All plasmid DNA constructs and amounts used for co-transfection in this study are listed in Supplementary Table 1 . Plasmid DNA was amplified in DH5α Escherichia coli, purified with an EndoFree plasmid kit (Qiagen, Valencia, CA, USA), and resuspended in sterile PBS as previously described [13 (link),14 (link)].
Endofree plasmid kit
Manufactured by Qiagen
Sourced in United States, Germany
The EndoFree Plasmid Kit is a laboratory product designed for the purification of endotoxin-free plasmid DNA. It utilizes a proprietary endotoxin removal technology to ensure the isolated plasmid DNA is suitable for transfection and other sensitive applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Gen elute hpselect plasmid giga prep columns, by Merck Group (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Ecor1, by Roche (2 mentions)
Pcr isolation kit, by Qiagen (2 mentions)
Taqman probes for ifn λ1, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
C1000 thermal cycler, by Bio-Rad (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Sc 108080, by Santa Cruz Biotechnology (2 mentions)
Plko 1 shrna vectors, by Horizon Discovery (2 mentions)
Plvx ires zsgreen vector, by Takara Bio (2 mentions)
Plasmid maxi kit, by Qiagen (2 mentions)
Ecm 830 electroporation unit, by Harvard Apparatus (2 mentions)
Jetprime kit, by Polyplus Transfection (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
P3xflag myc cmv 26, by Merck Group (1 mentions)
α mem, by Merck Group (1 mentions)
67 protocols using endofree plasmid kit
1
Plasmid DNA Purification and Co-Transfection Protocol
2
Cloning and Expression of CagA and Akt
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The full-length cagA gene was amplified from H. pylori strain 26695 by polymerase chain reaction (PCR) with specific primers containing unique KpnI sites (5′-GGGGTACCCACTAACGAAACTATTGATCAAACAAG-3′; 5′-GGGGTACCCTTAAGATTTTTGGAAACCACCTTTTG-3′). The PCR product was cloned into the KpnI site of the expression vector p3XFLAG-Myc-CMV™-26 (Sigma, Saint Louis, Missouri, USA) to generate p3XFLAG-CagA where upstream of full-length cagA gene is tagged with three adjacent FLAG epitopes (Asp-Tyr-Lys-Xaa-Xaa-Asp). Akt was tagged with HA (YPYDVPDYA) at its C-terminal end to generate Akt-HA (wt). Dominant-negative Akt (DN-Akt) was constructed by site-directed mutagenesis at the ATP binding site (K179M) from Akt-HA to express a kinase-dead Akt. All cloned plasmids were purified using Endofree plasmid kit (Qiagen, Hilden, Germany).
3
Murine Cytotoxic T-Cell Analysis
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Six-to-eight week old female C57BL/6 mice (owner bred) were kept under SPF isolation conditions and standard diet at the animal facilities of the German Cancer Research Center, Heidelberg, Germany. Agarose-gel verified plasmids (>95% supercoiled, QIAGEN EndoFree Plasmid Kit; preparations contained less than 0.1 endotoxin units/µg plasmid DNA as tested earlier by Limulus endotoxin assay) were injected. For CTL analysis, animals were immunized once (100 µg DNA/per animal [50 µg DNA in 50 µl PBS per musculustibialis anterior i.m.]).Ten to 12 days after vaccination, animals were sacrificed and spleens were isolated.
Two×107 spleen cells (pretreated with ACT lysis buffer [17 mMTris/HCl, 160 mM NH4Cl, pH 7.2] to deplete erythrocytes) were used directly for Elispot-assays or co-cultured with 2×106 irradiated (100 Gy) RMA-S cells loaded either with E6 or E7 peptide in 25 cm2 culture flasks in αMEM (Sigma, Deisenhofen, Germany) supplemented with 10% FCS, 0.1 mMβ-mercaptoethanol, 4 mM glutamine and antibiotics for 5–6 days at 37°C and 7.5% CO2 in a humidified incubator.
Two×107 spleen cells (pretreated with ACT lysis buffer [17 mMTris/HCl, 160 mM NH4Cl, pH 7.2] to deplete erythrocytes) were used directly for Elispot-assays or co-cultured with 2×106 irradiated (100 Gy) RMA-S cells loaded either with E6 or E7 peptide in 25 cm2 culture flasks in αMEM (Sigma, Deisenhofen, Germany) supplemented with 10% FCS, 0.1 mMβ-mercaptoethanol, 4 mM glutamine and antibiotics for 5–6 days at 37°C and 7.5% CO2 in a humidified incubator.
4
MHC-I and MHC-II Induction in Tumor Cells
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Overall, 100,000 tumor cells were cultured in T2 medium containing the specified stimulation agents. For MHC-I upregulation, cells were treated with 10 ng/mL recombinant human Interferon Gamma (rhIFN)-γ (Peprotech) for 24 hours. For MHC-II induction, cells were incubated with 30 ng/mL rhIFN-γ for 3 days or transduced with human Class II Major Histocompatibility Complex Transactivator (hCIITA)+/- lentiviral vectors. The appropriate controls of unstimulated and untransduced cells were also included in the experiments. MHC-I and/or MHC-II expression was detected by flow cytometry using the antibodies specified on online supplemental table 2 .
hCIITA was cloned into pHIV-Luc-ZsGreen (Addgene) backbone and DNA was isolated using EndoFree Plasmid Kit (Qiagen). hCIITA-expressing lentiviral particles were produced by co-transfecting HEK293T cells with the pHIV-CIITA-ZsGreen plasmid, the pMD2.G envelope plasmid and the psPAX2 packaging plasmid. Virus-containing supernatants were concentrated by ultracentrifugation and TCLs were transduced by spinoculation with hCIITA-expressing lentiviral particles.
hCIITA was cloned into pHIV-Luc-ZsGreen (Addgene) backbone and DNA was isolated using EndoFree Plasmid Kit (Qiagen). hCIITA-expressing lentiviral particles were produced by co-transfecting HEK293T cells with the pHIV-CIITA-ZsGreen plasmid, the pMD2.G envelope plasmid and the psPAX2 packaging plasmid. Virus-containing supernatants were concentrated by ultracentrifugation and TCLs were transduced by spinoculation with hCIITA-expressing lentiviral particles.
