X tremegene hp dna transfection reagent

After transformation into E. coli DH5α competent cells, pcDNA 3.1(+)-GPIHBP1 and pcDNA 3.1(+) were isolated with the E.Z.N.A. Endo-Free Plasmid Mini Kit I (Omega, USA). The cells were passaged and plated in 6-well plates for 24 hours before transfection at 80% to 90% confluence. The cells were divided into two groups: a transfection reagent+pcDNA3.1(+) (Control) group and a transfection reagent+pcDNA 3.1(+)-GPIHBP1 group (Over expression). The recombinant pcDNA 3.1(+)-GPIHBP1 eukaryotic expression vector or the pcDNA3.1(+) plasmid was transiently transfected into bovine primary mammary epithelial cells according to the instructions for the Roche X-treme GENE HP DNA transfection reagent. Transfection was performed using a 12:1 ratio of the Roche X-treme GENE HP DNA transfection reagent (Roche, USA) (µl) to the vector (µg). At 36 hours after transfection, the cells were collected to detect GPIHBP1 mRNA expression levels via real-time quantitative PCR.

To examine the effects of Faslg overexpression, we transfected the SCs with a mixture of X-treme GENE HP DNA Transfection Reagent (Roche, Germany) and pcDNA3.1(+)-Faslg plasmid, or a mixture of X-treme GENE HP DNA Transfection Reagent and an empty vector for 48 h. We performed real-time quantitative polymerase chain reaction (qPCR) and Western blot analyses. The above experiments were all repeated three times.

LX-2 cells were reverse transfected using siRNA/Lipofectamine RNAi/MAX reagent (Life Technologies, Thermo Fisher Scientific) for siRNA-mediated knockdown and plasmid/X-tremeGENE HP DNA transfection reagent (Roche) for plasmid-mediated overexpression. The cells were seeded into 6-well culture plates at approximately 80% confluence, and a preformulated transfection complex was added at the time of seeding. Scrambled siRNAs and siRNAs targeting KLF10 and ATF3 were purchased from Genolution (Seoul, Republic of Korea). The siRNA sequences were as follows: siKLF10 forward 5′-CAACAAGUGUGAUUCGUCAUU-3′, reverse 5′-UGACGAAUCACACUUGUUGUU-3′; siC forward 5′-CCUCGUGCCGUUCCAUCAGGUAGUU-3′; reverse 5′-CUACCUGAUGGAACGGCACGAGGUU-3′. For plasmid transfection, X-tremeGENE HP DNA transfection reagent (Roche) was used, with 1 µg of total plasmid and 2 µL of X-tremeGENE reagent in serum-free DMEM for each well of a 6-well culture plate. Human Flag-KLF10 expression vector was purchased from Sino Biological (HG18322-NF, Beijing, China). The full-length human ATF3 were amplified by PCR and were cloned into the pcDNA3-2xFlag vector.

Hsu cells were seeded in a 24-well plate and incubated at 27 °C and 5% CO₂ overnight. At the day of transfection, the complete cell media was replaced with 200 µL of Opti-MEM (Thermo Fisher Scientific, #31985062, Waltham, MA, USA) and placed back in the incubator while the lipoplex was prepared. The cells were then transfected with pUb_Piwi4 or pUb_EGFP plasmids using X-tremeGENE™ HP DNA transfection reagent (Roche, #6366236001, Burlington, MA, USA) per the manufacturer’s protocol. Briefly, plasmid DNA concentration was normalized to 100 ng/μL. Per well, 500 ng plasmid (5 µL) was combined with 44 μL of Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific, #31985062, Waltham, MA, USA) prior to the addition of 1 μL of X-tremeGENE™ HP DNA transfection reagent (Roche, #6366236001, Burlington, MA, USA). After 20 min of incubation, the diluted transfection reagent complex was added to the cells. After 1 h of incubation, 1 mL of complete cell medium was added to the cells. Mock-transfected cells were treated with X-treme GENE Transfection reagent diluted in Opti-MEM alone. All experiments were performed in triplicates. The expression of CqPiwi4 was confirmed through immunostaining using a monoclonal ANTI-FLAG M2 antibody (Sigma Aldrich #F3165, Burlington, MA, USA) at two days post-transfection.

SCs were cultured in DMEM (GIBCO, Grand Island, NY) with 100 IU/mL penicillin, 10% fetal calf serum, and 100 g/mL streptomycin at 37 °C and 5% CO₂. The SCs were identified by examining the immunofluorescence of an antibody to the marker S100, and the final cells were found to comprise 98% SCs. The Spp1 overexpression plasmid pcDNA3.1-Spp1 was constructed as previously described [21 (link)]. A mixture of pcDNA3.1-Spp1 plasmid and X-treme GENE HP DNA Transfection Reagent (Roche, Mannheim, Germany), or X-treme GENE HP DNA Transfection Reagent and an empty vector were then transfected into SCs for 48 h. After that, real-time quantitative (q)PCR and Western blot analyses were conducted. The pcDNA3.1-Spp1 overexpression experiments were repeated three times.

COL17A1 siRNA 1-3 were designed according to the siRNA design principles. The siRNA molecules were synthesized at GenePharma in China. COL17A1 knockdown was achieved following transient transfection using three different human fascin siRNA duplexes. In all experiments, we used a non-targeting oligonucleotide to exclude the nonspecific off-target effects of RNA interference (Table 1). siRNA Transfection Reagent was used for all transfections (Polyplus-transfection Inc, New York, NY, USA). The expression vector (pcDNA3.1) that express COL17A1 (pcDNA3.1- COL17A1) and a negative control (pcDNA3.1-NC) were transfected into H4 cells or U87 cells using X-tremeGENE HP DNA Transfection Reagent (Roche, Mannheim Germany) according to the manufacturer’s instructions. The cells were cultured 24 h prior to transfection, and cells transfected with pcDNA3.1- COL17A1 and pcDNA3.1-NC were harvested after 48 h. The expression vector (pcDNA3.1) that express 3’UTR (pcDNA3.1-3’UTR) and a negative control (pcDNA3.1-NC) were transfected into U251 cells or U87 cells using X-tremeGENE HP DNA Transfection Reagent (Roche, Mannheim Germany) according to the manufacturer’s instructions.