The EGFP-FOXM1B fusion plasmid was a gift from Dr M. Teh (Queen Mary University of London) and was cloned into the pcDNA4/TO plasmid (Invitrogen) for transient expression in HeLa TRex cells or into the pcDNA5FRT (Addgene) to generate stable cell lines. HEK293 Flp-In cells (Invitrogen) were first transfected with a pcDNA6/TR plasmid (Invitrogen) and selected with 5 μg/mL blasticidin to generate a stable HEK293tetR Flp-In cell line. This cell line was co-transfected with the GFP-FOXM1 and pOG44 (Flp recombinase vector) and selected with 100 μg/mL hygromycin (Invitrogen).
Pcdna6 tr plasmid
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA6/TR plasmid is a DNA vector used for the regulated expression of genes in mammalian cell lines. It contains a tetracycline-regulated promoter system that allows for tight control of gene expression.
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Lab products found in correlation
Pcdna4 to plasmid, by Thermo Fisher Scientific (2 mentions)
Pentr h1 to vector, by Thermo Fisher Scientific (2 mentions)
Td 09761, by Inotiv (2 mentions)
Facsaria flow cytometer, by BD (2 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Hek293 flp in cells, by Thermo Fisher Scientific (1 mentions)
Pcdna4 to myc his vector, by Thermo Fisher Scientific (1 mentions)
Pcdna4 to mychis, by Thermo Fisher Scientific (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
T rex system, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
9 protocols using pcdna6 tr plasmid
1
Inducible EGFP-FOXM1B Fusion Plasmid
2
Tetracycline-Regulated Expression of NF-κB and PYCARD
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E. coli strain DH5αF′ was used to propagate all the plasmids. pcDNA6/TR plasmid(Invitrogen, Carlsbad, CA, USA) was used to establish cell lines that stably express the Tet repressor. To allow tetracycline-regulated expression, we used three constructs. One contains a 0.9 kb fragment of NFκB (AD)-MBD with a 3xFLAG tag at the N-terminus [16 (link)] that was cloned into the EcoRI/XhoI sites of pcDNA4/TO/myc-His vector (Invitrogen) and named as pcDNA4/TO/NFκB (AD)-MBD. The other two constructs contain cDNA fragments corresponding to the entire coding regions of PYCARD variants 1 and 2, a 0.61 kb fragment of PYCARDv1 and a 0.56 kb fragment of PYCARDv2. These two fragments were cloned into the HindIII/XbaI sites of pcDNA4/TO/myc-His vector and named as pcDNA4/TO/PYCARDv1 and pcDNA4/TO/PYCARDv2, respectively. We PCR amplified these two variant cDNA fragments using the pooled human cDNA mix [20 (link)] as the template. Nucleotide sequences of the primers used for cDNA cloning are described in TableS1 .
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E. coli strain DH5αF′ was used to propagate all the plasmids. pcDNA6/TR plasmid(Invitrogen, Carlsbad, CA, USA) was used to establish cell lines that stably express the Tet repressor. To allow tetracycline-regulated expression, we used three constructs. One contains a 0.9 kb fragment of NFκB (AD)-MBD with a 3xFLAG tag at the N-terminus [16 (link)] that was cloned into the EcoRI/XhoI sites of pcDNA4/TO/myc-His vector (Invitrogen) and named as pcDNA4/TO/NFκB (AD)-MBD. The other two constructs contain cDNA fragments corresponding to the entire coding regions of PYCARD variants 1 and 2, a 0.61 kb fragment of PYCARDv1 and a 0.56 kb fragment of PYCARDv2. These two fragments were cloned into the HindIII/XbaI sites of pcDNA4/TO/myc-His vector and named as pcDNA4/TO/PYCARDv1 and pcDNA4/TO/PYCARDv2, respectively. We PCR amplified these two variant cDNA fragments using the pooled human cDNA mix [20 (link)] as the template. Nucleotide sequences of the primers used for cDNA cloning are described in Table
3
Tetracycline-Regulated IRX4 Expression
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Escherichia coli strain DH5αF’ was used to propagate all of the plasmids. The plasmid to induce MeTA, pcDNA6‐3xFLAG‐NF‐κB (AD)‐MBD, was constructed previously.10 pcDNA6/TR plasmid (Invitrogen) was used to establish cell lines that stably express the Tet repressor protein. The IRX4, CRYAB, CD69, and IL32 cDNA clones were PCR amplified by using the pooled human cDNA mix22 or HPDE‐1 cDNA as the templates and were cloned into the pcDNA6/myc‐His vector for colony formation assay. Nucleotide sequences of the primers used for cDNA cloning are described in Table S1 . To allow tetracycline‐regulated expression of IRX4, the 70‐bp fragment of 3xFLAG and the 1.6‐kbp fragment of IRX4 were cloned into the BamHI and XhoI sites of pcDNA4/TO/myc‐His (Invitrogen) vector to generate pcDNA4/TO/3xFLAG‐IRX4.
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Escherichia coli strain DH5αF’ was used to propagate all of the plasmids. The plasmid to induce MeTA, pcDNA6‐3xFLAG‐NF‐κB (AD)‐MBD, was constructed previously.
