Jurkat cells were seeded into 6-well plates (1x106 cells/well) and cultured at 37˚C for 24 h. Then, cells were transfected with 100 nM inhibitor control (the negative control of miR-221 inhibitor; 5'-CAGUACUUUUGUGUAGUACAA-3'; Guangzhou Ribobio Co., Ltd.), 100 nM miR-221 inhibitor (miR-221 antagonist' 5'-GAAACCCAGCAGACAAUGUAGCU-3'; Guangzhou Ribobio Co., Ltd.), 50 nM mimic control (the negative control of miR-221 mimic; 5'-CGGUACGAUCGCGGCGGGAUAUC-3'; Guangzhou Ribobio Co., Ltd.), 50 nM miR-221 mimic (miR-221 agonist; 5'-AGCUACAUUGUCUGCUGGGUUUC-3'; Guangzhou Ribobio Co., Ltd.), 100 nM miR-221 inhibitor + 0.2 µM control-small interfering (si)RNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.) or 100 nM miR-221 inhibitor + 0.2 µM PTEN-siRNA (cat. no. sc-29459; Santa Cruz Biotechnology, Inc.) using Lipofectamine 3000 reagent, according to the manufacturer's instructions. The transfection efficiency was detected 48 h later using RT-qPCR.
Pten sirna
Manufactured by Santa Cruz Biotechnology
Sourced in United States
PTEN siRNA is a small interfering RNA (siRNA) designed to target the PTEN gene. PTEN is a tumor suppressor gene that plays a crucial role in regulating cell growth and division. The PTEN siRNA is intended to temporarily silence the expression of the PTEN gene, which may be useful for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mir 221 inhibitor, by RiboBio (1 mentions)
Mir 221 mimic, by RiboBio (1 mentions)
Control small interfering si rna, by RiboBio (1 mentions)
Lipofectamine 3000, by Santa Cruz Biotechnology (1 mentions)
Inhibitor control, by RiboBio (1 mentions)
Mimic control, by RiboBio (1 mentions)
Non targeting sirna, by Santa Cruz Biotechnology (1 mentions)
Nqo1 sirna, by Santa Cruz Biotechnology (1 mentions)
Gfp pten, by Addgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
P3000, by Thermo Fisher Scientific (1 mentions)
Nonspecific sirna, by Santa Cruz Biotechnology (1 mentions)
Sall4 sirna, by Santa Cruz Biotechnology (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
X tremegene sirna transfection reagent, by Roche (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
9 protocols using pten sirna
1
Modulating miR-221 Expression in Jurkat Cells
2
miR-19b Regulation of Apoptosis Pathway
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miR-19b mimics and inhibitors (Dharmacon, Inc. 50 nM) were added to the culture media when the cells reached 50-60% confluence. The cells were transfected with the vehicle alone, an oligo control for the miR-19b mimic or an oligo control for the miR-19b inhibitor. Transfection efficiency was assessed using a GFP-labeled oligo control. Changes in miRNA expression were determined by qRT-PCR at 24 h following transfection. The Apaf1-siRNA, PTEN-siRNA, Casp3-siRNA and Casp7-siRNA were obtained from Santa Cruz Biotechnology.
3
Transfection of GBM cells
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The NQO1-siRNA (#sc-37139), PTEN-siRNA (#sc-29459), and nontargeting siRNA (#sc-37007) as a control were purchased from Santa Cruz Biotechnology. GFP-PTEN (#13039) plasmid was purchased from Addgene. GBM cells were transfected with 20 nM siRNA or 2 μg plasmid using the Lipofectamine 3000 and P3000 (#L3000075, Invitrogen) according to the manufacture's protocol with opti-MEM (#31985070, Gibo) as a transfection solution. Treated cells were incubated at 37°C for 48 h and then harvested for further experiments.
4
Knockdown and Overexpression in ICC-9810 Cells
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In order to knock down Sall4 or PTEN, or to overexpress Bmi-1, we transfected the siRNA or expressed vector into ICC-9810 cells by using lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instruction. Sall4 siRNA, PTEN siRNA, and nonspecific siRNA were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA); Bmi-1 expressed vector was purchased from Qiagen NV (Venlo, the Netherlands). The transfected cells were used for further analysis.
5
Lung Cancer Cell Line Knockdown
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The A549 human lung cancer cell line and the BEAS2B normal lung epithelial cell line were purchased from Shanghai Institute of Cell Biology, the Chinese Academy of Sciences. The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO2.
SHCBP1 siRNA was purchased from Abbexa Ltd. (cat. no. abx933286) and PTEN siRNA from Santa Cruz Biotechnology, Inc. (cat. no. sc-29459) Shanghai GenePharma Co., Ltd. synthesized the negative control (NC) siRNA. The siRNA-NC sequence was as follows: Forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3. According to the manufacturer's protocols, A549 cells were seeded in 6-well plates (1×105 cells/well) and starved in serum-free medium for 24 h prior to transfection for 72 h at room temperature using X-treme GENE siRNA transfection reagent (Roche Applied Science, Penzberg, Germany). The final concentration of SHCBP1 siRNA and siRNA-NC was 100 nM, and of PTEN siRNA was 200 nM.