5
In Utero Plasmid Electroporation
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Plasmids were purified using the EndoFree Plasmid Kit (QIAGEN Plasmid plus maxi kit, 12965). About 1 μl DNA solution, including the indicated plasmids (with each plasmid 1 μg/μl) and 0.1% fast green, was injected into the medial region of the lateral ventricles of the embryonic murine brain with a glass micropipette after the pregnant mouse had been anesthetized with 3% isoflurane. The electrical pulses were delivered to the embryo under the following conditions: 25 V, 50 ms on, 950 ms off, and five pulses (BTX ECM 830). After electroporation, the uterus was returned to the abdominal cavity, followed by suture of the abdominal wall and skin. Animals were kept warm at 37 °C until recovery from anesthesia.
6
Electrotransfer-based Gene Delivery in Mice
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Expression plasmids used for electrotransfer studies were purified using a Endofree plasmid kit (Qiagen) and dissolved in 0.9% NaCl. 45 min before electrotransfer, muscles were pretreated with hyaluronidase (10 U/muscle). Afterwards, naked plasmids (60 μg DNA) were injected into the tibialis anterior muscle and 10 pulses of 20 ms each were applied to each hindlimb at 175 V/cm and 1 Hz using an electroporator (ECM 830; BTX). Empty vector was used as control.
AAV for in vivo expression of Sesn1 and Sesn2 were generated and provided by the Virus Production Unit (UPV, UAB, Barcelona). AAVs were diluted in 0.9% NaCl at 0.25*1013 gc/ml and directly injected into muscles (40 µl/TA and 10 µl/EDL and soleus muscles). AAV-GFP was used as a control. Four days after transduction mice were subjected to the immobilization protocol.
AAV for in vivo expression of Sesn1 and Sesn2 were generated and provided by the Virus Production Unit (UPV, UAB, Barcelona). AAVs were diluted in 0.9% NaCl at 0.25*1013 gc/ml and directly injected into muscles (40 µl/TA and 10 µl/EDL and soleus muscles). AAV-GFP was used as a control. Four days after transduction mice were subjected to the immobilization protocol.
7
In Utero Electroporation of Lateral Ventricles
8
Cloning and Transfection of BLH6a in Protoplasts
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The fragment of 3× FLAG was amplified from the pCM1307-N-Flag-HA vector (Zhou et al., 2012 (link)), cloned into the pENTR/D TOPO Vector (Invitrogen), and LR ligated into pUC19-35S-RfA-35S-sGFP (Li et al., 2012 (link)) to generate pUC19-35S-FLAG-35S-sGFP. BLH6a coding sequences were amplified from P. alba × P. glandulosa xylem cDNA and cloned into pUC19-35S-FLAG-35S-sGFP, generating pUC19-35S-Flag:BLH6a-35S-sGFP. Primers are listed in Supplementary Table 1 . Plasmid DNA for the protoplast transfection was prepared using the EndoFree Plasmid Kit (QIAGEN Inc., Valencia, CA, USA). Leaf protoplasts were prepared from mesophyII of leaves in tissue culture bottles following the published protocol (Lee et al., 2017 (link)), and xylem protoplasts were prepared as described previously (Lin et al., 2014 (link)). Plasmids were transferred into protoplasts, and each chromatin immunoprecipitation (ChIP) assay was performed using 5 × 106 protoplasts with anti-FLAG antibodies as described before (Yan et al., 2019 (link)). One-fiftieth of the supernatants before adding antibodies were used as input in the qPCR. qPCR was conducted using the Green Premix Ex Tag II (TaKaRa, Dalian, China) and detected by the Roche LightCycler 480 II (Roche Co., Basel, Switzerland), with 18S as the reference.
9
Quantifying Interferon Response to Plasmid DNA Transfection
10
Quantifying Interferon Response to Plasmid DNA Transfection
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A non-coding plasmid DNA (pCR2.1, Life Technologies) was isolated using Endofree plasmid kit (Qiagen) and digested with EcoR1 (Roche) to make linearized DNA. The digested DNA was purified using PCR isolation kit (Qiagen). DNA transfection was performed using Lipofectamine 2000 (Life Technologies). Cells (3 million at 1×106/ml) were transfected with 3μg of linearized DNA for 22 hours in RPMI-1640 (Invitrogen) with 10 % FBS (Hyclone) in a 6 well plate. After incubation, cellular RNA was extracted using the RNeasy Mini kit (Qiagen) and real time qRT-PCR was performed using Taqman probes for IFN-λ1, IFN-β, RANTES and GAPDH (Applied Biosystems) with C1000 Thermal Cycler (BioRad). Fold change of gene products was calculated using the delta-delta CT method [11 (link)].
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