FOXA3-WT cDNA (pCMV6-XL5-FOXA3) and vector (pCMV6-XL5) were obtained from Origene (Rockville, MD, USA). FOXA3 mutants at nucleotides 185 (c.185C>T) and 731 (c.731C>T) were generated by site-directed mutagenesis of the FOXA3-WT plasmid (Origene). 10T1/2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (CellGro, Manassas, VA, USA) supplemented with Penicillin/Streptomycin and 10% fetal bovine serum (Hyclone, Logan, UT, USA). A total of 5.0 × 105 10T1/2 cells were transfected using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Cologne, Germany) with either 1.2 μg of control plasmids or with 1.2 μg FOXA3-WT, FOXA3 c.185C>T or FOXA3 c.731C>T. pmaxGFP plasmid (0.3 μg, Amaxa Biosystems) was co-transfected for transfection normalization. Twenty-four hours after nucleofection, cells were treated with 5 μg ml−1 insulin and with 10 μM troglitazone for additional 3 days before cells were harvested for RNA analysis.
Pmaxgfp plasmid
Manufactured by Lonza
Sourced in Switzerland, Germany
The PmaxGFP plasmid is a recombinant DNA molecule that contains the coding sequence for the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. This plasmid is designed to express GFP under the control of a strong promoter, allowing for the visualization of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nucleobond xtra maxi endotoxin free kit, by Macherey-Nagel (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Yoyo 1 iodide, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Pcmv6 xl5, by OriGene (1 mentions)
Dulbecco s modified eagle s medium, by Corning (1 mentions)
Nucleofector 96 well shuttle system, by Lonza (1 mentions)
Giga prep kit, by Qiagen (1 mentions)
Nucleofector system, by Lonza (1 mentions)
Nucleofector 2, by Lonza (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Chemically modified sgrna, by Synthego (1 mentions)
Nucleofection, by Lonza (1 mentions)
33 protocols using pmaxgfp plasmid
1
FOXA3 Mutant Functional Analysis
2
Plasmid-Mediated Bleomycin Delivery
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The pMAX-GFP plasmid (3486bp) (Amaxa, Cologne, Germany) coding for enhanced green fluorescent protein was used in the experiments. Plasmid concentrations of 0–400 µg/mL were used to enhance the transfer of the chemotherapeutic drug, bleomycin, and to evaluate the percentage of transfected cells. The plasmid was purified from competent DH5α cells using endo-free plasmid Giga Prep kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The concentration and the quality of the plasmid was checked using a spectrophotometer (Nanodrop 2000, Thermo Fisher, Washington, DC, USA) and gel electrophoresis.
3
Optimizing Cardiomyocyte Progenitor Nucleofection
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Cardiomyocyte progenitor cells were nucleofected using the Lonza Amaxa 2b Nucleofector Device and the Human Stem Cell Nucleofector I Kit according to manufacturers’ protocol. For protocol optimization, GFP was nucleofected using the pmaxGFP plasmid (Amaxa, Cologne, Germany). Briefly, 1 × 106 cells were resuspended in 100 μL of nucleofection buffer and 2 μg of pmaxGFP. To determine the optimal nucleofection condition, several programs were tested. Each program differs in duration and intensity of the pulsation as described by the manufacturer. After nucleofection, cells were seeded in six well plates with SP++ medium. Program X001 was selected and used in further nucleofections. For the comparison of GFP expression with or without Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), and for FACS measurements, cells were nucleofected with 1 μg of plasmid.
For the optimization of the construct, 0.5 μg pmaxGFP, 2 μg mouse(m)Hcn2-IRES-DsRed, and 2 μg rat(r)SkM1-IRES-GFP plasmid was used. For the combination of mHcn2 and rSkM1, 2 μg of each plasmid was co-nucleofected in the CPCs. Four days after nucleofection, cells were seeded on coverslips for immunocytochemistry.
For patch-clamp experiments, 2 μg of mHcn2 or 2 μg of rSkM1 plasmid (both without reporter markers) was co-nucleofected with 0.5 μg of GFP plasmid.
For the optimization of the construct, 0.5 μg pmaxGFP, 2 μg mouse(m)Hcn2-IRES-DsRed, and 2 μg rat(r)SkM1-IRES-GFP plasmid was used. For the combination of mHcn2 and rSkM1, 2 μg of each plasmid was co-nucleofected in the CPCs. Four days after nucleofection, cells were seeded on coverslips for immunocytochemistry.
