Transient gene expression in HeLa or HEK293T cells was performed with transfection reagent Polyethylenimine (PEI MAX 40K, Polysciences Catalog# 24765) according to the manufacturer’s instructions. For induction of DNA damage, cells were exposed to 1 μM MNNG or 2 mM H2O2 for 20–30 min. In case of PARylation analysis, cells were pre-treated with 1 μM PARG inhibitor PDD00017273 (Sigma Aldrich) or PARP1 inhibitor AZD2281 or PJ34 for 1 hr. Cells were lysed in the following buffer: 50 mM HEPES pH7.9, 350 mM NaCl, 5 mM EDTA, 1% NP-40, 3 mM DTT, and 0.1 mM PMSF. After centrifugation for 10 min at 14,000 rpm, the supernatant was collected as whole cell extracts (WCE). The procedure for making nuclear extract (NE) has been described previously49 (link).
Pei max 40k
Manufactured by Polysciences
Sourced in United States
PEI MAX 40K is a polyethylenimine (PEI) product with a molecular weight of approximately 40,000 Daltons. It is used as a transfection reagent for the delivery of nucleic acids into mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Hek293f cell, by Thermo Fisher Scientific (5 mentions)
Protein g affinity chromatography, by GE Healthcare (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Gibson assembly master mix, by New England Biolabs (3 mentions)
Pmd2 g, by Addgene (2 mentions)
Quicktiter lentivirus titer kit, by Cell Biolabs (2 mentions)
Fetal bovine serum (fbs), by Lonsera (2 mentions)
Phelper, by Cell Biolabs (2 mentions)
Pei max 40k, by Cell Biolabs (2 mentions)
E8263 25ku, by Merck Group (2 mentions)
Z648043, by Merck Group (2 mentions)
Pei max 40k, by Thermo Fisher Scientific (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dual luciferase detection kit, by Promega (1 mentions)
Cytation 3 image reader, by Agilent Technologies (1 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
42 protocols using pei max 40k
1
Induction of DNA Damage in Cells
2
Dual-Luciferase Assay for Evaluating Transcriptional Regulation
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Cells were seeded at 60% confluency in 6-well plates and incubated in hormone-deprived medium for 72 h. Using the transfection reagent PEI MAX 40 K (Polysciences Inc. # 24765-100), cells were co-transfected with 50 ng of the Renilla luciferase transfection control plasmid pRL-CMV (Promega #E2261) and 1 μg of either the ERα reporter plasmid XETL (here referred to as ERE-Luc) [20 (link)] or the HIF1α reporter plasmid HRE-Luc [152 (link)] (a gift from Navdeep Chandel (Addgene #26731)). 24 h later, cells were harvested and seeded into 96-well plates. 24 h after seeding, cells were treated with the corresponding drugs for another 24 h (Table S1). For assays using HRE-Luc, cells were incubated in 1% O2 using a hypoxia incubator during the drug treatments. For assays in HEK 293 T cells, 1 μg of the plasmid HEG0 [153 (link)], which expresses full-length ERα, was added to the PEI MAX transfection mixture mentioned above. In parallel, a group of cells were transfected with the plasmid pSG5 [154 (link)] as the empty vector control. Using the dual-luciferase detection kit (Promega #E1910), cells were lysed in the lysis buffer for 30 min, and luciferase activities were measured with Cytation 3 Image Reader (Agilent). Firefly luciferase activities were normalized to those of the Renilla luciferase.
3
Rabbit Antibody Generation and Purification
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Hybridomas were cultured in medium E (Clonacell) at 37 °C with 5% humidity. When cells achieved appropriate density, total RNA was extracted using the Qiagen RNeasy mini kit, per the manufacturer’s protocol. cDNA was generated using the Invitrogen SuperScript IV First Strand Synthesis System using random hexamers following the manufacturer’s protocol. The variable regions of heavy and κ chains were PCR-amplified using previously described rabbit (63 (link)) primers and PCR conditions. PCR products were purified and cloned into an expression plasmid (63 (link), 64 (link)) adapted from the pFUSE-rIgG-Fc and pFUSE2-CLIg-rK1 vectors (InvivoGen) using the Gibson Assembly Master Mix (New England Biolabs) under ampicillin selection following the manufacturer’s protocol. Antibody heavy and light plasmids were cotransfected at a 1:1 ratio into HEK293F cells (ThermoFisher) using PEI Max 40K (linear polyethylenimine hydrochloride, Polysciences). Antibody supernatant was harvested 4 d following transfection and purified using protein G affinity chromatography following the manufacturers protocol (GE Healthcare).
