siRNAs for TONSL and MMS22L were purchased from IDT (Coralville, IA, USA) and Genolution (Seoul, Republic of Korea), respectively. The sequences of each siRNA are listed in Supplementary Table S2 . siRNA transfections were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Cells were assayed 3–5 days after transfection. TONSL-specific shRNAs in the lentiviral pLKO.1 vector (TRCN0000424634, #1; TRCN0000424077, #2; and TRCN0000424443, #3) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and the pLKO.1 vector was used as a control. Lentiviruses were produced in HEK293T cells by co-transfecting the shRNA-expressing vector, pMD2.G (Addgene, Watertown, MA, USA, #12259), and psPAX2 (Addgene) using jetPRIME (Polyplus-transfection, Illkirch, France) according to the manufacturer’s protocol. Cells were transduced with 5 μg/mL hexadimethrine bromide (Sigma). Two days later, the cell extract or RNA was used for western blotting or quantitative reverse transcription PCR to confirm the knockdown effect (Supplementary Figure S5 ).
Plko 1 vector
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, China
The PLKO.1 vector is a plasmid-based tool commonly used in molecular biology and genetic engineering applications. It serves as a backbone for the construction and manipulation of recombinant DNA. The vector provides essential genetic elements, such as a promoter, multiple cloning site, and selection marker, which facilitate the insertion and expression of foreign DNA sequences in various host cell systems.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Merck Group (16 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions)
Pspax2, by Addgene (9 mentions)
Polybrene, by Merck Group (9 mentions)
Pmd2 g, by Addgene (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pez lv151, by GeneCopoeia (2 mentions)
Pspcas9n bb 2a puro vector, by Addgene (2 mentions)
Lenticrisprv2.0 vector, by Addgene (2 mentions)
Gateway cloning system, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Plko 1 puro empty vector shc001, by Merck Group (2 mentions)
In fusion hd cloning kit, by Takara Bio (2 mentions)
Powerplex16hs assay, by Promega (2 mentions)
Gateway recombination cloning technology, by Thermo Fisher Scientific (2 mentions)
Hif 1α specific inducible luciferase reporter construct, by Thermo Fisher Scientific (2 mentions)
90 protocols using plko 1 vector
1
Targeted Knockdown of TONSL and MMS22L
2
Silencing Mouse Fxyd5 with shRNAs
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Short hairpin RNAs (shRNAs) against mouse Fxyd5 (accession no. NM_001111073; shFxyd5-1 and shFxyd5-2) were designed using the online design software (https://portals.broadinstitute.org/gpp/public/seq/search ) and then cloned into the pLKO.1 vector (MilliporeSigma; cat. no. SHC001) to silence Fxyd5 expression. A non-targeting shRNA clone was used as the negative control (shNC). Briefly, ATDC5 cells were seeded into 6-well plates at a density of 1×106 cells/well and cultured overnight at 37°C. Cells were then transfected with 50 pmol shRNAs using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h according to the manufacturer's recommendation. Transfection efficiency was detected via western blot at 48 h after transfection. The sequences for shRNAs were as follows: shFxyd5-1, forward 5′-CCGGCGCCTGTGTCTCCTCACTATTCTCGAGAATAGTGAGGAGACACAGGCGTTTTTG-3′, reverse 5′-AATTCAAAAACGCCTGTGTCTCCTCACTATTCTCGAGAATAGTGAGGAGACACAGGCG-3′; shFxyd5-2, forward 5′-CCGGGCTGTTCATCACGGGAATTATCTCGAGATAATTCCCGTGATGAACAGCTTTTTG-3′, reverse 5′-AATTCAAAAAGCTGTTCATCACGGGAATTATCTCGAGATAATTCCCGTGATGAACAGC-3′; shNC, forward 5′-CACCGCAATTTTTTTTTTTGATTCACGAATGAATCAAAAAAAAAAAAATGC-3′, reverse 5′-AAAAGCAATTTTTTTTTTTGATTCATTCGTGAATCAAAAAAAAAAAAATGC-3′.