For patch-clamp experiments, 2 μg of mHcn2 or 2 μg of rSkM1 plasmid (both without reporter markers) was co-nucleofected with 0.5 μg of GFP plasmid.
4
Fluorescent Labeling of MSC and Muscle-Derived Cells
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For the assessment of interactions in a direct co-culture, MSC were transfected with pMAX-GFP plasmid (Amaxa Biosystems) using the Nucleofector system (Amaxa Biosystems). Typically, 2x106 cells were in 100 μl Cell Line Nucleofector Human MSC Solution and mixed with 2 μg plasmid DNA. Nucleofections were performed using the Amaxa Nucleofector II device with a U-023 program. Post-nucleofection cells were incubated overnight in a RPMI medium supplemented with 20% FBS and used for the following procedures. In one experiment, muscle-derived cells were labeled with red membrane fluorochrome PKH26, and MSC were labeled with green PKH67 (both from Sigma Aldrich). Staining was performed according to the manufacturer instructions with a 4 nM of PKH solution for 4 min in RT. The labeling efficiency was evaluated with fluorescence microscopy (Olympus IX51) and is demonstrated in the supplementary material (S1 Fig ).
5
Genome Editing in VACV-Infected Cells
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On day 1, 5 × 106 CV-1 cells were infected with VACV at an MOI of 0.02 in DMEM supplemented with 0.5% v/v FCS for 1 h at 37 °C in a 5% CO2 atmosphere. After infection, cells were electroporated using the Neon Transfection System (Thermo Fisher Scientific, Courtaboeuf, France) as follows: cells were washed once in 1X phosphate-buffered saline (PBS), harvested by trypsinization, and resuspended in 100 µL electroporation R buffer (1 × 107 cells/mL). Cells (100 µL) were mixed with 2.25 µg of each plasmid: pCMV-Cas9ΔNLS, the pGEM-T-E9L vector, and the pU6-gRNA of interest. In control experiments (i.e., transfection without pCMV-Cas9ΔNLS and pU6-gRNA), 4.5 µg of empty pmaxGFP plasmid (Lonza, Basel, Switzerland) was co-transfected together with the pGEM-T-E9L vector. The cell/DNA mixture was aspirated into a 100 µL Neon tip and submitted to two electric pulses at 1050 volts for 30 ms. Cells were then seeded in six-well plates containing warm DMEM supplemented with 10% FCS and incubated for 3 days at 37 °C and 5% CO2.
6
Western Blot Analysis and Compound Stimulation
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Western blot analysis was performed as previously described (92 (link)) using primary antibodies and secondary Horseradish peroxidase-coupled antibodies listed in Table S3 . Chemiluminescence was detected on a Chemidoc Touch Imaging System (Bio-Rad). Data was processed with ImageLab (Bio-Rad) and normalized to total protein (stain-free detection) (94 (link)).
For the compound stimulation cells were grown in 6-well plates and treated as indicated (24 h) with chloroquine (Sigma-Aldrich, #C6628) or EDME. For Rab11-overexpression cells were transiently transfected with pEGFP-C1-Rab11-WT (Addgene, #12674) or pmaxGFP-plasmid (Lonza, #VCA-1001) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, #100022057) according to manufacturer’s instructions and incubated (48 h). For cathepsin B release cells were grown overnight, medium was exchanged to FCS-free medium, and treated with ionomycin (5 μM) (Sigma, #10634) for 10 min (95 (link)). Supernatant was concentrated using Merck Amicon centrifugal units (Thermo Fisher Scientific, #10341782).
For the compound stimulation cells were grown in 6-well plates and treated as indicated (24 h) with chloroquine (Sigma-Aldrich, #C6628) or EDME. For Rab11-overexpression cells were transiently transfected with pEGFP-C1-Rab11-WT (Addgene, #12674) or pmaxGFP-plasmid (Lonza, #VCA-1001) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, #100022057) according to manufacturer’s instructions and incubated (48 h). For cathepsin B release cells were grown overnight, medium was exchanged to FCS-free medium, and treated with ionomycin (5 μM) (Sigma, #10634) for 10 min (95 (link)). Supernatant was concentrated using Merck Amicon centrifugal units (Thermo Fisher Scientific, #10341782).
