Human RIG-I complementary DNA (cDNA) was purchased from Sino-Biological; the cDNA was amplified and clone into viral vector Plvx-IRES-Puro (Clontech). The clustered regularly interspaced short palindromic repeats (CRISPR)-related plasmid PX458 and lentivirus packaging plasmids Pspax2 and PMD2.G were purchased from Addgene. Fibrinogen and thrombin were purchased from Searun Holdings. Methotrexate (MTX) and paclitaxel (PAX) were purchased from Selleck. STAT3 inhibitor Stattic, c-SRC inhibitor dasatinib and ITGB3 inhibitor cilengitide were purchased from MedChemExpress. Puromycin and Lipofectamine 2000 were purchased from Invitrogen (California, USA). Murine and human IFNA were purchased from Novoprotein. Polybrene, collagenase I and collagenase IV were purchased from Sigma.
Methotrexate mtx
Manufactured by Selleck Chemicals
Sourced in United States
Methotrexate (MTX) is a chemical compound used in various laboratory applications. It is a folate antagonist that inhibits the enzyme dihydrofolate reductase, which is essential for cell division and growth. MTX is commonly used in research settings to study cellular processes and the effects of folate metabolism on biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Thermo Fisher Scientific (1 mentions)
Stattic, by MedChemExpress (1 mentions)
Cilengitide, by MedChemExpress (1 mentions)
Plvx ires puro, by Takara Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Sphingosine 1 phosphate (s1p), by Cayman Chemical (1 mentions)
Verteporfin, by Selleck Chemicals (1 mentions)
5 fluorouracil 5 fu, by Selleck Chemicals (1 mentions)
Bromodeoxyuridine (brdu), by Abcam (1 mentions)
Dhcr24, by Abcam (1 mentions)
Dpp 4, by Abcam (1 mentions)
Pcmv3 plasmids, by Sino Biological (1 mentions)
Sitagliptin, by Selleck Chemicals (1 mentions)
β actin, by Abcam (1 mentions)
Cytarabine ara c, by Selleck Chemicals (1 mentions)
Vincristine vcr, by Selleck Chemicals (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
5 protocols using methotrexate mtx
1
Establishment of RIG-I Overexpression Cell Line
2
Optimized In Vitro and In Vivo Drug Preparation
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S1P (CAS26993-30-6; Cayman Chemical, Michigan, USA), Verteporfin (S1786; Selleckchem, Houston, Texas, USA), TY52156 (HY-19736; MedChem Express, Monmouth, NJ, USA) and Methotrexate (MTX) (S1210; Selleckchem, Houston, Texas, USA) were diluted and stored in accordance with manufacturer recommendations. As for in vitro experiments, drug stocks were diluted in the base media. While stocks were diluted in saline immediately prior to use in vivo experiments. 2 μM Verteporfin, 10 μM TY52156 and 1 μM MTX were used in in vitro experiments.
3
Cytotoxicity Evaluation of Anti-cancer Drugs
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Cells were seeded into 96-well plates at a density of 5 × 103 cells per well. After one day of growth, the cells were treated with different concentrations of Etoposid (VP – 16) (Selleck, USA) 5-Fluorouracil (5 – FU) (Selleck, USA), Methotrexate (MTX) (Selleck, USA), Paclitaxel (TAXOL) (Selleck, USA), or ActD (Selleck, USA) and incubated at 37°C for 72 hours. Cell viability was detected by CCK – 8 viability assay at 72 hours after drug treatment [9 (link)]. The absorbance at 490 nm was measured with a Varioskan Flash microplate reader (Thermo Fish Scientific, Waltham, MA, USA) and the results were calculated using SPSS software.
The concentrations of drugs used in MTX-resistant cells were listed below:
VP-16:0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 ng/ml;
5-FU:0.05, 0.1, 0.5, 1, 5, 10, 20, 50 μg/ml;
TAXOL:0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30 nM;
MTX:1, 4, 10, 40, 100, 400, 1000 μM;
ActD:0.1, 0.4, 0.8, 1, 4, 8, 10, 40 μg/ml.
The concentrations of drugs used in original cells were listed below:
VP-16:0.005, 0.01, 0.05, 0.1, 0.5, 1, 2 ng/ml;
5-FU:0.05, 0.1, 0.5, 1, 5, 10, 20 μg/ml;
TAXOL:0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30 nM;
MTX:0.01, 0.04, 0.1, 0.4, 1, 4, 10, 40 μM;
ActD:0.1, 0.4, 0.8, 1, 4, 8, 10, 40 μg/ml.
(Note: It is converted into a uniform concentration unit for subsequent statistics)
The concentrations of drugs used in MTX-resistant cells were listed below:
VP-16:0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 ng/ml;
5-FU:0.05, 0.1, 0.5, 1, 5, 10, 20, 50 μg/ml;
TAXOL:0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30 nM;
MTX:1, 4, 10, 40, 100, 400, 1000 μM;
ActD:0.1, 0.4, 0.8, 1, 4, 8, 10, 40 μg/ml.
The concentrations of drugs used in original cells were listed below:
VP-16:0.005, 0.01, 0.05, 0.1, 0.5, 1, 2 ng/ml;
5-FU:0.05, 0.1, 0.5, 1, 5, 10, 20 μg/ml;
TAXOL:0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30 nM;
MTX:0.01, 0.04, 0.1, 0.4, 1, 4, 10, 40 μM;
ActD:0.1, 0.4, 0.8, 1, 4, 8, 10, 40 μg/ml.
(Note: It is converted into a uniform concentration unit for subsequent statistics)
4
Cell Culture and Transfection Reagents
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Primary antibodies were provided by following sources: DPP4, DHCR24, BrdU, and β-Actin (Abcam, Cambridge, MA, USA); Methotrexate (MTX) and DPP4 inhibitor Sitagliptin were obtained from Selleckchem (Houston, TX, USA). pCMV3 plasmids encoding empty vector (EV) or DCHR24 were obtained from Sinobiological Inc. (Beijing, China). Lentiviral shRNA plasmids encoding scramble control, shDHCR24, and shDPP4 were provided by Genecopoeia Inc. Human GTN cell lines JEG3 JAR, and BEWO were provided by American Type Culture Collection (Manassas, VA, USA). These GTN cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 4 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin.
5
Cytarabine, Methotrexate, and Vincristine Protocol
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Cytarabine (Ara-C) (Selleckchem, Cat # S1648), Methotrexate (MTX) (Selleckchem, Cat # S1210), Vincristine (VCR) (Selleckchem, Cat # S1241), MG132 (Selleckchem, cat # S2619), Caffeine (Sigma-Aldrich, Cat # C0750), and 79-6 (Calbiochem, Cat # 197345) were diluted and stored per manufacturer recommendations. For in vitro experiments drug stocks were diluted in base media and for in vivo experiments stocks were diluted in saline immediately prior to use. In vitro concentrations of Ara-C [1 μM], MTX [50 μM], VCR [25 μM], MG-132 [1-5 μM], Caffeine [2.5-10 mM], and 79-6 [125 μM] were used to approximate clinically relevant doses in ALL or published in vitro concentrations [27 (link), 57 (link)- 63 (link)].
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