Stable knockdown cell lines were generated by transfecting PC3 cells with pGIPZ vectors (Open Biosystems) for RB1CC1 (Clone ID #1 V3LHS-307113; Clone ID #2 V3LHS-229368), non-silencing (NS), and GAPDH using Arrest-In Transfection Reagent (Open Biosystems), followed by puromycin selection. These cell lines were named PC3-NS, PC3-GAPDH, PC3-RB1CC#1, and PC3-RB1CC1#2. Expression of RB1CC1 and GAPDH in knockdown cells was analyzed by qPCR. RB1CC1 knockdown was also confirmed by western blot using a polyclonal antibody raised against the c-terminal region (Sigma; SAB4200135). HEK293T and HEK293T transfected with p3XFLAG-CMV10-hFIP200 expression vector (Addgene) were used as controls.
Arrest in transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Arrest-In Transfection Reagent is a lipid-based formulation designed for efficient delivery of nucleic acids into mammalian cells. It facilitates the uptake of plasmid DNA, siRNA, and other nucleic acids into the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Pgipz vector, by Thermo Fisher Scientific (2 mentions)
Sab4200135, by Merck Group (1 mentions)
Pcdna3.1 his c, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Sartorius (1 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (1 mentions)
Hif 1α sirna, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
9 protocols using arrest in transfection reagent
1
Generation of RB1CC1 Knockdown Cell Lines
2
Lentiviral Knockdown of Nox4 in Cells
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A lentiviral plasmid vector expressing a short hairpin RNA in a microRNA scaffold targeting both rat and mouse Nox4 (pGIPZ-Nox4, oligo ID V2LMM_32043) was obtained from Open Biosystems. Lentiviral vector pGIPZ-Nox4 was cotransfected with viral packaging vectors according to the manufacturer’s protocol into HEK-293T cells at 80% to 90% cell density using Arrest-In transfection reagent (Open Biosystems). Following 24 h of incubation, fresh DMEM with 10% fetal bovine serum was applied to the transfected cells. The medium containing viral particles was harvested after 24 h, 48 h, 60 h and 72 h. The supernatant was collected and passed through a 0.45 μm filter and concentrated by ultracentrifugation at 35,000 rpm for 4 h. The pellets were resuspended in serum-free DMEM and flash frozen.
3
Silencing REST in HeLa Cells for Sterol Analysis
4
Establishment and Characterization of EOC Cell Lines
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The EOC cell line OVTW59 P0 and P4 sublines (human, established in our lab)21 (link) were maintained in a DMEM solution with 5% FBS. P0 was the original OVTW59 cell line, while P4 was a subline selected sequentially from P0, was more invasive than P0. The A549 cell line (human, kindly, provided by Dr. Lin from National Taiwan University Hospital, NTUH), which expresses HIF-2α under hypoxic conditions, was used as the study control.
In the inducible IGFBP3-expressing cell line, which was used only in the in vivo study, the full-length human IGFBP3 cDNA was constructed in a doxycycline-induced expression plasmid pBIG2i (provided by Dr. Lin from NTUH) and was labeled as -pBIG2i-hIGFBP3. Transfectants with plasmids without the IGFBP3 cDNA were labeled as -pBIG2i. IGFBP3 expression was induced by 2 mg/mL of doxycycline in drinking water during heterotransplantation. In the constant IGFBP3-expressing cell line, the full-length human IGFBP3 cDNA was constructed in the expression vector pKG3226 that contains the human β-actin promoter and was labeled as -pKG3226-hIGFBP3 (-I)48 (link) . Transfectants with plasmid without the IGFBP3 cDNA was labeled as -pKG3226 (-V). The transfection of the plasmids followed the protocol of Arrest-In Transfection Reagent (Open Biosystems, Inc. Huntsville, AL, USA).
In the inducible IGFBP3-expressing cell line, which was used only in the in vivo study, the full-length human IGFBP3 cDNA was constructed in a doxycycline-induced expression plasmid pBIG2i (provided by Dr. Lin from NTUH) and was labeled as -pBIG2i-hIGFBP3. Transfectants with plasmids without the IGFBP3 cDNA were labeled as -pBIG2i. IGFBP3 expression was induced by 2 mg/mL of doxycycline in drinking water during heterotransplantation. In the constant IGFBP3-expressing cell line, the full-length human IGFBP3 cDNA was constructed in the expression vector pKG3226 that contains the human β-actin promoter and was labeled as -pKG3226-hIGFBP3 (-I)48 (link) . Transfectants with plasmid without the IGFBP3 cDNA was labeled as -pKG3226 (-V). The transfection of the plasmids followed the protocol of Arrest-In Transfection Reagent (Open Biosystems, Inc. Huntsville, AL, USA).
