SK-LU-1 cells were cultured in RPMI 1640 medium with 10% FBS. All cell lines were maintained at 37°С in a 5% CO2 incubator. Cell lines were immediately expanded upon receipt and aliquots frozen to allow the cell lines to be restarted every three to four months from the same batch of cells. Cell phenotypes were verified in every experiment.
4
Culture of LUAD Cell Lines
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The LUAD cell lines A549, PC9, and H2030 were purchased from ATCC (MD, USA). These LUAD cell lines were maintained in the RPMI 1640 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA) at 37 °C in a humid incubator with 5% CO2.
5
Heat Stress Induction in Cell Lines
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HeLa (ATCC, CCL-2, RRID: CVCL0030), U2OS (ATCC HTB-96, RRID:CVCL0042), H2030 (ATCC CRL-5914, RRID:CVCL1517), and HCC827 (ATCC CRL-2868, RRID:CVCL2063) were purchased from ATCC. HEKT293 (Thermo R70007, RRID: CVCL6911) were purchased from Thermo Scientific. HeLa and U2OS cells were cultivated in Dulbecco’s Modified Eagle’s Medium (Biochrom), containing 10% fetal calf serum, 2 mM L-glutamine, and 100 U penicillin/streptomycin. H2030, HCC827: RPMI 1640 Medium, containing 10% fetal calf serum, 2 mM L-glutamine, and 100 U penicillin/streptomycin. HEK T293: DMEM GlutaMAX™ Medium, containing 10% fetal calf serum and 100 U penicillin/streptomycin. All cell lines were tested negative for mycoplasma contamination. Cell line data were collected from Cancerrxgene (Wellcome Sanger Institute) and RNA-Seq data were obtained from Klijn et al.21 (link).
For heat stress induction, cells were incubated at 44 °C with 5% CO2. Preliminary experiments in HeLa cells and U2OS cells revealed no substantial difference between 42 °C for 4 h and 44 °C for 1 h on RNA level in our hands13 (link). Thus, the latter conditions were applied for subsequent experiments, as they induced SatIII foci in a comparable or even stronger fashion.
For heat stress induction, cells were incubated at 44 °C with 5% CO2. Preliminary experiments in HeLa cells and U2OS cells revealed no substantial difference between 42 °C for 4 h and 44 °C for 1 h on RNA level in our hands13 (link). Thus, the latter conditions were applied for subsequent experiments, as they induced SatIII foci in a comparable or even stronger fashion.
6
Lung Cancer Cell Line Authentication
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The human lung cancer cell lines A549, H2030, H2009 and Calu1 (Table 2 ) were purchased from ATCC and grown at 37 °C in RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 μg/ml penicillin and 100 units/ml streptomycin (complete medium). Cell lines were authenticated at the RTSF Genomics Core of Michigan State University using the Promega GenePrint 10 System. All cell lines included in this study were negative for Mycoplasma as regularly tested with the Mycoplasma Plus PCR Primer Set (Agilent). Selumetinib (Cat#S1008), trametinib (Cat#S2673) and silmitasertib (Cat#S2248) were purchased from SelleckChem.
7
Malin overexpression and stable knockdown in lung cancer cells
8
Cultivation of LUAD Cell Lines
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All LUAD cell lines (A549, Calu-1, H1792, H2030, MOR, and SW1573) used in this study were obtained from ATCC or Sigma-Aldrich (MOR only). The 634T cells were a generous gift from the laboratory of Kwok Wong (NYU). Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Corning 10-040-CV) supplemented with penicillin–streptomycin and 10% fetal bovine serum. Spheroids were grown in medium supplemented with methylcellulose (Thermo Fisher Scientific M352-500). To prepare 100 mL of methylcellulose-containing medium, 1.2 g of methylcellulose was autoclaved in a bottle containing a stir bar. One hundred ml of medium was warmed to 37 °C and then added to the methylcellulose. The methylcellulose medium was then shaken vigorously and allowed to dissolve at 4 °C overnight, with stirring. Cells were maintained at 37 °C in a humidified incubator containing 5% CO2. All cell lines (and their derivatives) were confirmed to be mycoplasma-free using the MycoAlert mycoplasma detection kit (Lonza LT07-218) at the beginning and upon completion of all experiments.
9
Authenticated Lung Cancer Cell Lines
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A panel of authenticated human lung carcinoma cell lines used in the study, including A549, H460, H520, H1299, SK-MES-1, H2030, H2122, H1975, HCC827, and PC9, along with 16BHE, an immortalized human bronchial epithelial cell line, were obtained from ATCC (Manassas, VA, United States). All cell lines were cultured in RPMI 1604 or DMEM (Invitrogen) supplemented with 5–10% FBS (Sigma-Aldrich, St; Louis, MO, United States) under 37°C and 5% CO2. Mammary epithelium medium was purchased from Lonza (Basel, Switzerland). During the study all cell lines were periodically examined for Mycoplasma contamination by PCR analysis (Cheung et al., 2011 (link)).
The sources of antibodies and chemical inhibitors are described in a prior study (Xu et al., 2017 (link)). Additional antibodies, including BRD4 and phosphorylated or γH2AX, were purchased from Cell Signaling Technology (Danvers, MA, United States). Osimertinib was obtained from Selleckchem (Houston, TX, United States).
The sources of antibodies and chemical inhibitors are described in a prior study (Xu et al., 2017 (link)). Additional antibodies, including BRD4 and phosphorylated or γH2AX, were purchased from Cell Signaling Technology (Danvers, MA, United States). Osimertinib was obtained from Selleckchem (Houston, TX, United States).
10
Cell Culture and Treatment Protocols
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NIH3T3-E1 fibroblasts were obtained from ATCC (Manassas, VA) and cultured, as previously described [35 (link),36 (link)]. CAPAN-1 and PANC-1 human pancreatic cancer cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT, USA) and antibiotics, as previously described [37 (link)]. The human lung cancer cell lines A549 and H2030 were obtained from ATCC and cultured in RPMI-1640 containing 10% FBS. The human hepatoma cell line HepG2 was obtained from ATCC and cultured in DMEM containing 10% FBS. Mouse embryonic fibroblasts (MEFs) from suppressor of fused (Sufu) null mice (Sufu−/−) were provided by Dr. Philip Beachy of Stanford University and cultured in DMEM containing 10% FBS. Gant61, SB431542 and vismodegib (GDC0449) were obtained from Cayman Chemical, HPI-1 was obtained from Abcam. For experiments, cells were treated in a medium containing 5% FBS.
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