3
Lentiviral Knockdown of ASAH1 in GBM Cells
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Knockdown cells were generated by lentiviral infection using a non-targeting control for the PLKO.1 vector (Millipore Sigma, St. Louis, MO, USA, cat# SHC001) or Precision LentiORF positive control (Horizon Discovery, Cambridge, UK, cat# OHS5832) as a fluorescent positive control for infection and two different ASAH1-directed shRNA constructs (Millipore Sigma, St. Louis, MO, USA, cat# TRCN0000235585 and TRCN0000219048). Constructs were transfected into CSC293T cells using Fugene HD (Promega, Madison, WI, USA, cat# PRE2312). After 48, 72, and 96 h, virus was collected for infection. Viral RNA was isolated using NucleoSpin RNA (Takara Bio, San Jose, CA, USA, cat# 740956.50) and titered using the Lenti-X™ qRT-PCR Titration Kit (Takara Bio, San Jose, CA, USA, cat# OHS6085). To infect GBM cells, a multiplicity of infection of 10 was used. For migration assays, infected GBM cells were growth factor-deprived 24 h after infection. GBM cells were collected and plated for experiments 48 h after infection. For each infection, RNA was collected at the time of plating for the experiment to confirm knockdown.
4
Pooled shRNA Screening Protocol
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Each shRNA was cloned into the pRSI-U6-(sh)-UbiC-TagRFP-2A-Puro vector (Cellecta Inc.) and as pool of two shRNAs was used to infect target cells. Complete sequences of shRNAs used for validation experiments are reported in Supplementary Table S1 . shRNAs targeting mouse genes were engineered into the pLKO.1 vector (Sigma). 3 scrambles shRNAs were pooled together and used as neutral control (SCR). The shRNAs targeting Chd4 were used as pool of two distinct shRNAs. Complete sequences of shRNAs and control are provided in Supplementary Table S1 .
5
Genetic Manipulation of XPO6 and PFN1 in Cells
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Three shRNAs targeting human XPO6, a shRNA targeting luciferase (Zhu et al., 2020), and a shRNA targeting human ENL in the lentiviral pLKO.1 vector were purchased from Sigma. XPO6 cDNAin the lentiviral vector pEZ-Lv151 was purchased from GeneCopoeia (# EX-W2830-Lv151). Silent mutations resistant to shXPO6 #3 (GGAAAGGTTGGTC to AGAGCGCCTCGTG) were introduced by Genewiz between nucleotides 1861-1877 in theXPO6 cDNA (numbering after ATG). Two shRNAs targeting human Pfn1 and a scrambled shCTRL were cloned in the lentiviral pFLRu-FH vector (Diamond et al., 2015 (link)). Silent mutations resistant to shPFN1 #1 and #2 were introduced in human Pfn1 cDNA by QuickChange: AGC to TCG (nucleotide 253-255) and TTG to CTC (nucleotide 367-369). Paired guide RNAs specific for human XPO6 gene used with Cas9 (D10A) nickase (Cas9n) were designed using E-CRISP (http://www.e-crisp.org/E-CRISP/ ). Oligonucleotides for four pairs of sgRNAs were synthesized, each pair consisting of two annealed oligonucleotides whose target sequences in XPO6 are ≤ 20 base pairs apart. Following oligo annealing, they were inserted into the BbsI sites of the pSpCas9n(BB)-2A-Puro vector (PX462, Addgene 48141) (Ran et al., 2013 (link)). Single guide RNAs for human XPO6 used with wild-type Cas9 were similarly designed, and cloned into the BsmBI site in the lentiCRISPRv2.0 vector (Addgene #52961).