5
Transient Expression of SARS-CoV PLpro in A549 Cells
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Human alveolar basal epithelial A549 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone Laboratories, Logan, Utah, USA) with 100 U/mL of penicillin and streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel). SARS-CoV PLpro gene, nt 4507–5840 of the SARS-CoV TW1 strain (GenBank accession no. AY291451 ) was amplified by RT-PCR, and then cloned into expression vector pcDNA3.1/His C (Invitrogen), as described in our prior reports12 (link)13 (link). The empty vector pcDNA3.1 or pSARS-PLpro at the concentrations of 0, 0.5, 1, 2, 5, and 10 μg/ml was transfected into A549 cells with Arrest-In transfection reagent (Thermo scientific). After 5-h incubation, transfected cells were maintained in DMEM medium containing 20% FBS. Transient expression of recombinant PLpro in A549 cells 2 days post transfection was analyzed using immunefluoresce staining and Western blotting with mouse polyclonal antibodies against anti-E. coli synthesized PLpro, as described in our prior reports12 (link)13 (link).
6
HIF-1α Knockdown via Arrest-In Transfection
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We used Arrest-In Transfection Reagent (Thermo, MA, USA) to transfect cells according to the manufacturer’s protocol. HIF-1α siRNA (ID: s6541) was obtained from ThermoFisher Scientific (Waltham, MA, USA).
7
Dual-Luciferase Assay in Nasopharyngeal Carcinoma
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The plasmid pGL3-Basic or pGL3-MMP-1 promoter was co-transfected with pRL-TK into nasopharyngeal carcinoma cells using Arrest-In Transfection Reagent (Thermo, Waltham, MA, USA) Two different luciferase activities were then measured 24 h after the transfection using Dual-Glo® Luciferase Assay System (Promega). After subtracting the background value, data were normalized to the activity of Renilla Luciferase before statistical analyses.
8
Transfection and RNAi Reagents Protocol
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We used Arrest-In Transfection Reagent (Thermo, MA, USA) to transfect cells according to the manufacturer’s protocol. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, supported by the National Core Facility Program for Biotechnology Grants of NSC (NSC 100-2319-B-001-002). The mouse library is referred to as TRC-Mm 1.0. Individual clones are identified as shRNA TRCN0000072184, shRNA TRCN0000009691, TRCN0000099432, TRCN0000099433, TRCN0000375819 and shRNA TRCN0000229458. BNIP3 plasmid was obtained from OriGene Technologies Inc. (Rockville, MD, USA).
9
Frzb Overexpression and Knockdown in MC3T3-E1 Cells
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MC3T3-E1 cells were transfected with control pcDNA3.1+, pcDNA3.1-Frzb, pcDNA3.1-Frzb CRD , or pcDNA3.1-Frzb NTN constructs using lipid-based agent Fugene HD (Roche Diagnostic, Vilvoorde, Belgium). Selection was initiated 24 h after transfection by supplementing the maintenance medium with 0.1 mg/ml geneticin (Invitrogen) for pcDNA3.1-Frzb CRD and 1 mg/ml for all other constructs. Clones were picked after 14 days of selection. Three different antibiotic-resistant clonal colonies of each condition were isolated and grown independently. Overexpression levels were assessed by quantitative RT-PCR (Q-PCR). Silencing of Frzb was performed using a pGIPZ-shmiRNA construct directed against mouse Frzb (Thermo Scientific, Aalst, Belgium); and a non-interfering pGIPZ vector (Thermo Scientific) was used as a control. MC3T3-E1 cells were transfected using Arrest-In transfection reagent (Thermo Scientific) and after 24 h, selection with 1 μg/ml puromycin (Invitrogen) was initiated and continued for 7 days. Three different antibiotic-resistant clonal colonies of each condition were isolated and grown independently. Knockdown efficiency was assessed by Q-PCR. Antibiotic pressure was maintained for the whole duration of the experiments.
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