To analyse the effect of MTH1 on the sensitivity of cells to the alkylating agent Temozolomide, which exerts its toxicity primarily through methylation of O6-guanine, we made use of two glioblastoma cell lines, U251 and U251-MTH1 in which the MTH1 gene had been deleted using CRISPR/Cas9. These two cell lines were kind gifts from Dr Massimo Squatrito. MTH1 knockout was verified using western blotting using rabbit anti-MTH1 (Novus Biologicals). Cells were seeded in MEM medium (Gibco), 10% FBS (Gibco), 50 μg/ml Pen/Strep (Gibco), 1% MEM Non-Essential Amino Acids Solution (100×) (Gibco) and 1 mM Sodium pyruvate (Gibco) and treated with DMSO, Temozolomide (15 μM, Sigma-Aldrich) or Lomeguatrib (20 μM, selleckchem.com) or with both Temozolomide (15 μM) and Lomeguatrib (20 μM). Caspase 3/7 activity was monitored as a measure of early apoptosis after 48 h of treatment (800 cells were seeded in each well of a 384-well plate) at eight different Temozolomide and Lomeguatrib concentrations ranging from 0 to 100 μM, using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's recommendations. For assessment of cell viability, 400 cells were seeded in each well of a 384-well plate and Temozolomide and Lomeguatrib were added as described for the apoptosis assay. Cell viability was measured after 96 h using Resazurin as previously described (22 (link)).
Lomeguatrib
Manufactured by Selleck Chemicals
Sourced in United States
Lomeguatrib is a laboratory reagent used in biochemical research. It is a small organic compound that functions as a DNA repair inhibitor. The core function of Lomeguatrib is to disrupt the activity of a specific enzyme involved in DNA repair processes.
Automatically generated - may contain errors
Lab products found in correlation
Temozolomide, by Promega (1 mentions)
Temozolomide, by Selleck Chemicals (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Temozolomide, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Rabbit anti mth1, by Novus Biologicals (1 mentions)
Mem non essential amino acids solution, by Thermo Fisher Scientific (1 mentions)
Mem medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
Recombinant human epidermal growth factor, by R&D Systems (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
Temozolomide tmz, by Merck Group (1 mentions)
Human neurocult ns a proliferation supplements, by STEMCELL (1 mentions)
Anti β actin igg hrp, by Santa Cruz Biotechnology (1 mentions)
Neurocult ns a basal medium human, by STEMCELL (1 mentions)
5 azacitidine 5 aza, by Selleck Chemicals (1 mentions)
Berzosertib, by Selleck Chemicals (1 mentions)
4 protocols using lomeguatrib
1
Modulation of Temozolomide Sensitivity by MTH1
2
Culturing Patient-Derived Glioblastoma Stem Cells
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The patient-derived GSCs were well characterized (Table 1 ) and cultured in NS-A medium (90% NeuroCult NS-A Basal Medium Human plus 10% Human NeuroCult NS-A proliferation Supplements, StemCell Technologies, Vancouver, British Columbia, Canada in 3D sphere as we described before [45 (link)]. Complete medium was supplied with recombinant human epidermal growth factor (R&D System, Minneapolis, MN, USA) and 100 units/mL penicillin-100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). Anti-TUSC3 and anti-MGMT antibodies were obtained from Abcam (Cambridge, United Kingdom); anti-β-actin IgG-HRP was obtained from Santa Cruz Biotech (Dallas, TX, USA) and anti-DNMT1, Goat anti-Rabbit IgG-HRP and Goat anti-mouse IgG-HRP were obtained from CellSignaling Technology (Danvers, MA, USA). Temozolomide (TMZ) was obtained from Sigma (St. Louis, MO, USA). 5-Azacitidine (5-Aza) and Lomeguatrib were obtained from Selleckchem (Houston, TX, USA).
3
Glioblastoma Stem Cell Line Establishment
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The GSC lines were established by isolating neurosphere-forming cells from fresh surgical specimens of human GBM tissue obtained from patients at The University of Texas MD Anderson Cancer Center from 2005 to 2008, as described previously.21 (link) Eight GSC lines [4 with MGMT-unmethylated/MGMT expression (MGMT+)] and [4 with MGMT-methylated/no MGMT expression (MGMT-)] were cultured in DMEM/F12 medium containing B27 supplement (Invitrogen), basic fibroblast growth factor, and epidermal growth factor. Short tandem repeats using the Applied Biosystems AmpFISTR Identifier kit were used to authenticate cells. The last authentication test was performed in July 2017. All cell lines tested negative for mycoplasma contamination using the MycoAlert Detection Kit.
TMZ was from Sigma–Aldrich and lomeguatrib, elimusertib, ceralasertib, and berzosertib were from Selleckchem. For in vitro use, all inhibitors were dissolved in dimethyl sulfoxide. For irradiation experiments, we performed photon irradiation using an X-RAD 320-Precision X-Ray Biological Radiator to deliver a precise dosage of 2 Gy to 12 Gy to the cultured cells. After irradiation, cells were transferred to a cell culture–grade incubator for downstream applications. Sham-treated cultures were kept in close proximity to the X-RAD device for the same amount of time without exposure to X-rays.
TMZ was from Sigma–Aldrich and lomeguatrib, elimusertib, ceralasertib, and berzosertib were from Selleckchem. For in vitro use, all inhibitors were dissolved in dimethyl sulfoxide. For irradiation experiments, we performed photon irradiation using an X-RAD 320-Precision X-Ray Biological Radiator to deliver a precise dosage of 2 Gy to 12 Gy to the cultured cells. After irradiation, cells were transferred to a cell culture–grade incubator for downstream applications. Sham-treated cultures were kept in close proximity to the X-RAD device for the same amount of time without exposure to X-rays.
4
Inhibition of Sonic Hedgehog Pathway
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Anti-Smo, anti-Gli1, and anti-MGMT antibodies were purchased from Abcam (Cambridge, UK). Anti-Cyclin D1 and anti-CDK-6 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-β-actin antibody was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), and GANT61, GDC0449, and lomeguatrib were obtained from Selleck Chemicals (Houston, TX, USA). The TMZ was purchased from J&K Scientific (Beijing, People’s Republic of China).
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