U87 cells were respectively transfected with miR-431-5p mimics (miR-mimics), miR-431-5p inhibitor (anti-miR), miR-431-5p inhibitor negative control (anti-NC), and miR-431-5p mimics negative control (miR-NC) using Lipofectamine 2000 reagent (GenePharma Co., Ltd., Shanghai, China). The lentiviral vector (plenti-GIII-UbC) was used to construct a recombinant vector (plenti-GIII-Ubc-EPB41L1) which was then transfected into U87 cells (plenti-EPB41L1). Meanwhile, U87 cells transfected with pLenti-GIII-UbC-Null (plenti-Null) were the negative control group.
Lipofectamine 2000 reagent
Manufactured by GenePharma
Sourced in China
Lipofectamine 2000 reagent is a cationic lipid-based transfection reagent used for the delivery of nucleic acids, such as DNA or RNA, into mammalian cells. It facilitates the uptake of these molecules by the cells, enabling researchers to study gene expression, perform gene knockdown experiments, and other applications involving the introduction of genetic material into cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pcdh cmv mcs ef1 puro, by System Biosciences (2 mentions)
Mimics control, by GenePharma (1 mentions)
Mir 182 5p mimics, by GenePharma (1 mentions)
Mir 203 inhibitor, by GenePharma (1 mentions)
Shrna nc, by GenePharma (1 mentions)
Inhibitor nc, by GenePharma (1 mentions)
Hfob cell line, by American Type Culture Collection (1 mentions)
Pcdna3.1 pdk1, by GenePharma (1 mentions)
Si usp22 2, by GenePharma (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
U6 gfp neo plasmid, by GenePharma (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Mrc 5, by Thermo Fisher Scientific (1 mentions)
Nih3t3 cells, by Thermo Fisher Scientific (1 mentions)
Zyg11b mus 1520, by GenePharma (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
21 protocols using lipofectamine 2000 reagent
1
EPB41L1 Overexpression in U87 Cells
2
Investigating miR-182-5p and RAB27A in Gastric Cancer
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To investigate the role of miR-182-5p on GC cell activities, HGC-27 cells in the logarithmic growth phase were transfected with 50 nM miR-182-5p mimics or 8 ng mimics control (GenePharma, Shanghai, China) using 1 μl of the Lipofectamine 2000 reagent. The groups were designed as follows: control group, miR-mimics group, anti-miR group, anti-negative control (NC) group, and miR-NC group. To study the role of RAB27A on GC cell activities, HGC-27 cells were transfected with lentiviral vectors recombined with human RAB27A gene sequence (the constructed vector was plenti-GIII-Ubc-RAB27A). These were assigned to the plenty-RAB27A group and cells transfected with the empty vector plasmid were designated as the NC group (named plenti-Null group).
3
Overexpression and Knockdown of β-Catenin in HMCs
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HMC was cultured in groups on a six-well plate. After the cells reached approximately 80% confluence, 2.5 µg of plasmids expressing CTNNB1 (HG11279-M-F, Sino Biological Inc., Beijing, China) were transfected into the HMCs to overexpress β-catenin using Lipofectamine 2000 reagent (11668-027, Invitrogen-BRL, Carlsbad, CA, USA) following the manufacturer’s protocol. For β-catenin knockdown, 75 pmol of small interfering RNA (siRNA) against CTNNB1 (GenePharma Corporation, Shanghai, China) was transfected into the HMCs using Lipofectamine 2000 reagent. The siRNA sequences are as follows: si-CTNNB1 (Human) sense 5′-GGGUUCAGAUGAUAUAAAUTT-3′ and antisense 5′-AUUAUAUCAUCUGAACCCAG-3′. The siRNA/liposome and pcDNA/liposome complexes were added to the serum-free medium and the complexes were formed at room temperature for 20 min. After 4 h of incubation, the medium containing HG (30 mM) was added and the pregnancy continued. After 48 h of incubation, cells were harvested to detect β-catenin expression and related targets.
