The following cells and reagents were used: HEK-293 (ATCC® CRL-1573™) and MCF-7 cells were purchased from ATCC. LCC9 cells are ERα+/progesterone receptor (PR) + cells resistant to antiestrogens (tamoxifen and fulvestrant) and were kindly provided by Dr. Robert Clarke, Georgetown University54 (link). Transfection reagents included Anti-miR-29a (Anti-miRTMs, Ambion), pre-miR-29b-1–3p or pre-miR-29a-3p precursor (Pre-miRTMs, Ambion), miR-29a mimic or miR-29b-1 mimic (miR-VanaTM, Ambion), Lipofectamine RNAiMAX (Invitrogen), FuGENE® HD (Promega) Pre-miR™ negative control #1 (Ambion), Anti-miR™ negative control #1(Ambion), miR-VanaTM negative control (Ambion), pEZX-MT06_ATP5G1 wild type (WT), pEZX-MT06_ATPIF1 (WT), pEZX-MT06_negative control (miTargetTM miRNA 3′ UTR, GeneCopoeia).
Hek 293 atcc crl 1573
Manufactured by American Type Culture Collection
Sourced in United States
HEK-293 (ATCC® CRL-1573™) is a cell line derived from human embryonic kidney cells. It is a widely used host cell line for the production and study of recombinant proteins and viral vectors.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Mcf 7 cell, by American Type Culture Collection (1 mentions)
Mirvanatm, by Thermo Fisher Scientific (1 mentions)
Pre mirtms, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Fugene hd, by Promega (1 mentions)
Roti mark, by Carl Roth (1 mentions)
β mercaptoethanol, by Serva Electrophoresis (1 mentions)
Mcf 7 atcc htb 22, by American Type Culture Collection (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
Celltiter blue reagent, by Promega (1 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by GE Healthcare (1 mentions)
Wst 1 reagent, by Roche (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Penicillin, by Sartorius (1 mentions)
Streptomycin, by Sartorius (1 mentions)
13 protocols using hek 293 atcc crl 1573
1
Modulating miR-29 in Antiestrogen-Resistant Cells
2
Cytotoxicity of L-dopa, M. pruriens, and Nanogel on HEK293 Cells
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The human embryonic kidney cell line HEK293 ATCC CRL-1573 was obtained from ATCC (Manassas, VA, USA) and maintained in EMEM supplemented with 10% FBS and 1% antibiotic–antimycotic solution at 37 °C in 5% CO2/95% air [20 (link)]. The cytotoxicity of 0.01–1000 µg/mL L-dopa, 0.01–100 µg/mL M. pruriens seed extract, and 0.02–5000 µg/mL nanogel were investigated by XTT assay using the above-mentioned method [20 (link)].
3
Cell Viability Assay Protocol
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Glass beads (0.25–0.50 mm), GSH, protein marker Roti®-Mark, Rotiphorese Gel 30 SDS Ultra-Pure and Trishydroxyaminoethane (Tris) were from Roth (Karlsruhe, Germany), Cell Titer-Glo® reagent and Cell Titer-Blue reagent® were from Promega, (Mannheim, Germany), Coommassie Brilliant Blue R250, N,N,N′,N′-tetramethylethylenediamine (TEMED), 3.3-diaminobenzidin-4-hydrochloride (DAB) and β-mercaptoethanol were from Serva (Heidelberg, Germany) and WST-1 reagent was from Roche (Mannheim, Germany). Transfection reagent TurboFect™ was from Fermentas (St. Leon-Rot, Germany), RPMI 1640, Dulbecco’s modified Eagle Medium (DMEM), Opti-MEM®, fetal calf serum (FCS) and Zeocin were from Invitrogen (Karlsruhe, Germany), hematoxylin was from Merck (Darmstadt, Germany), milk powder from Heirler Cenovis (Radolfzell, Germany), and bovine serum albumin was from PAA Laboratories (Linz, Austria). Cell lines used were the human breast adenocarcinoma cell line MCF-7 (ATCC HTB-22) and the human embryonic kidney cell line HEK 293 (ATCC CRL-1573). All additional cell culture material was from Greiner Bio-One (Frickenhausen, Germany). All other chemicals were from Sigma-Aldrich (Taufkirchen, Germany).
4
Culturing Human Epithelial and Kidney Cells
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HBECs (Cascade Biologics, Portland, OR, USA) were cultured in epithelial cell growth medium (medium 200; Cascade Biologics, USA) at 37°C in an incubator with 5% CO2. The medium contained 10% fetal bovine serum (FBS), 1X low serum growth supplement, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B (Biological Industries, Kibbutz Beit Haemek, Israel). Human embryonic kidney 293 cells (HEK293, ATCC® CRL-1573™) were cultured in Dulbecco's modified Eagle's medium (Cascade Biologics, USA) at 37°C in an incubator with 5% CO2. The medium was supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B.
