Human lung adenocarcinoma cell lines PC9 and HCC827 were obtained from ATCC (Manassas, VA, USA). The cells were grown in RPMI-1640 medium (Hyclone, UT, USA) with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 100 U/mL penicillin/streptomycin at 37 °C in a 5% CO2 culture chamber. Escalating doses of gefitinib (Selleckchem, TX, USA) ranging from 0.01 to 10.0 mM were added to the medium in order to induce drug resistance, and 1 µM gefitinib was added every 2 weeks to maintain resistance of gefitinib-resistant PC9 (PC9R) cells. Lentiviral particles were collected by transfecting 293T cells with plasmids (GeneChem, Shanghai, China) expressing negative control short hair RNA (shNEG) or short hair RNA against PLAT (shPLAT, RNAi Consortium). Lentivirus overexpressing human PLAT (PC9-PLAT, GenBank accession number NM_000930) was obtained from GeneCopoeia (Germantown, MD, USA). After cells reached 60% confluence in 6-well plates, they were infected with lentiviral particles at a multiplicity of infection of 10 and incubated with 4 µg/mL polybrene in medium.
Gefitinib
Manufactured by Selleck Chemicals
Sourced in United States, Germany, China, France
Gefitinib is a tyrosine kinase inhibitor used in laboratory research. It functions by inhibiting the epidermal growth factor receptor (EGFR) tyrosine kinase.
Automatically generated - may contain errors
Lab products found in correlation
Erlotinib, by Selleck Chemicals (66 mentions)
Afatinib, by Selleck Chemicals (50 mentions)
Osimertinib, by Selleck Chemicals (42 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (27 mentions)
Lapatinib, by Selleck Chemicals (26 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (21 mentions)
Crizotinib, by Selleck Chemicals (19 mentions)
Trametinib, by Selleck Chemicals (17 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (17 mentions)
Hcc827, by American Type Culture Collection (13 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (13 mentions)
Sorafenib, by Selleck Chemicals (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Penicillin, by Thermo Fisher Scientific (11 mentions)
Cisplatin, by Merck Group (10 mentions)
Cisplatin, by Selleck Chemicals (10 mentions)
Dacomitinib, by Selleck Chemicals (10 mentions)
Ly294002, by Selleck Chemicals (10 mentions)
Dasatinib, by Selleck Chemicals (10 mentions)
322 protocols using gefitinib
1
Inducing Gefitinib Resistance in Lung Cancer Cell Lines
2
Development of Gefitinib-resistant Lung Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human lung carcinoma H292 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Embryonic lung fibroblast (MRC-5) cells were provided from the Korean Cell Line Bank (Seoul, Korea). H292 cells were cultured in RPMI 1640 medium, and MRC-5 cells were cultured in MEM medium supplemented with 10% FBS and antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B). Cells were incubated at 37 °C with 5% CO2 in a humidified atmosphere. Gefitinib-resistant H292 cells (H292-Gef) were developed from the parental H292 cells through continuous exposure to gradually increasing concentrations of Gefitinib (Selleckchem, Houston, TX, USA) and were maintained in RPMI 1640 medium containing 1 μM Gefitinib.
3
DRAM1 Overexpression Enhances Gefitinib Efficacy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five-week-old male nude BALB/c mice were obtained from Soochow University. Mice were housed in SPF conditions with food and water ad libitum, a 12 h light/dark cycle and controlled (22–23 °C) temperature. Animal welfare and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA) and the related ethical regulations of Soochow University. Mice were randomly injected subcutaneously with 5 × 105 PC9 or 5 × 105 DRAM1-overexpressing PC9 cells in 30 μl of RPMI-1640 on the right back flank. Two weeks later, tumor size was measured with electronic calipers every 4 days through blind outcome assessment. Tumor volumes (V) were calculated by the formula V = (X2Y)/2, where X and Y are the shortest and longest diameters of the tumor, respectively. Mice were sacrificed on day 41 after tumor cell injection. For determination of a role of DRAM1 in gefitinib chemotherapy, mice bearing PC9 cells or DRAM1-overexpressing PC9 cells were orally administered gefitinib (S1025, Selleck), dissolved in 0.5% CMC-Na, at dose of 50 mg/kg/day for 13 days. Tumor size and weight were measured. Tumors were collected and fixed with 4% formaldehyde or stored at −80 °C.
