For proliferation assays cells were plated in 384-well plates at a density of 800 cells per well in 40 µl total volume. One day later, a serial dilution of each inhibitor was performed using a D300e dispenser (Tecan). 96 h post-treatment, cell viability was determined using CellTiterGlo reagent (Promega) and luminescence quantified on an Envision MultiLabel Plate Reader (PerkinElmer). To calculate the fraction cell viability drug-treated cells were normalized to average cell viability of DMSO-only treated cells. Curve fitting was performed using GraphPad Prism v9.1.1 four-parameter inhibitor response with variable slope. AUC values were calculated by GraphPad Prism v9.1.1. Inhibitors were obtained from SelleckChem: Erlotinib-OSI-744 (S1023), Osimertinib-AZD9291 (S7297), Torin 1 (S2827), Alisertib-MLN8237 (S1133), Barasertib-AZD1152 (S1147). DMSO (Sigma-Aldrich).
Erlotinib osi 744
Manufactured by Selleck Chemicals
Sourced in United States
Erlotinib-OSI-744 is a laboratory compound used for research purposes. It is a tyrosine kinase inhibitor that targets the epidermal growth factor receptor (EGFR). This compound is commonly used in scientific studies to investigate cellular signaling pathways and potential therapeutic applications.
Automatically generated - may contain errors
Lab products found in correlation
Alisertib mln8237, by Selleck Chemicals (2 mentions)
Envision multilabel plate reader, by PerkinElmer (2 mentions)
Torin1, by Selleck Chemicals (2 mentions)
Osimertinib azd9291, by Selleck Chemicals (2 mentions)
D300e dispenser, by Tecan (2 mentions)
Barasertib azd1152, by Selleck Chemicals (2 mentions)
Celltiter glo reagent, by Promega (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Afatinib bibw2992, by Selleck Chemicals (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Crl 2868, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using erlotinib osi 744
1
Cell Viability Assay for Inhibitor Screening
2
Small Molecule Inhibitors in Cancer
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Afatinib (BIBW2992) and erlotinib (OSI-744) were purchased from Selleck Chemicals (TX, USA). The protein assay kit was from Bio-Rad (Hercules, CA, USA). The E-cadherin antibody was a gift from Dr. Keith R. Johnson (University of Nebraska Medical Center). All other antibodies used are listed in Supplementary Table 1 .
3
Developing Erlotinib-Resistant Lung Cancer Cell Lines
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The human lung cancer HCC827 cell line was purchased from ATCC (ATCC® CRL-2868™). The PC9 cell line, which was derived from a human adenocarcinoma of lung tissue, was preserved in our laboratory. The lung cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Gibco BRL, Carlsbad, CA, USA) and 100 U/ml penicillin/streptomycin at 37°C in a humidified incubator containing 5% CO2. Erlotinib (OSI-744) was purchased from Selleck Chemicals (Houston, TX, USA). Insulin-like growth factor 1 human recombinant was obtained from ProSpec (ProSpec, Rehovot, Israel). Two erlotinib-resistant lines, namely HCC827/ER and PC9/ER, were developed by applying high-dose (1–5 μM) pulses of erlotinib combined with continuous low-dose (0.01 μM) administration for >8 months (13 (link)). To avoid the effects of the drugs, resistant cell lines were cultured in a drug-free medium for ≥2 weeks prior to further experiments.
4
Proliferation Assay with Kinase Inhibitors
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For proliferation assays cells were plated in 384-well plates at a density of 800 cells per well in 40 µl total volume. One day later, a serial dilution of each inhibitor was performed using a D300e dispenser (Tecan). 96 hours post-treatment, cell viability was determined using CellTiterGlo reagent (Promega) and luminescence quantified on an Envision MultiLabel Plate Reader (PerkinElmer). To calculate the fraction cell viability drug-treated cells were normalized to average cell viability of DMSO-only treated cells. Curve fitting was performed using GraphPad Prism four parameter inhibitor response with variable slope. AUC values were calculated by GraphPad Prism (Graphpad). Inhibitors were obtained from SelleckChem: Erlotinib-OSI-744 (S1023), Osimertinib-AZD9291 (S7297), Torin 1 (S2827), Alisertib-MLN8237 (S1133), Barasertib-AZD1152 (S1147). DMSO (Sigma-Aldrich).
5
Establishment of Erlotinib-Resistant Lung Cancer Cell Lines
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The human lung cancer HCC827 cell line was purchased from ATCC (ATCC ® CRL-2868™). HCC827/ER cells were cultured by Dr Jing Han by applying high-dose (1-5 µM) pulses of erlotinib combined with continuous lowdose (0.01 µM) administration for >8 months (12). H1650/ ER cells were presented by Dr Li Li (Department of Respiratory Disease, Daping Hospital, Third Military Medical University, Chongqing, China). The lung cancer cells were grown in RPMI-1640 medium containing 10% fetal bovine serum (Gibco BRL, Carlsbad, CA, USA) and 100 U/mL penicillin/streptomycin at 37°C in a humidified incubator containing 5% CO 2 . Erlotinib (OSI-744) was purchased from Selleck Chemicals (Houston, TX, USA).
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