6
Lentiviral Knockdown of Clock, Bmal1, and Postn
7
Lentiviral shRNA and ORF Constructs
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SH002 shScramble (shScr) control and constructs targeting PPFIA1 and CTTN were purchased from TRC.1 library in pLKO.1 vector (Sigma-Aldrich, St. Louis, MO, USA). Five shRNA constructs for both genes were tested and those giving efficient knockdown were selected for further analyses. These constructs were TRCN0000002969 for PPFIA1 and TRCN0000040274 and TRCN0000040275 for CTTN. Open reading frame (ORF) for PPFIA1 (clone ID 4794300) was ordered from the ORFeome Collection (Open Biosystems, Pittsburgh, PA, USA) and cloned from donor pENTR221 vector into lentiviral destination expression vector pLenti6/V5 DEST (Invitrogen) using Gateway cloning system. Empty pLenti6/V5 DEST vector was used as a control. Gateway cloning was done at the Genome Biology Unit, University of Helsinki, Finland.
8
Overexpression and Knockdown of USP5 in FLS Cells
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All of the lentivirus and plasmids were produced by MDL Technology Company (MDL Biotech, Beijing, China). Lentivirus containing vector plasmid (control) and overexpression plasmid of USP5 was constructed by using a pLenti6/V5 vector (Life Technologies); lentivirus containing negative control plasmid (sh-NC) and shRNA plasmid of USP5 (sh-USP5) was constructed by using a pLKO.1 vector (Sigma-Aldrich). Lentivirus was obtained and enriched according to the manufacturer's instruction. FLS cells were infected at 50 MOI of lentivirus, and jetPEI reagents (Polyplus-transfection) were used for the transfection of Myc-USP5, Flag-TRAF6, and HA-ub plasmids.
9
Cloning and Silencing of CASC9 and NF-κB
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The full-length sequences of NF-κB (NM_001165412.2) and CASC9 (NR_103848.1) were cloned into pcDNA3.1(+) expression vector. The small hairpin RNA targeting CASC9 was synthesized and cloned into the pLKO.1 vector (Sigma, USA). All the plasmids were validated by sequencing. CASC9, NF-κB, and TK1 siRNAs were purchased from the Ambion (USA). MiR-195-5p mimics and inhibitors were synthesized by the Ribobio (China). The plasmid vectors and siRNAs were transfected into BC cells using Lipofectamine 3000 (Invitrogen, USA) as per the manufacturer's protocol. For generation of CASC9-depleted 5637 cell lines, the 5637 cells were transfected with control shRNA (sh NC) or CASC9-targeting shRNA (sh CASC9) and selected in the presence of 2 μg/mL puromycin.
10
Knockdown of NF-YA and NF-YB in Cell Lines
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Scrambled control (shSC), NF-YA (shNF-YA) and two NF-YB (shNF-YB-1 -gctatgtctactttaggcttt-; shNF-YB-2 -ccaaagaatgtgttcaagaatc-) shRNAs were cloned into pLKO.1 vector (Sigma Aldrich), and viral production and transduction were carried out as previously described [13 (link)]. H322 and HCT116 cells were transduced with shSC or shNF-YA viral supernatants, in triplicate, and cells collected after 72 hrs of incubation. Hela-S3 cells were infected with shSC or shNF-YB or shNF-YA and collected at 72 hrs after infection. In the experiments shown in Figure S11 , transient DNA transfections with shSC and shNF-YB-2 were performed in triplicate with Lipofectamine 2000 (Invitrogen 11668027). Cells were collected after 72 hours. Knockdown efficiency was assayed by PCR on cDNAs and by Western Blots on whole cell protein extracts using anti-NF-YA, anti NF-YB and anti-Actin antibodies. Total RNA was prepared by Trizol extraction and retrotrascribed with Iscript cDNA Synthesis kit (BIORAD 170-8890). For arrays, RNA was prepared according to Affymetrix standard protocol and hybridized to Hu-Gene 2.0 expression arrays.
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