4
Lentiviral Particle Production and Titration
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Two days prior to transfection, 293T cells were plated on 10 cm plates. The cells were transfected with plasmids: 1.3 pmol psPAX2, 0.72 pmol pMD2.G (Addgene plasmids #12260 and #12259; both were gifts from Didier Trono) and 1.64 pmol respective pLKO plasmid using PEI MAX 40K (Polysciences, Inc, Warrington, PA) at a ratio of DNA to PEI 1:3. Medium was changed 4 h after transfection. Cell culture supernatants containing pseudoviral particles were collected 48 h later, filtered through 0.45 µm PES filters and concentrated by overnight centrifugation at 8500 g, 4°C.
Pellets containing pseudoviral particles were resuspended in equal volumes of serum-free DMEM.
Initially, vectors were titrated using QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24; Cell Biolabs, San Diego, CA); the viral titers for SHC002 and SHC016 were similar, therefore in subsequent experiments equal volumes of concentrated viruses were used.
Pellets containing pseudoviral particles were resuspended in equal volumes of serum-free DMEM.
Initially, vectors were titrated using QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24; Cell Biolabs, San Diego, CA); the viral titers for SHC002 and SHC016 were similar, therefore in subsequent experiments equal volumes of concentrated viruses were used.
5
Purification of GFP-tagged SHANK Proteins
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Full-length rat GFP-Shank1A was already described (Romorini et al., 2004 (link)), as well as full-length rat GFP-tagged Shank2 (Boeckers et al., 2005 (link)) and full-length rat GFP-Shank3a (Grabrucker et al., 2011a (link)). HEK293T cells were cultivated in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37°C in 5% CO2. For transient transfection, cells were plated on 10-cm petri dishes and cultivated until 70–80% confluence and were then transfected with either full-length rat GFP-Shank1A, rat GFP-tagged Shank2 or rat GFP-Shank3a. Therefore, an appropriate amount of DNA was mixed with DMEM with 10% FBS and PEI MAX 40K (Polysciences) transfection reagent and spread onto the cells. The cells were lysed 24 h after the transfection (lysis buffer of the μMACS GFP Isolation Kit (Miltenyi Biotec) + PhosphoSTOP and Complete Mini EDTA-free Protease Inhibitor Cocktail (both Roche) and purification of the recombinant GFP-tagged SHANK1, 2, and 3 proteins was performed using the μMACS GFP Isolation Kit (Miltenyi Biotec) according to manufacturer’s instructions. Purified recombinant proteins were stored at –20°C until usage.
6
Generation of Recombinant AAV Vectors
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HEK293T cells were seeded in 150-mm plate, plasmids (pAAV-HBV1.04 or pAAV-HBV1.2, pAAV2/8-RC, and pHelper in 1:1:1 molar ratio) were cotransfected by using PEI MAX 40K (PolySciences, Warrington, PA). At 72 hours posttransfection, the cell culture supernatants and cells were harvested and filtered with 0.22-μm filter, then concentrated with Amicon Ultra-15 centrifugal filter unit (MilliporeSigma, Burlington, MA). The titer of recombinant AAV was determined by quantitative polymerase chain reaction (qPCR) as described previously.42 (link) Primer sequences are listed in Table 1 .
For AAV-HBV transduction experiments, HepG2 and AML12 cells were seeded in 24-well plates and transduced with 5 × 104 vg per cell diluted in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Lonsera), 1% penicillin/streptomycin (Gibco), and 4% PEG8000. One day posttransduction, cells were washed 3 times with PBS and treated further as indicated in the following experiment.
For AAV-HBV transduction experiments, HepG2 and AML12 cells were seeded in 24-well plates and transduced with 5 × 104 vg per cell diluted in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Lonsera), 1% penicillin/streptomycin (Gibco), and 4% PEG8000. One day posttransduction, cells were washed 3 times with PBS and treated further as indicated in the following experiment.
7
Cell Culture and Transfection Protocol
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Huh7, HepG2, AML12, HepAD38, and HEK293T cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Lonsera, Montevideo, Uruguay) and 1% penicillin/streptomycin (Gibco, Waltham, MA). The cell cultures were maintained in a 5% CO2 atmosphere at 37°C. PEI MAX 40K (PolySciences, Warrington, PA) was used for DNA transfection according to the manufacturer's instructions.