SHCBP1 siRNA was purchased from Abbexa Ltd. (cat. no. abx933286) and PTEN siRNA from Santa Cruz Biotechnology, Inc. (cat. no. sc-29459) Shanghai GenePharma Co., Ltd. synthesized the negative control (NC) siRNA. The siRNA-NC sequence was as follows: Forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3. According to the manufacturer's protocols, A549 cells were seeded in 6-well plates (1×105 cells/well) and starved in serum-free medium for 24 h prior to transfection for 72 h at room temperature using X-treme GENE siRNA transfection reagent (Roche Applied Science, Penzberg, Germany). The final concentration of SHCBP1 siRNA and siRNA-NC was 100 nM, and of PTEN siRNA was 200 nM.
6
Luciferase Assay for IL2 Promoter Activity
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The luciferase assay was performed as described previously (Kim et al., 2016 (link)). In brief, a pGL3-Il2 promoter vector, a pRL Renilla luciferase control reporter vector, and PTEN siRNA (Santa Cruz Biotechnology, Inc.) were cotransfected into EL4 mouse lymphoma cells using a Gene Pulser (Bio-Rad Laboratories, Inc.) at 950 μF and 270 V. Transfected cells were then allowed to recover in complete medium for 18 h before stimulation with anti-CD3ε (10 µg/ml) and anti-CD28 (10 µg/ml) antibodies for 24 h. Cells were then harvested and examined using the Dual Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions. Relative luciferase activity was calculated by dividing Firefly luciferase activity by Renilla luciferase activity.
7
Nephroblastoma Cell Line WiT49 Transfection
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Nephroblastoma cell line WiT49 was provided by The Hospital for Sick Children (Toronto, Canada) and was subsequently grown in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin streptomycin. These cells were cultured in a humidified chamber at 37°C with 5% CO2. miR-130b-3p inhibitor (miR-130b-3p antagomir; sequence: 5′-UGCCAACCUUGCAAGCCGAAG-3′) and inhibitor control (antagomir-negative control; sequence: 5′-CAGUACUUUUGUGUAGUACAA-3′) were purchased from GenePharma Co. Ltd. WiT49 cells were transfected with either 100 nM inhibitor control, 100 nM miR-130b-3p inhibitor, 2 µl control-siRNA (cat. no. Sc-36869; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 2 µl PTEN-siRNA (cat. no. Sc-29459; Santa Cruz Biotechnology, Inc.), 100 nM miR-130b-3p inhibitor + 2 µl control-siRNA or 100 nM miR-130b-3p inhibitor + 2 µl PTEN-siRNA by using the Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells without any treatment were considered as the control group. The cells were subjected to the following experiments 48 h after transfection.
8
Palbociclib and Ribociclib Cytotoxicity Assay
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Palbociclib and ribociclib were from Elleckchem. They were prepared to 10 mM with dimethyl sulfoxide (DMSO) as stock solution and stored at −20°C. Then they were freshly diluted with cell culture medium to certain concentrations. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazol-iumbromide (MTT), antibody against β-actin (Cat# A2228), and monoclonal anti-HA-peroxidase antibody (Cat# H6533) were purchased from Sigma. Anti-Myc-peroxidase antibody (Cat# R951-25) was from Thermo Fisher. BrdU incorporation assay kit (#6831) and antibodies against PTEN (Cat# 9188), phospho-Rb (Ser280) (Cat# 8181), Rb (Cat# 9309), phospho-Akt (Ser473) (Cat# 4060), Akt (Cat# 4685), phospho-ERK (Thr202/Tyr204) (Cat# 4370), ERK (Cat# 4695), phospho-GSK-3β (Ser9) (Cat# 5558), GSK-3β (Cat# 12456), phospho-Elk-1 (Ser383) (Cat# 9186), and Elk-1 (Cat# 9182) were from Cell signaling (Beverly, MA). The horseradish peroxidase linked IgG secondary antibodies were obtained from GE Healthcare. Plasmids of constitutively active Akt (CA, Cat# 14751) (Scheid et al., 2002 (link)), ERK (CA, Cat# 39194) (Robinson et al., 1998 (link)), and PTEN (#78776) were from Addgene. PTEN siRNA (Cat# sc-29459) and control siRNA (Cat# sc-36369) were purchased from Santa Cruz. All chemicals and reagents not described above were from Sigma-Aldrich (St. Louis, MO).
9
PTEN Knockdown and Overexpression
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To knockdown the gene of phosphatase and tensin homologue (PTEN), PTEN small interfering RNA (PTEN siRNA, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. To overexpress PTEN, the PTEN eukaryotic expression plasmid was generated by cloning the open reading frame of the PTEN gene into the pcDNA3.1 plasmid (Life Technologies, Carlsbad, CA, USA). For transfection, cells were plated at 30-50% confluence. 24 h later, 50 pmol/ml RNA oligonucleotides or 2 μg/mL plasmids were transfected by using the Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction.
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