Human kidney epithelial cells (HK-2) were purchased from the National Platform of Experimental Cell Resources for Science (Beijing, China) and cultured in DMEM/F12 medium (Thermo Fisher Scientific, Pittsburgh, PA, USA) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 1% penicillin–streptomycin (Gibco Life Technologies, Carlsbad, CA, USA) and 1% insulin–transferrin–selenium (Sigma) at 37 °C in a humidified chamber with 5% CO2. HK-2 cells were grown for 24 h to reach approximately 90% confluence before being randomly divided into three groups: control group, LPS group (LPS 10 μg/ml), LPS + GA group (LPS 10 μg/ml + GA 50 μg/ml). After 24 h treatment, the cells were harvested for various analyses. For LPS + GA + si-SIRT1 group, cells were transiently transfected with 50 nM si-SIRT1 (GenePharma, Shanghai, China) by Lipofectamine 2000 (Invitrogen) prior to LPS and GA treatment.
Si sirt1
Manufactured by GenePharma
Sourced in China
The Si-SIRT1 is a laboratory equipment product designed to facilitate the study of the SIRT1 protein. SIRT1 is an important regulator of cellular processes and is involved in various physiological and pathological conditions. The Si-SIRT1 product provides researchers with the necessary tools to investigate the role and function of SIRT1 in their research studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pcdna sirt1, by GenePharma (2 mentions)
Si control, by GenePharma (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Si nc, by GenePharma (2 mentions)
Si nc duplexes, by GenePharma (1 mentions)
Mir 494 mimic, by GenePharma (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Si sirt3, by GenePharma (1 mentions)
Pcdna3.1 nc, by GenePharma (1 mentions)
Inhibitor nc, by GenePharma (1 mentions)
Mimics nc, by GenePharma (1 mentions)
Antagomir nc, by RiboBio (1 mentions)
Mir 181a mimic, by RiboBio (1 mentions)
Small interfering si rna, by GenePharma (1 mentions)
18 protocols using si sirt1
1
LPS-Induced Injury in Human Kidney Cells
2
SIRT1 Silencing in Rat Cells
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si-SIRT1 and si-NC duplexes were purchased from Genepharma (Shanghai, China) and have the following sequences: Rat SIRT1 (forward 5′-GAAGUUGACCUCCUCAUUGUdTdT-3′ and reverse 5′-ACAAUGAGGAGGUCAACUUCdTdT-3′) using lipofectamine 2000 according to manufacturer's protocol.
3
miR-494 Modulation in H9c2 Cells
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H9c2 cells were seeded into 6-well plates (3.5×105 cells/well) or 96-well plates (1.5×104 cells/well). At 80% confluence, cells were transfected with 50 nM miR-494 mimic (5′-UGAAACAUACACGGGAAACCUCU-3′), 50 nM miR-494 mimic negative control (NC; 5′-UUCUCCGAACGUGUCACGUTT-3′), 100 nM miR-494 inhibitor (5′-AGAGGUUUCCCGUGUAUGUUUCA-3′), 100 nM miR-494 inhibitor NC (5′-CAGUACUUUUGUGUAGUACAA-3′), 50 nM small interfering (si)RNA targeting SIRT1 (siSIRT1; 5′-CCCUGUAAAGCUUUCAGAATT-3′) or 50 nM siRNA negative control (5′-UUCUCCGAACGUGUCACGUTT-3′ for 4 h at 37°C; all purchased from Shanghai GenePharma Co., Ltd.) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 28 h post-transfection, cells were collected, exposed to H/R treatment and used for subsequent experiments.
4
Modulating SIRT1 expression in colorectal cancer
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LOVO and SW480 cells (1 × 105) were seeded onto 6-well plates. Short interfering RNA of SIRT1 (siSIRT1) 5′- CCAAGCAGCUAAGAGUAAUTT-3′ (sense), 5′- AUUACUCUUAGCUGCUUGGTT-3′ (antisense) (Genepharma, China) and pcDNA-SIRT1 (Genepharma, China) were transiently transfected into LOVO and SW480 cells when cells reached 70–80% confluence using Lipofectamine™ 3000 Reagent (Invitrogen, USA). After 48 h transfection, the cells were collected for further experiments.
5
siRNA-Mediated SIRT1 Knockdown
6
Ischemic Injury Cell Treatment
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For cell treatment, H/R-injured H9c2 cells were treated with either dabigatran (1 nM) or thrombin (5 U/ml) with or without dabigatran (1 nM). In addition, H/R-injured H9c2 cells were treated with thrombin (5 U/ml) with or without 3-MA. The dose of thrombin was determined according to our experimental experience and previous reference [17 (link)].