8
Rabbit Monoclonal Antibody Production
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The rabbit Ab variable regions of heavy and kappa chains were PCR amplified using previously described primers and PCR conditions (Table S4 ) (McCoy et al., 2016 (link)). PCR products were purified and cloned into an expression plasmid adapted from the pFUSE-rIgG-Fc and pFUSE2-CLIg-rK1 vectors (InvivoGen) using the Gibson Assembly® Master Mix (NEB) under ampicillin selection following the manufacturer’s protocol. Ab variable regions were sequenced by Sanger sequencing.
Ab heavy and light plasmids generated through B cell sorting were co-transfected at a 1:1 ratio into HEK293F cells (Thermofisher) using PEI Max 40K (linear polyethylenimine hydrochloride, Polysciences, Inc.). Ab supernatants were harvested four days following transfection and purified using protein G affinity chromatography following the maunfacturers protocol (GE Healthcare).
Ab heavy and light plasmids generated through B cell sorting were co-transfected at a 1:1 ratio into HEK293F cells (Thermofisher) using PEI Max 40K (linear polyethylenimine hydrochloride, Polysciences, Inc.). Ab supernatants were harvested four days following transfection and purified using protein G affinity chromatography following the maunfacturers protocol (GE Healthcare).
9
Cell Culture and Transfection Protocols
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HEK293T, TIVE, MDA-MB-231, ONS76, and U87MG cells were cultured in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich) with 5% CO2 at 37°C. HCT116 cells were cultured in McCoy's 5A medium (Hyclone). All media contained 1% penicillin and streptomycin (Gibco) and 10% fetal bovine serum (FBS; Gibco). HEK293T cells were transfected with polyethylenimine (PEI) MAX 40K (Polysciences) or Lipofectamine 3000 (Invitrogen) according to the manufacturers’ protocols. Briefly, 5×105 cells/well were seeded in six-well plates for 24 h. Doxycycline-inducible HCT116 cells expressing the BCL2L11 TDMD trigger were seeded at 2.5 × 105 cells/mL in tetracycline-depleted McCoy's 5A media (Atlanta Biologicals S10350). Twenty-four hours later, the medium was supplemented with doxycycline at 500 ng/mL. Doxycycline (250 ng/mL) was replenished after 24 h. Cells were harvested for analysis 72 h after seeding.
For protease inhibitor treatment, 12 different cell lines (HEK293T, MCF7, MDA-MB-231, TIVE, HCT116, MED8A, DAOY, ONS76, MOLT4, RPMI-8402, K562, and U87MG) were cultured to 50% confluency and treated with 1 µM MLN4924 in the medium for 48 h, or 10 µM MG-132 for 24 h, and then the total RNA was collected for Northern blot and RT-qPCR.
For protease inhibitor treatment, 12 different cell lines (HEK293T, MCF7, MDA-MB-231, TIVE, HCT116, MED8A, DAOY, ONS76, MOLT4, RPMI-8402, K562, and U87MG) were cultured to 50% confluency and treated with 1 µM MLN4924 in the medium for 48 h, or 10 µM MG-132 for 24 h, and then the total RNA was collected for Northern blot and RT-qPCR.
10
Lentiviral Transduction of 4T1 Cells
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All lentivectors were obtained from the stem cell technology research center (Tehran, Iran). Lentiviruses were produced by the three-plasmid system and transient cotransfection of HEK 293T cells using polyethyleneimine (PEI MAX 40K, Polysciences) transfection reagent.14 (link) The mixture of the packaging helper plasmids, psPAX and pMD.2G, with miR342/scrambled recombinant pCDH-GFP-Puro lentivectors, was prepared in a total of 15 µg/mL and DNA/PEI at a 1:2 ratio. 72 hours post-transfection, lentivirus-containing supernatant was collected every 12 hours and centrifuged at 2000 × g/4 °C for 10 minutes to eliminate packaging cells collected during harvesting. The seeded 4T1 cells at a confluency of 60% were transduced with miR342/scrambled lentiviruses and examined by fluorescent microscope for GFP expression after 96 hours post-infection. The stable cells were selected using the puromycin (Sigma, USA) treatment protocol. The control and treatment groups were considered the 4T1-scramble and 4T1-miR342 stable cells.
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