7
CRISPR-Cas9 Knockout Protocol for T-ALL Cells
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Briefly, the sgRNA was designed with at least 3 bp of mismatch to any other site in the human genome to mitigate the risk of off-target editing. To generate the modified cells, 400,000 cells were transiently co-transfected with/without 0.5 μg pmaxGFP plasmid (Lonza), with 33 pmol spCas9 protein, 100 pmol chemically modified sgRNA (Synthego), and 100 pmol of blocking ssODN (IDT) via nucleofection (Lonza), using solutions and programs according to the manufacturer’s recommended protocol. Five days post-nucleofection, cells were single-cell sorted by fluorescence-activated cell sorting into 96-well plates. Cells were clonally expanded and verified the desired modification via targeted deep sequencing followed by CRIS.py analysis27 (link). Selected homo/hetero knock-out clones were validated by MiSEQ, Sanger sequencing, RNAseq as well as western blotting to confirm the expression of USP7. We note that T-ALL cells tend not to tolerate Cas9 that well, which could explain our difficulty in generating viable CRISPR clones in HSB-2 cells.
8
Transfection and Transcriptional Activation Analysis
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U87MG cells or PFAs were seeded in 6, 12 or 24 well plates at a density designed to yield 90% confluency within 24 hours. Cells were transfected using Transfectin (Bio-Rad) or Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. pUC19 plasmid was used to normalize total transfection input DNA across all conditions. pmaxGFP plasmid (Lonza) was included to monitor transfection efficiency by fluorescence microscopy. M50 Super 8x TOPFlash β-catenin plasmid [77 (link)] (a gift from Randall Moon (Addgene plasmid # 12456; http://n2t.net/addgene:12456 ; RRID:Addgene_12456) and the Cignal TCF/LEF β-catenin plasmid (Qiagen #336841) are luciferase reporter constructs under the control of β-catenin-mediated transcriptional activation. Flag-Tat86WT and Flag-Tat101WT are HIV-1 Tat expression vectors that have been previously described [78 (link), 79 (link)]. Flag-Tat101WT was used to generate Tat mutants, Tat101C31R, Tat101W11F, and Tat101W11Y utilizing the GeneArt Site-Directed Mutagenesis System (Invitrogen).
9
Comprehensive SARS-CoV-2 Protein Expression Constructs
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Wild-type (Wu-han strain) SARS-CoV-2 N proteins (Genbank: NC_045512.2 ), R203K/G204R, and Omicron N protein were synthesized by MISSION BIOTECH CO., LTD, Taiwan, and ligated to the pcDNA3.1 backbone using Gibson Assembly (NEB, E2611L). The sequence of other coronavirus nucleocapsid proteins including SARS-N (Genbank: AYV99827.1 ), MERS-N (Genbank: AHE78101.1 ), NL63-N (Genbank: AFV53152.1 ), 229E-N (Genbank: AGW80953.1 ), HKU1-N (Genbank: ARU07581.1 ), OC43-N (Genbank: YP_009555245.1 ) were constructed to the pcDNA3.1 backbone by Gibson Assembly. GFP-DNA used in this study, pmax-GFP plasmid was supplied by Lonza; in vitro transcribed (IVT) single strand mRNAs: SARS-CoV-2 RBD, SARS-CoV-2 Spike and Luciferase-P2A-GFP used in this study were provided by National Biotechnology Research Park (NBRP), Taipei, Taiwan. FAM-labeled siRNA, RNA#1 and RNA#12 RNA used in this study is purchased from MDBio, Inc. Taiwan and the sequence is 5′-UUCUCCGAACGUGUCACGUTT-3′.
10
Reverse transfection of siRNAs against polyamine synthesis enzymes
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Reverse transfection of siRNA was performed using Lipofectamine RNAiMAX (Invitrogen) as previously described [90 (link)]. Following Silencer Select siRNAs (Invitrogen) were used: ornithine decarboxylase 1 (ODC1) (s9821, s9822, s9823 as si1-3), agmatinase (AGMAT) (s379, s381 as si1-2), spermidine synthase (SMS) (s13173, s13175 as si1-2), spermine synthase (SRM) (s13430, s13432 as si1-2), deoxyhypusine synthase (DHPS) (s91, s92 as si1-2), and non-targeting control (Cat#AM4641). Turbofect (ThermoFisher) was used for vector transfection following manufacturer’s recommendations. Flag-tagged KSHV ORF50/RTA vector was a gift from Dr. Pinghui Feng (University of South California) [91 (link)], pSG5-BZLF1 was a gift from Dr. Diane Hayward (Addgene # 72637) [92 (link)], and pA3M-LANA was a gift from Dr. Erle Robertson (University of Pennsylvania). The pmaxGFP plasmid was obtained from Lonza.
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