4
NF-κB and p53 Regulation in H/R Injury
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NF-κB p65 and/or p53 were knocked down and overexpressed in H/R-injured IEC-6 cells according to the manufacturer’s instructions. IEC-6 cells were seeded in 6-well plates, and then a specific NF-κB p65 siRNA, p53 siRNA, and control siRNA (GenePharma, Shanghai, China) or a specific cDNA encoding NF-κB p65, p53, and control cDNA (Genecopia, Guangzhou, China) were transfected with Lipofectamine 2000 reagent (GenePharma, Shanghai, China). Cotransfection of NF-κB p65 and p53 siRNA or NF-κB p65 and p53 cDNA was also performed according to the manufacturer’s instructions. After 48 h, CA was added to the incubation for 24 h before H/R, and then the protein levels of NF-κB p65, p53 and Bax were estimated as previously prescribed [50 (link)].
5
Hypoxic Regulation of circPVT1 and SOCS3 in BSMCs
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BSMCs were cultured in DMEM containing 10% FBS, antibiotics, and 5% CO2 at 37°C. In the hypoxia trial, the cell culture was placed in a 37°C incubator with 1% O2. The cells were plated in DMEM containing 10% FBS at a density of 5 × 105 cells/mL on the day prior to transfection of shRNA-circPVT1 (300 ng/50 μL), shRNA-suppressor of cytokine signaling 3 (SOCS3) (300 ng/50 μL), shRNA-NC (300 ng/50 μL), miR-203 inhibitor (500 ng/50 μL), or NC inhibitor (500 ng/50 μL) (Shanghai GenePharma Co., Ltd., Shanghai, China) in Lipofectamine 2000 reagent according to the manufacturer’s instructions. Six hours after transfection, the medium was replaced with DMEM containing 10% FBS, and transfection continued for an additional 36–48 h.
6
Investigating HAND2-AS1 in Osteosarcoma Cell Lines
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The human osteosarcoma MG-63 and SAOS-2 cell lines, and the normal bone hFOB cell line were obtained from the American Type Culture Collection (ATCC). The MG-63 and SAOS-2 cells were cultured in Eagle's minimum essential medium (cat. no. 30-2003; ATCC) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). hFOB cells were cultured in McCoy's 5a modified medium (cat. no. 30-2007; ATCC) containing 10% FBS. All cells were cultured at 37°C with 5% CO2. HAND2-AS1 small interfering RNA (siRNA; 5′-CCGAGGUGCUCCAAUAUUATT-3′) and negative control siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′); were purchased from Shanghai GenePharma Co., Ltd. All three cell lines were cultured to 80–90% confluence, and 5×105 cells/sample were transfected with 50 nM siRNA using Lipofectamine® 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure the expression levels of HAND2-AS1, and to confirm a <50% reduction prior to subsequent experiments. Subsequent experiments were performed at 24 h after transfections. The control group for transfection was un-transfected cells, and the negative control group was negative control siRNA-transfected cells.
7
Modulating TCRP1 in Breast Cancer
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TCRP1 shRNA (forward primer, GGAAAUACAUAGACCUACA and reverse primer, UGUAGGUCUAUGUAUUUCC) oligonucleo-tides with 3′dTdT overhangs were synthesized by QIAGEN. Control shRNA in the experiments refers to a non-short hairpin (NSh) shRNA (NSF, UUCUCCGAACGUGUCACGU; NSR, ACGUGACACGUUCGGAGAA), which was designed and synthesized by QIAGEN. The TCRP1 overexpression vector pcDNA3.1-TCRP1 was constructed as previously described (13 (link)). The vector for overexpressing PDK1 (pcDNA3.1-PDK1) and SGK1 (pcDNA3.1-SGK1) were purchased from Shanghai GenePharma Co., Ltd. MCF7-R cells were transfected with shRNA or plasmids by using Lipofectamine 2000 reagent (cat. no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.). The expression levels of TCRP1, PDK1 and SGK1 were determined by western blotting and q-PCR.