5
Cell Line Acquisition and Cultivation
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The human embryonic kidney cells HEK293 (ATCC® CRL-1573™), the colorectal adenocarcinoma cell line HT-29 (ATCC® HTB-38), the melanoma SK-MEL-28 (ATCC® HTB-72™), and breast adenocarcinoma MDA-MB-231 (ATCC® HTB-26™) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).
6
Scoulerine Inhibits Ovarian Cancer
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Scoulerine was procured from Herbest Bio-Tech, China (CAS 6451-73-6) (purity > 98 %). Human ovarian epithelial adenocarcinoma cells (OVCAR3; ATCC ® HTB161™) and HEK293; ATCC ® CRL-1573™) were obtained from the ATCC, USA. Antibodies, anti-GAPDH, anti-N-Cadherin, anti-E-Cadherin, anti-Vimentin, anti-CD44, anti-Oct-4, anti-Sox2, anti-Ald-h1A1, anti-Akt and anti-p-Akt were obtained from Cell Signaling Technology (USA). While other antibodies, anti-CD24, anti-PI3K, anti-mTOR and anti-p-mTOR were purchased from Santa Cruz Biotechnology (USA). Clarity Western ECL substrate luminol assay kit was obtained from (BioRad Laboratories, Inc. Tumorsphere medium was obtained from Stem Cell Technologies. All chemicals are purchased from Sigma or stated accordingly.
7
Pediatric Medulloblastoma Cell Lines
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The pediatric MB cell lines D283 Med and USP-13-MED [15, 16] (Grp3/4-MBs), and the Human Embryonic Kidney 293 cell (HEK93T) were used in this study. D283 Med (ATCC HTB-185) and HEK-293 (ATCC CRL-1573) cells were obtained from the American Type Culture Collection, and the USP 13-MED cell line was kindly provided by Prof. Dr. Oswaldo Keith Okamoto -Biosciences Institute of the University of São Paulo. The cell lines authentications were performed to validate the Short Tandem Repeat (STR) prolife. All cells were maintained in DMEM/F12 medium (Gibco™, Thermo Fisher®, Carlsbad, CA, USA), supplemented with 10% FBS, 100U/ml penicillin, 100 µg/ml streptomycin and kept in a humid atmosphere containing 5% CO2 at 37ºC.
8
Evaluating Small Molecule Modulators in MM Cells
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The human MM cell lines, U266 (ATCC® TIB-196™) and HEK293 (ATCC® CRL-1573™) were purchased from the American Type Culture Collection, KMS12 were purchased from DSMZ collection of microorganisms and cell cultures. U266 and KMS12 were grown in RPMI1640 (Lonza, Italy) and HEK293T cell line was grown in DMEM (Lonza, Italy); both media were supplemented with 10% heat-inactivated FBS (Euroclone, Italy), 2 mM L-Glutamine (Euroclone, Italy), 100 U/ml Penicillin and 100 μg/ml Streptomycin (Sigma-Aldrich, Germany).
DAPT (GSI-IX, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) was purchased from Sigma-Aldrich, reconstituted in DMSO and administered to cells at a final concentration of 25 μM. Three compounds selected from the docking procedure, namely BAS 00693376, BAS 00327971 (named from here on as IGOR1 and IGOR2, respectively), and ASN13406980 (used as negative control), were purchased from Asinex (www.asinex.com ) and reconstituted in DMSO at a concentration of 50 mM.
DAPT (GSI-IX, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) was purchased from Sigma-Aldrich, reconstituted in DMSO and administered to cells at a final concentration of 25 μM. Three compounds selected from the docking procedure, namely BAS 00693376, BAS 00327971 (named from here on as IGOR1 and IGOR2, respectively), and ASN13406980 (used as negative control), were purchased from Asinex (
9
Cell Line Cultivation Protocols
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Human gastric cancer cell line MKN74 was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB025, JCRB Cell Bank, Tokyo, Japan). HeLa (ATCC® CCL-2™, derived from human cervix carcinoma) and human embryonic kidney 293 (HEK293, ATCC® CRL-1573™) cell lines were purchased from the American Type Culture Collection (ATCC®, Rockville, MD, USA). MKN74 cells were maintained in RPMI medium 1640 Glutamax I (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone, Thermo Fisher Scientific) and 1% (v/v) Penicillin/Streptomycin (Gibco). HeLa and HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific, Inc, UK) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone, Thermo Fisher Scientific, Inc, UK) and 1% (v/v) Penicillin/Streptomycin (Gibco). All cultured cells were maintained at 37 °C in a humidified 5% CO2 incubator and the medium was substituted every 2–3 days.
10
HEK293 and HK-2 Cell Culture Protocol
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The cell lines used in this study, HEK293 ATCC® CRL‐1573 (human embryonic kidney cell line) and HK‐2 ATCC® CRl‐2190 (kidney proximal tubule epithelial cell line), were purchased from the American Type Culture Collection (ATCC). They were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotic‐antimycotic (Gibco) in 25‐ and 75‐cm3 culture flasks and were maintained in a humidified atmosphere with 5% CO2 at 37°C.
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