Metformin, LPS, AICAR, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma (St. Louis, MO). Recombinant murine IL-13 was purchased from PeproTech (Rocky Hill, NJ). Mouse recombinant M-CSF and antibodies against AMPKα1 and p-AMPKα1 were from Cell Signaling Technology (Beverly, MA, USA). Antibody against Actin was purchased from Santa Cruz Biotechnology (CA, USA). Antibodies for flow cytometry including PE-conjugated anti-mouse CD206, PE-conjugated anti-mouse CD86 and FITC-conjugated anti-mouse F4/80 were purchased from Biolegend (San Diego, CA, USA). For immunofluorescence, first antibodies including anti-mouse F4/80, anti-CD31, anti-αSMA, and FITC-conjugated anti-mouse CD206 were from eBioscience, Abcam, Sigma and Biolegend respectively, while secondary antibodies including anti-Rat and anti-mouse were from life technology. FITC Annexin V Apoptosis Detection Kit I and matrigel were purchased from BD (San Jose, CA, USA). JetPrime transfection agent was obtained from Polyplus. Clodronate liposomes and PBS liposomes were purchased from ClodronateLiposomes.com (Amsterdam, Netherlands).
Jetprime transfection agent
Manufactured by Polyplus Transfection
Sourced in France, United States
JetPrime is a transfection reagent that facilitates the delivery of nucleic acids, such as plasmid DNA, siRNA, or mRNA, into mammalian cells. It is designed to efficiently complex and protect the nucleic acids, enabling their uptake by the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Immobilized anti myc antibody, by Merck Group (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Anti mouse f4 80, by Thermo Fisher Scientific (1 mentions)
Anti cd31, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti mouse cd206, by BioLegend (1 mentions)
Pe conjugated anti mouse cd86, by BioLegend (1 mentions)
Recombinant murine il 13, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse f4 80, by BioLegend (1 mentions)
Anti α sma, by Thermo Fisher Scientific (1 mentions)
Aicar, by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Matrigel, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Antibody against actin, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Cell Signaling Technology (1 mentions)
Lsm 700, by Zeiss (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
13 protocols using jetprime transfection agent
1
Macrophage Polarization Dynamics
2
Immunoprecipitation of Myc-tagged Proteins
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HEK293T cells were transfected with the expression vectors using jetPRIME Transfection Agent (Polyplus, Cat. number PT-114-15) according to the manufacturer's instructions. Transfected cells were washed with phosphate-buffered saline (PBS) 24 hours after transfection and lysed in ice-cold lysis buffer consisting of 150 mM NaCl, 50 mM Tris at pH 7.5, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1 × protease inhibitor cocktail (Roche). After centrifugation at 4°C, the supernatant was collected and incubated with immobilized anti-Myc antibody (Sigma-Aldrich, Cat. number E6654) at 4°C overnight. After washing five times with washing buffer (a modified lysis buffer containing 500 mM NaCl instead of 150 mM), the immunoprecipitated proteins were separated by polyacrylamide gel electrophoresis (PAGE) and then transferred to PVDF membrane. The blot was incubated with corresponding primary antibodies, followed by incubation with secondary antibodies (Bio-Rad), and the signals were detected with the ECL system (Cell Signaling Technology).
3
Transfection of Liver Cancer Cells with siRNA and Lentivirus
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For transfection, the cells were seeded in 6‐well plates at a density of 3 × 105 cells/well. The anti‐SERPINE2 small interfering RNAs (siRNAs) were constructed by Sunya Biological (Hangzhou, Zhejiang, China) and all plasmids were constructed by REPOBIO (Hangzhou, Zhejiang, China). Liver cancer cells or HEK‐293T cells were transfected with a JetPRIME transfection agent (Polyplus, Illkirch, France) according to the manufacturer's instructions. SERPINE2‐overexpression and short hairpin RNA (shRNA) knockdown lentivirus (Zorin Biological Company, Shanghai, China) were used to infect liver cancer cell lines, and 10 μg/mL puromycin (Thermo Fisher Scientific, Waltham, MA, USA) was used to screen stable liver cancer cell lines. The specific sequences of the siRNAs and shRNAs are listed in Supplementary Table S7 .
4
Nrf2 Gene Silencing with shRNA
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Small hairpin RNA (shRNA) was used to silence Nrf2 gene expression. Nrf2 siRNAs were obtained from Invitrogen (Nrf2-si-1: siRNA ID 68238; Nrf2-si-2: siRNA ID 156499; and Nrf2-si-3: siRNA ID 156501). For effective gene silencing, siRNAs were transfected twice. In short, according to the manufacturer’s instructions, use jetPRIME ® Transfection Agent (Polyplus) to transfect cells with 30 nM negative control (NC) siRNA and Nrf2-siRNA. After 24 h of incubation, a second transfection was performed.