8
Hypoxia/Reoxygenation and Sevoflurane Treatment in H9c2 Cardiomyocytes
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Rat cardiomyocytes H9c2 (China Center for Type Culture Collection, CCTCC, Wuhan, Hubei, China) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Gibco Company, Grand Island, NY, USA) and incubated at 37°C and 95% humid air with 5% CO2. Subsequent experimentation was carried out when cells reached 80%–90% confluence. H/R cell models were established according to a previously published method [22 (link)]. Briefly, H9c2 cells were subjected to hypoxia/reoxygenation (H/R; 3 h of hypoxia followed by 6 h of reoxygenation). Sevo treatment was administered for 15 min at the start of reoxygenation process (H9c2 cells were placed in a 37°C airtight container, then the Sevo evaporation tank was opened and 2% Sevo was allowed to flow through the airtight container with O2 for 15 min at the start of reoxygenation). In accordance with the manufacturer’s instructions of the Lipofectamine 2000 reagent (Shanghai GenePharma Co, Ltd, Shanghai, China), small interfering (si)-USP22-1, si-USP22-2, si-KDM3A-1, si-KDM3A-2, si-YAP1-1, and si-YAP1-2 plasmids (all procured from GenePharma) were transfected into H9c2 cells 6 h prior to H/R.
9
Transfection of CLDN7 and IRF2 in CAL-27 Cells
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CAL-27 cells in the logarithmic growth phase were collected and plated into 6-well plates at a density of 1x106 cells/well at 37˚C. When cells reached 85% confluence, cells were transfected with 2 µg CLDN7 plasmid (pc-CLDN7), 2 µg IRF2 plasmid (pc-IRF2) or 2 µg empty vector plasmid (pcDNA3.1) using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C according to the standard protocol (16 (link)). The aforementioned plasmids were purchased from Shanghai GenePharma Co., Ltd. Additionally, 2 µg short hairpin RNA (shRNA) targeting CLDN7 (sh-CLDN7) was constructed in a U6/GFP/Neo plasmid (GenePharma, Shanghai, China) and transfected into CAL-27 cells using Lipofectamine® 2000 reagent at 37˚C. Western blot analysis was employed to evaluate the transfection efficiency 48 h post-transfection.
10
Serum Starvation Induces Cilium Formation
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HEK293T, MRC-5, and NIH-3T3 cells (FuHeng Cell Center) were cultured in DMEM (c11995500bt; Gibco) with 10% FBS and 1% penicillin/streptomycin (15140122; Gibco) under 5% CO2 and detached by incubating in trypsin (25200072; Gibco). Expression plasmids were transfected using Lipofectamine 2000 reagent (11668019; Thermo Fisher Scientific) according to the manufacturer’s instructions. For knockdown experiments, siRNA duplexes (150 pmol) siRNA with 6 μl Lipofectamine 2000 reagent targeting Zyg11b were used in 6-well plates along with nontargeting control siRNA duplexes, to transfect NIH-3T3 cells for 24 h. The sequences of siRNA duplexes were as follows: siZyg11b#1 sense, 5′-GCUUGUCAUGCAGUGGCUUTT-3′ (Zyg11b-Mus-1520; GenePharma) and siZyg11b#1 antisense, 5′-AAGCCACUGCAUGACAAGCTT-3′ (Zyg11b-Mus-1520; GenePharma). Nontargeting siRNA duplex (GenePharma, Negative control) served as the negative control. MRC-5 and NIH-3T3 cells were serum-starved for 24 h to induce cilium formation (Li et al., 2020b (link); Nandadasa et al., 2019 (link)). After cilium formation upon serum starvation for 24 h, MRC-5 and NIH-3T3 cells were harvested 2 d after transfection of plasmid DNA or siRNA duplexes for fluorescence microscopy and preparation of lysates.
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