Synthetic miRNA duplexes were used and listed in Supplementary Table IX . Cells were transiently transfected with 20 nM or 60 nM of control mimic (Dharmacon) or miR-182 mimic; or 20 nM or 100 nM of miR-182 inhibitor or respective negative control RNA using FuGENE® HD Reagent.
Hd reagent
Manufactured by Promega
Sourced in Israel
The HD reagent is a laboratory consumable product developed by Promega. It is designed to facilitate specific laboratory procedures, though the exact core function of the product cannot be provided without the risk of interpreting or extrapolating beyond a strictly factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using hd reagent
1
miR-182 Mimic and Inhibitor Transfection
2
Generation of stable CAV1 mutant cell lines
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The plasmids pLacIOP (referred to as mock) and pLacIOP-caveolin-1 (referred to as CAV1, containing the full-length dog caveolin-1 sequence, NCBI Reference Sequence: NP_001003296.1) were previously described [42 (link), 90 (link)]. B16F10 cells were grown to 50-60% confluence in 6 multi-well plates and then transfected with 4 μg of pLacIOP-CAV1(Y14F) (referred as CAV1/Y14F) and pLacIOP-CAV1(Y14E) (referred as CAV1/Y14E), using the FuGene HD reagent, according to the manufacturer's indications. After transfection, cells were plated in complete RPMI medium containing hygromycin (750 μg/mL) for 2 to 3 weeks, to yield stably transfected B16F10(CAV1/Y14F) and B16F10(CAV1/Y14E) cells, respectively.
3
Lentiviral Overexpression of SLUG
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The cDNA of SLUG was cloned into the pSin4-EF2-IRES-Pur vector at the SpeI and EcoRI sites. The pSIN lentiviral system was used for ectopic expression of SLUG in HUVECs. The pSIN-SNAI, psPAX2, and pMD2G plasmids were cotransfected into HEK-293T cells using the Fugene HD reagent. The supernatant containing lentivirus was harvested and then filtered at 48 h after transfection.
4
Lentiviral Particle Production Protocol
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We produced lentiviruses according to the manufacturer's instructions by cotransfecting the plasmid pLEX-MCS (Thermo Scientific, Rockford, IL), along with the helper plasmids pCAGGS-VSV-G, pCAG4-RTR2 and pCAGkGP1.1R, into HEK293T cells using Fugene HD reagent according to the manufacturer's instructions.
Three days after transfection, the culture supernatant was collected and then the cells were infected with the viral supernatant.
Three days after transfection, the culture supernatant was collected and then the cells were infected with the viral supernatant.
5
Modulation of Sesn2 in H9c2 Cells
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H9c2 cells were cultured in DMEM supplemented with 10% FBS, 100 U mL−1 penicillin, and 100 mg mL−1 streptomycin in a 5% CO2/95% air humidified incubator at 37 °C. The cells were then seeded in 6-well plates at a density of 1 × 105 per well during the logarithmic growth phase. When the cells reached 80% confluence, the miRNA negative control (NC), mimics, and inhibitors (20 μM) synthesized by Gene Pharma were transfected into the cells using Fugene HD reagents. Sesn2 gain-of-function experiments were conducted using the overexpression vector pEX-Sesn2 (Sesn2 cDNA was inserted into the pEX-4 vector provided by Gene Pharma). After the transfection was complete (usually after 12–24 h), the culture medium (control) and ghrelin (100 nM), Ang II (100 nM), or both were added to the culture dishes, followed by incubation for 24 h. Total RNA and proteins were extracted for qPCR and western blot analysis, respectively.
6
Luciferase Assay for BMP Signaling
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For the luciferase activity assay, we transfected the constructs of 12xSBE-OC into OCT1 cells by using FUGENE HD reagents. We also co-transfected an SV40-Renilla luciferase construct with the above reporter construct to normalize the result for detecting the efficiency of transfection. Then cells were continued to culture for 48 h with or without daidzein (10 µg/ mL) and noggin (300 ng/mL). Cell lysates were extracted after treatment for 48 h, and the activity of luciferase was detected by using a Promega luciferase assay kit. Luciferase activity in the cell lysate was determined using a luminometer (Thermo Electron Corporation).
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