The lentivirus vector down-expressing KDM4B was prepared for MFE-296 cells using shRNA targeting KDM4B (pLKO.1, TRCN0000018013; Open Biosystems, Lafayette, CO, USA) or a negative control vector (pLKO.1, Open Biosystems) according to the manufacturer's protocol. The lentivirus vector down-expressing KDM4A was prepared for AN3CA cells using a shRNA targeting KDM4A (pLKO.1, TRCN0000013493; Open Biosystems) or a negative control vector (pLKO.1, Open Biosystems) according to the manufacturer's protocol. shRNA lentiviral particles were treated in six-well plates in the presence of polybrene (6 μg/ml). Cells were treated with puromycin (2 μg/ml) to generate stable KDM4B and KDM4A knockdown clones, and several puromycin-resistant KDM4B and KDM4A knockdown clones were harvested by ring selection. The shRNA oligo sequences are provided in Table 2 .
Plko 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PLKO.1 is a laboratory equipment product designed for research and scientific applications. It serves as a lentiviral transfer vector that enables the expression of genes of interest in target cells. The core function of the PLKO.1 is to facilitate the delivery and integration of genetic material into the host cell's genome, providing a tool for researchers to study gene function and expression.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Plko 1, by Addgene (3 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Silentfect transfection reagent, by Bio-Rad (2 mentions)
Virapower, by Thermo Fisher Scientific (2 mentions)
Hiv 1 gag p24 duoset elisa kit, by R&D Systems (2 mentions)
Sigenome smartpool, by Horizon Discovery (2 mentions)
Lentiviral lkb1 short hairpin rna shrna constructs, by Thermo Fisher Scientific (2 mentions)
Rhs4696 101315918, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Pspax2, by Addgene (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Pgreenfire1 nf κb ef1α puro lentivector, by System Biosciences (1 mentions)
Effectene, by Qiagen (1 mentions)
Quikchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Ni nta, by Qiagen (1 mentions)
Anti his tag antibody, by Proteintech (1 mentions)
46 protocols using plko 1
1
Generation of KDM4B and KDM4A knockdown cell lines
2
Lentiviral Knockdown of Human VDR
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Plasmid to knockdown human VDR (clone ID: TRCN0000019507), with each hairpin sequence of the short hairpin RNA (shRNA) construct cloned into the lentiviral vector, pLKO.1, was purchased from Open Biosystems. Plasmid that expresses a super-repressor form of the NF-κB inhibitor IκBα was constructed as follows. Briefly, the insert of the plasmid pcDNA3-IκBα S32A/S36A [38 (link)] which expresses a super-repressor of NF-κB was subcloned via gateway technology into the lentiviral plasmid pDSL (ATTC) lacking the SV40-GFP cassette. Production of infective lentiviral particles was done as previously described [39 (link)]. Filtered supernatant was added to the growing culture of EA.hy926 cells and incubated overnight. Next day, fresh medium was replaced and the cells were left to grow for additional 2–3 days before starting puromycin selection (pLKO.1 constructs). The stable cell line was selected using 1 μg/ml puromycin selectable marker. Western blot was performed to check for VDR gene knockdown that was achieved after five passages.
3
Lentivector-mediated shRNA and overexpression
4
Lentiviral shRNA Expression Vectors
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IFI6, E2F2, STAT1, and RelA (the p65 subunit of NF-κB), NF1 shRNA sequences in either pLKO.1 or pZIPZ lentiviral expression vectors were obtained from Open Biosystems. For retrovirus or lentivirus production viral expression, constructs, and viral packaging plasmids were co-transfected into 293T cells using Effectene (Qiagen), following the manufacturer’s recommendations. Supernatant fractions were collected 48 hr after the transfection, and purified virus particles were used to infect primary or melanoma cell lines and were selected by growth on puromycin or by sorting GFP-positive cells using a flow cytometer.
5
Lentiviral Knockdown of Dectin-1 in BMDMs
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Lentivirus generation and transduction of shRNA was performed as described previously [23 (link)]. The lentiviral construct vector pLKO.1 and three packaging plasmids (pMDLg/pRRE, pRSV-Rev and pMD2.VSV-G) were from Open Biosystems (Huntsville, AL, USA) and target dectin-1 (TRCN0000066928) was from Sigma-Aldrich, supplied as glycerol stocks. Lentivirus generation was achieved by transfecting with Lipofectamine 2000 into HEK 293 T cells with pLKO puro.1 or target shRNA plasmid and the packaging plasmids. After 72 h, the virus-containing HEK 293 T cell culture supernatants were collected and filtered. Lentivirus determination was performed as described previously [23 (link)]. The lentivirus particles mixed with 8 μg/mL Polybrene (Sigma-Aldrich) and non-specific shRNA or dectin-1 shRNA into BMDMs, according to the manufacturer’s protocol. After transduction, the BMDMs were harvested and the target gene-silencing efficiency was examined by RT-PCR analysis.
6
Lentiviral shRNA and Expression Vectors
7
LIN28B Knockdown and Overexpression
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The two independent shRNA sequences cloned into the lentiviral vectors targeting human LIN28B (pLKO.1, TRCN0000122191 and TRCN0000122599) were obtained from Open Biosystems. shRNA for enhanced green fluorescent protein (SHC005) and the nontarget shRNA (SHC002) served as negative controls. For studies using the constructed pMSCV-neo-hLIN28B retroviral expression vector (Addgene), the pMSCV-neo empty vector was used as a control.
8
Knockdown of PKC Isoforms in BMMCs
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shRNA constructs targeting mouse PKCα, PKCβ were purchased from Open Biosystems. The constructs were cloned into a lentiviral vector (pLKo1, Open Biosystems, Huntsville, AL) and used to generate infectious particles with a lentiviral packaging mix (Virapower, Invitrogen, Waltham, MA), according to the manufacturer's protocol.23 Viral stocks were titered using HIV‐1 Gag p24 DuoSet® ELISA Kit (R & D Systems, Minneapolis, MN). For viral transfection, for control, and single knockdowns (PKCα or PKCβ), viral stocks of 100 transfection units (TU), and for double knockdowns, viral stocks of total 100 TU (50 TU PKCα and 50 TU PKCβ) along with protamine sulfate (5 µg/ml) were added to 1 × 106 BMMCs suspended in RPMI medium, and the cells were incubated for 48 h at 37°C. Knockdown of PKCs was determined via western blotting. We did not observe a significant difference in PKC knockdown between 50 TU and 100 TU (not shown). For siRNA transfection, BMMCs were transfected with siGENOME SMART pool (a mix of 4 pre‐made siRNA; Horizon Discovery, Lafayette, CO) of 50 nM PKCα‐specific siRNA to block PKCα (Cat# D‐040348‐0), or non‐specific siRNA (negative control; Cat# D‐001206‐14‐05). Transfection was carried out with siLentFect transfection reagent (Bio‐Rad, Hercules, CA) according to manufacturer's instructions.
9
Thrap3 Mutant Generation and Knockdown
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Various deletion mutants of Thrap3 were generated and subcloned into HA-pcDNA3.1 vector. The sequence used for the lentivial shRNA expression vector (pLKO.1, Open Biosystems) targeting Thrap3 was 5′-AATGGTCACTATGTGCTGAGC-3′. For lentiviral production, HEK-293T cells (ATCC) were transfected with 10 µg of the lentiviral vectors. Following infection of the cells with the viral vectors, cells were selected by incubation with 2 μg/mL puromycin.
10
Lentiviral Knockdown of PKCα and PKCβ
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