4
Caspase-3/7 Assay for Gefitinib Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at equal densities into culture plates and allowed to adhere overnight. Prior to gefitinib treatment, cells were serum deprived in 1% FBS-containing media for 2 h and then treated with 1 μM gefitinib (Selleckchem, cat #S1025) for 24-72 h. Reconstituted Caspase-Glo 3/7 assay reagent (Promega, cat #G8093) was then added into each well, mixed via orbital shaker, and incubated at room temperature for 45 min. Luminescence was quantified with a BioTek Synergy H1 plate reader. The values represented were normalized to the caspase value for untreated cells. At least two independent experiments were conducted for each cell line.
5
Induction of Gefitinib Resistance in Lung Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human lung adenocarcinoma cell line PC9 and NCI-H1975 (H1975, intrinsically harbor EGFR L858R/T790 M) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). To induce gefitinib resistance, PC9 cells were exposed to increasing concentrations of gefitinib [39 (link)]. The gefitinib-sensitive PC9 and -resistance PC9R and H1975 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin. The gefitinib resistance in the PC9R and H1975 cells was maintained by adding 1 μΜ gefitinib (Selleckchem, TX, USA). The cells were grown as monolayers in a humidified atmosphere containing 5% CO2 at 37 °C.
6
In Vivo Evaluation of CHIR-99021 and Gefitinib in Lung Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung cancer cells (NCI-H460) were cultured with HUVECs seeded at a density of 1 × 106 cells/well in an ultra-low attachment 6-well plate (Corning B.V. Life Sciences, Amsterdam, Netherlands). After 2 days, MCTSs were implanted subcutaneously in male BALB/c-nu mice and allowed to grow to a tumor volume of 100–200 mm. Tumor-bearing mice were randomized into the following four groups of six mice each; Group 1, control with normal saline (N/S); Group 2, treated with CHIR-99021 (Selleckchem, Houston, TX, USA), 16 mg/kg, intraperitoneal injection (i.p.); Group 3, treated with Gefitinib (Selleckchem, Houston, TX, USA), 50 mg/kg, oral injection (p.o.); Group 4, treated with CHIR-99021 and Gefitinib, respectively. All drugs were administered 3 days a week for 2 weeks. All treated mice were sacrificed on day 28, and tumors were resected for further histological evaluation.
7
Establishment of Gefitinib-Resistant NSCLC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human PC-9 (EGFR mutation-positive, exon 19 deletion) NSCLC cells were from Riken BioResource Research Center (Tokyo, Japan) and A549 (wild-type EGFR) NSCLC cells and HEK293 cells were from the American Type Culture Collection (ATCC, Manassas, VA, USA). PC9 and A549 cells were propagated in RPMI-1640 medium containing 10% fetal calf serum, and 1% antibodies (all from PAA-Laboratories, Cölbe, Germany). HEK293 cells were cultivated under standard protocols provided by ATCC. All cells were maintained at 37 °C, 85% humidity, and 5% CO2. We established gefitinib-resistant NSCLC cells (PC9/GR and A549/GR) by exposing PC9 and A549 cells to increasing concentrations of gefitinib (Selleck Chemicals, Houston, TX, USA) as reported [16 (link)] for 6 months.
8
Establishment of Gefitinib-Resistant Lung Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gefitinib-sensitive PC9 and HCC4006 human lung adenocarcinoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA). To induce Gefitinib resistance, the cells were exposed to increasing concentrations of Gefitinib (ranging from 0.01 to 10.0 mM; Selleck Chemicals, Houston, TX, USA). The viability of Gefitinib-resistant PC9R and HCC4006R cells was unaffected up to 10 µM Gefitinib (Fig. 1J and K ), whereas the viability of Gefitinib-sensitive PC9 and HCC4006 cells was significantly reduced in the presence of 0.1 µM Gefitinib (Fig. 1D ). Gefitinib-sensitive and -resistant cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin. In order to maintain Gefitinib resistance, 1 µM Gefitinib was added to the cells every 2 weeks. The cells were grown as monolayers in a humidified atmosphere of 5% CO2 at 37°C.
9
Protein Expression Modulation by Targeted Drugs
10
Establishment of Gefitinib-Resistant PC-9 Cell Line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PC-9 cells were obtained from Affiliated Cancer Hospital & Institute of Guangzhou Medical University. The gefitinib-resistant PC-9 cell line was established by continually exposing to a stepwise escalation of the concentration of gefitinib (Selleckchem, United States) from 0.1 to 15 μM over 8 months. gefitinib-sensitive and gefitinib-resistant PC-9 cell lines were maintained in RPMI 1640 (Gibco, United States) supplemented with 10% fetal bovine serum (Gibco, United States) and 1% penicillin and streptomycin (Thermo Fisher Scientific) in a humidified condition with 5% CO2 at 37°C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!