For cell transfection, H9c2 cells treated with thrombin (5 U/ml) were transfected with si-SIRT1 or si-NC (all purchased from GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
For cell transfection, H9c2 cells treated with thrombin (5 U/ml) were transfected with si-SIRT1 or si-NC (all purchased from GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
7
Modulation of MFF and SIRT Genes in PAMSCs
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The MFF-overexpressing vectors (pcDNA3.1-MFF), small interfering RNA (siRNA) targeting to MFF, SIRT1 and SIRT3 (si-MFF, si-SIRT1, si-SIRT3), miR-340-5p mimics, and miR-340-5p inhibitor as well as their corresponding negative controls (pcDNA3.1-NC, si-NC, inhibitor NC, mimics NC) were synthesized and purchased from Genepharma (Shanghai, China). PAMSCs were maintained in a 96-well plate and the transfection experiments were performed by using Lipofectamine™ 3000 (Invitrogen, CA, USA). After 48 h, the transfection efficiency was measured using qRT-PCR method.
8
ANRIL Overexpression and miR-181a Modulation
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ANRIL-overexpressing plasmids were constructed by inserting the ANRIL coding sequence into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). Oligonucleotides, including miR-181a mimic (5′-AACAUUCAACGCUGUCGGUGAGU-3′), negative control (NC) mimic (5′-UUCUCCGAACGUGUCACGUTT−3′), miR-181a antagomir (5′-ACUCACCGACAGCGUUGAAUGUU-3′) and NC antagomir (5′-CAGUACUUUUGUGUAGUACAA−3′), were purchased from Guangzhou RiboBio Co., Ltd. Small interfering (si)RNA targeting SIRT1 (siSIRT1, 5′-CCCUGUAAAGCUUUCAGAATT−3′) and NC siRNA (siNC; 5′-UUCUCCGAACGUGUCACGUTT−3′) were obtained from Shanghai GenePharma Co., Ltd. When the cell density reached 50–60% confluence, cell transfection was performed with Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Equal amounts of empty pcDNA3.1 vector (4 µg/well), NC mimic, NC antagomir (50 or 150 nM) or siNC (10 or 20 nM) diluted in the same volume of transfection reagents were transfected as controls, depending on the experimental purposes. The efficiency was examined 2 days after transfection. For H/R treatment, cells were cultured under normoxia or exposed to H/R (hypoxia for 4 h followed by reoxygenation for 8 h) at 2 days after transfection. The transfection efficiency for all transfections was effective under normoxic or H/R treatment conditions.
9
Prostate Cancer Cell Line Manipulation
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Human PCa PC-3 cells (androgen-independent) and LNCap cells (androgen-dependent) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were maintained in F12 (Gibco, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA) at 37°C in a 5% CO2 atmosphere.
The pcDNA3.1-empty vector (pEX-5), pcDNA3.1-hsa-miR-221 inhibitor sponges (miR-221 inhibitor), pcDNA3.1-hsa-miR-222 inhibitor sponges (miR-222 inhibitor), pGPU6-empty (pGPU6), and pGPU6-siSIRT1 (siSIRT1) were synthesized by GenePharma (Shanghai, China). The wild-type SIRT1 3'UTR region was constructed into psiCHECK-2 by GenePharma (Shanghai, China). PC-3 cells were grown to 80%–90% confluence and transfected with plasmids using Lipofectamine 2000 (DNA/Lipofectamine 2000 = 1/2) according to the manufacturer's instructions. Four hours after transfection, the culture medium was replaced with fresh F12 containing 10% FBS. A stable transfection expression of cell lines was established after cells had been incubated in complete F12 medium with G418 (1000 µg/ml) for 15 days. We verified the clones using western blot and real time PCR, and pooled the successful clones for the experiments. The verification results of western blot are shown inFigure S1 . In addition, all the experiments were also confirmed by transient transfection.
The pcDNA3.1-empty vector (pEX-5), pcDNA3.1-hsa-miR-221 inhibitor sponges (miR-221 inhibitor), pcDNA3.1-hsa-miR-222 inhibitor sponges (miR-222 inhibitor), pGPU6-empty (pGPU6), and pGPU6-siSIRT1 (siSIRT1) were synthesized by GenePharma (Shanghai, China). The wild-type SIRT1 3'UTR region was constructed into psiCHECK-2 by GenePharma (Shanghai, China). PC-3 cells were grown to 80%–90% confluence and transfected with plasmids using Lipofectamine 2000 (DNA/Lipofectamine 2000 = 1/2) according to the manufacturer's instructions. Four hours after transfection, the culture medium was replaced with fresh F12 containing 10% FBS. A stable transfection expression of cell lines was established after cells had been incubated in complete F12 medium with G418 (1000 µg/ml) for 15 days. We verified the clones using western blot and real time PCR, and pooled the successful clones for the experiments. The verification results of western blot are shown in
10
Regulation of Breast Cancer by miR-22 and SIRT1
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miR-22 mimics (miR-22), miR-22 inhibitor (anti-miR-22), scrambled negative control miRNA (miR-NC), siRNA specific targeting sirt1 (si-sirt1), scrambled negative control siRNA (si-NC), and plasmid encoding sirt1 (pcDNA-sirt1) were all synthesized by GenePharma Co. Ltd. (Shanghai, China). Breast cancer cells (1 × 105) were plated into 6-well plates and cultured in medium without antibiotics for approximately 24 h prior to transfection. The next day, cells were transiently transfected with miRNAs, siRNAs or plasmids using Lipofectamine 2000 (Invitrogen). Cells were collected 48 h post-transfection for functional analysis.
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