Human U-2 OS, HeLa, and HEK293T cells were obtained from ATCC. Cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% streptomycin/penicillin antibiotics (Wisent) and 10% fetal bovine serum (Gibco). Cells were grown at 37°C in a 5% CO2 humidified atmosphere. Cells were regularly tested to ensure the absence of mycoplasma contamination. For treatments, HU (Bioshop), CPT (Alfa Aesar), mitomycin C (MMC) (Tocris Bioscience), cycloheximide (CHX) (Biobasic), MG132 (Calbiochem), VE-821 (ATRi), KU55933 (ATMi), and NU7441 (DNA-PKi) (Selleck Chemicals) were used as indicated in the corresponding figure legends. γ-irradiation was performed in a Gamma cell 3000 Elan (Best theratronics) and UV-C irradiation was done using a luminometer-calibrated Stratalinker 2400 crosslinker (Stratagene).
Nu7441 dna pki
Manufactured by Selleck Chemicals
NU7441 is a DNA-dependent protein kinase (DNA-PK) inhibitor. It acts as a potent and selective inhibitor of DNA-PK, an enzyme involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. NU7441 has been used in various research applications to study the role of DNA-PK in cellular processes.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mitomycin c mmc, by Bio-Techne (1 mentions)
Gammacell 3000 elan, by Best Theratronics (1 mentions)
Parpi olaparib, by Selleck Chemicals (1 mentions)
5 iododeoxyuridine idu, by Merck Group (1 mentions)
Auxin iaa, by Merck Group (1 mentions)
Ab115730, by Proteintech (1 mentions)
Matrigel, by BD (1 mentions)
Ab18192, by Abcam (1 mentions)
Ht 29, by National Collection of Authenticated Cell Cultures (1 mentions)
Atri ve 821, by Selleck Chemicals (1 mentions)
Atmi ku 55933, by Selleck Chemicals (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hek293t, by National Collection of Authenticated Cell Cultures (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Hepg2, by National Collection of Authenticated Cell Cultures (1 mentions)
H1299, by National Collection of Authenticated Cell Cultures (1 mentions)
Hct116, by National Collection of Authenticated Cell Cultures (1 mentions)
Beas 2b, by National Collection of Authenticated Cell Cultures (1 mentions)
H1975, by National Collection of Authenticated Cell Cultures (1 mentions)
4 protocols using nu7441 dna pki
1
Cell Culture and Treatments for DNA Damage Studies
2
Inhibitor-Driven DNA Damage Response
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The inhibitors used in this study are VE-821 (ATRi, Selleckchem), AZD6738 (ATRi, Selleckchem), UCN-01 (CHK1i, Sigma Millipore), Mirin (MRE11i, Sigma Millipore), NU7441 (DNA-PKi, Selleckchem) and olaparib (PARPi, Selleckchem). 5-Iododeoxyuridine (IdU, Sigma Millipore) was used at a 25 μM concentration. Auxin (IAA, Sigma Millipore) was used at a 10 μg/ml concentration.
3
Evaluating DNA-PKi Therapy in C4-2 Xenografts
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C4–2 cells (5 × 106 cells per injection) were resuspended in a 1:1 mixture of 1X PBS and Matrigel (BD Biosciences 354234) and injected subcutaneously into the flank of 5- to 6-week-old SCID male mice following protocol approved by IACUC at Thomas Jefferson University. Upon tumor reaching 150 mm3, mice were treated with the DNA-PKi NU7441 (Selleck Chemicals, 25 mg/kg) or vehicle daily for 4 days via intraperitoneal injection. After 4 days, tumors were harvested and processed for pyruvate kinase activity and pyruvate levels as described in Supplemental Experimental Methods. Tissue was used for hematoxylin and eosin (H&E) staining, and immunohistochemistry staining for pDNA-PK (Abcam ab18192), DNA-PK (Abcam, ab168854), PKM2 (Cell Signaling, #4053S), GLUT-1 (Abcam, ab115730) and MCT1 (Proteintech, 20139–1-AP) performed by Thomas Jefferson Pathology Core.
4
DNA Damage Induction in Cancer Cell Lines
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Colorectal cancer (CRC) cell lines (HCT116, HT29, WIDR and T87), lung cancer cell lines (A549, H460, H1299 and H1975), a human bronchial epithelial cell line (BEAS-2B), human glioma cell lines (U251, U373 and U87), human hepatoma cell lines (HuH7, 7721, HepG2) and human embryonic kidney 293 cells (HEK 293T) were all purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The culture conditions used in this study are shown in Supplementary Table S1 . High glucose Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 medium were supplied by HyClone (Thermo Fisher, USA). All culture media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher), 100 U/mL penicillin and 100 µg/mL phytomycin (Gibco, Thermo Fisher). All cultures were maintained at 37 °C and 5% CO2. For induction of DNA damage, cells were treated with camptothecin (CPT, 1 µM, Selleck) for 1 h, etoposide (ETO, 100 μg/mL, Selleck) for 4 h, or γ-irradiation. ATM inhibitor (ATMi, KU-55933), ATR inhibitor (ATRi, VE-821) and DNA-PK inhibitor (DNA-PKi, NU7441) were purchased from Selleck. During DNA damage induction by ionizing radiation (IR), CPT or ETO, the corresponding inhibitors were maintained in complete medium continuously unless indicated.
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