4
Engineered Cell Lines for NOR Study
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HT1080 and 3T3 cells were cultured in DMEM with 10% fetal bovine serum (FBS) and antibiotics. HT1080 cells were transfected with the tetracycline repressor-expressing plasmid conferring blasticidin resistance (pcDNA6/TR plasmid, Invitrogen) to produce the HT1080 TetR cell line. The UBF-KD cell line was generated by transfection of HT1080 TetR cells with pTER+UBFshRNA. UBF-KD cells were selected and cultured in medium containing tetracycline-free FBS, 5 μg/mL blasticidin, and 200 μg/mL zeocin. The viability of UBF-KD cells was assessed by seeding 105 cells and counting surviving cells following 1–5 d of culture in the above medium with 2 ng/mL or 1 μg/mL Dox. Pseudo-NOR clone 3D cells (Mais et al. 2005 (link)) were grown in medium containing 5 μg/mL blasticidin. Ptk-2 cells were cultured in minimum essential medium (MEM) Eagle (+Earles salts, reduced NaHCO3, without L-glutamine) supplemented with 10% FBS, 200 mM 2% L-glutamine, and 1% MEM nonessential amino acid. Neo-NOR clones were obtained by cotransfection of HT1080 cells with the insert from pNeo-NOR and a plasmid conferring blasticidin resistance in a 200:1 molar ratio. Clones were maintained in medium containing 5 μg/mL blasticidin. AMD was used at 0.1 μg/mL for 2 h.
5
Generating Tetracycline-Inducible GPR30 Cell Line
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HFF11 cells were transfected with the pcDNA6/TR plasmid (Invitrogen, Carlsbad, CA) using TransIT-LT1 to create an HFF11 cell line expressing the tetracycline (TET) repressor, and stable clones were selected based on blasticidin resistance. FLAG-tagged GPR30 was ligated into the HindIII/XbaI sites of the pcDNA 4T/O plasmid (Invitrogen), the resulting plasmid transfected into HFF11 cells expressing the TET repressor to create T-REx HFF11 cells, and stable clones selected based on blasticidin and zeocin resistance. The cells were then grown in phenol red–free DMEM supplemented with 10% normal or charcoal-treated FBS (growth medium) in 5% CO2 at 37°C.
6
Culturing human CML and mouse stromal cell lines
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The human CML cell lines LAMA-84 and K562 (ATCC) and A1-K562 [26 (link)] were regularly tested for mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza, Basel, Switzerland, #LT07-418). A1-K562 cells expressing the tetracycline repressor (Tet-K562) were established by the stable transfection of the pCDNA6-TR plasmid (Invitrogen Life Technologies) as previously described [26 (link)]. The mouse M2-10B4 cell line, derived from bone marrow stromal cells, was purchased from STEMCELL Technologies (Vancouver, BC, Canada).
All the cell lines were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), 1 mM sodium pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin.
All the cell lines were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), 1 mM sodium pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin.
7
Tetracycline-Induced Expression of US21-HA Proteins
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The tetracycline (Tet)-induced expression of pUS21-HA, pUS21-HA D178N, and pUS21-HA D201N was attained using the T-REx system (Invitrogen, Waltham, MS, USA, and Life Technologies, Waltham, MA, USA). To this end, U2OS cells were co-transfected with pcDNA6/TR plasmid (Invitrogen, ) carrying the Tet repressor gene and pcDNA4/TO plasmids (Invitrogen, Waltham, MA, USA) containing the different US21-HA ORFs (16 (link)). Stably transfected T-REx-U2OS cell lines were selected using 2 µg/mL blasticidin and 500 µg/mL zeocin (Life Technologies, Carlsbad, CA, USA). To induce the expression of pUS21-HA proteins, tetracycline (1 µg/mL) was added for 24 or 48 h.
8
Conditional Lyn Knockdown in Mouse Mammary Tumors
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Pairs of complementary DNA oligonucleotides (Table S3 ), encoding shLyn#2 (shLyn) or shScr, were annealed and cloned into a pENTR™/H1/TO vector (ThermoFisher Scientific). The H1/TO -shLyn or -shScr cassette was then transferred into a Gateway-modified pSEW lentiviral vector (Regan et al., 2012 (link)) via LR recombination. ORF of Tetracycline repressor (TetR) was amplified from pcDNA™6/TR plasmid (ThermoFisher Scientific) (Table S3 ) and cloned into a pHIV-H2BmRFP lentiviral vector. Primary mouse BlgCre Brca1fl/flp53+/− mammary tumor cells (line #2) were transduced using pHIV-RFP-TetR and pSEW-GFP-TO-H1(-shScr or -shLyn) lentiviral vectors. Cells positive for both GFP and RFP expression were then sorted on a FACSAria flow cytometer (BD Biosciences) and assessed for Lyn knockdown in vitro in the absence or in the presence of doxycyline (0.5 ug/ml). 250,000 (shLyn- or shScr-) cells were orthotopically injected into the fourth right mammary fat pad of nude mice. Mice were randomized to either a control (DOX-) or a doxycicline (DOX+) diet (TD.09761, Harlan Teklad, Harlan, Indianapolis, USA). Tumor volumes were calculated from caliper measurements of tumor width (W) and length (L) using the formula (L x W2)/2).
9
Doxycycline-Inducible Lyn Knockdown in Murine Mammary Tumor Cells
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