5
Fluorescent Protein Localization in COS-7 Cells
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Cultured cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and transfected with expression vectors or siRNAs using jetPRIME transfection agent (Polyplus Transfection Inc., New York, NY, USA, Cat. No. PT-114–15) according to the manufacturer’s instructions. To visualize the localization of GFP- or mCherry-tagged proteins, transfected COS-7 cells that grown on glass coverslips were fixed with 4% PFA, then incubated with DAPI (Gen-View Scientific Inc., El Monte, CA, USA). Slides were mounted in mounting solution (50% glycerol/PBS) and imaged using a confocal microscope (LSM 700, Zeiss, Germany).
6
Dendritic Cell Transfection Optimization
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DCs were cultured in 60 mm dish containing DMEM with 10% FBS for 24 h at a density of 3 × 105 cells per dish. The miR-142-5p mimic and inhibitor and their negative control (NC) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CDK5 siRNAs was purchased from Shanghai Biotechnology Company (Shanghai, China). The cells were transfected with miR-142-5p mimic or inhibitor at a final concentration of 25 nmol/L or CDK5 siRNA at 20 nmol/L using jetPRIME® Transfection Agent (Cat# PT-114-15; Polyplus-transfection SA, Illkirch, FRANCE) according to the manufacturer’s instruction. The sequence of CDK5 siRNA was as follows: UAUGACAGAAUCCCAGCCCTT, GGGCUGGGAUUCUGUCAUATT.23 (link) DCs were transfected twice to improve transfection efficiency. The second transfection was performed 24 hours after the end of the first transfection. Transfection efficiency of the DC transfection system was monitored by using siGLO Cyclophilin B Control siRNA purchased from Thermo Scientific (Waltham, MA, USA) as described.24 (link)
7
siRNA Transfection in CAL27 and FaDu Cells
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CAL27 and FaDu cells (1 × 10 5 /well) were seeded in 24-well plates and immediately transfected with 40 nM siRNA using jetPRIME transfection agent (Polyplus, France), according to manufacturer's recommendations. For each gene, two distinct siRNA duplexes targeting different regions within the transcript sequence were used (Qiagen, Germany). Nontargeting siRNA was used as control. RNA was isolated 48 h post-transfection. The experiments were repeated twice.
8
RHOB Knockout in NSCLC Cell Lines
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The human NSCLC cell lines A549, H1299, H827, and PC9 were previously obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and were maintained at 37°C in a humidified chamber containing 5% CO2.
For the RHOB KO A549 and PC9, the TALEN sequences targeting RHOB were designed by CELLECTIS (Paris, France), and inserted into two plasmids comprising CMV and T7 promoters. Triple transfection of the two TALEN-encoding plasmids with a Puromycin selection cassette upstream RHOB gene was performed using the JetPrime® transfection agent (Polyplus transfection) according to the manufacturer's recommendations. Puromycin selection was performed for 48 h after transfection and the pool of surviving clones was subcloned by limit dilution in 96-well plates. For each subclone, RHOB DNA levels and RHOB protein expression were analyzed by PCR and Western Blot.
For the RHOB KO A549 and PC9, the TALEN sequences targeting RHOB were designed by CELLECTIS (Paris, France), and inserted into two plasmids comprising CMV and T7 promoters. Triple transfection of the two TALEN-encoding plasmids with a Puromycin selection cassette upstream RHOB gene was performed using the JetPrime® transfection agent (Polyplus transfection) according to the manufacturer's recommendations. Puromycin selection was performed for 48 h after transfection and the pool of surviving clones was subcloned by limit dilution in 96-well plates. For each subclone, RHOB DNA levels and RHOB protein expression were analyzed by PCR and Western Blot.
9
Modulating B7H3 Expression in Cancer Cells
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B7H3 expression plasmids (Origene, Rockville, MD) were delivered into U138 cells (which have low expression of B7H3) with JetPrime transfection agent (Polyplus, Berkeley, CA) for 6 h, then successfully transfected cells were selected using 800 μg/mL geneticin (Gibco-BRL, Grand Island, NY) for 4 weeks to obtain stable clones.
B7H3 shRNAs (Sigma Aldrich, St Louis, MI) and B7H3 siRNAs (Thermo Scientific Dharmacon; cat#J-007813-11, J-007813-12) were transfected into A172 and GBM2 cell lines (which have high expression of B7H3). After transfection for 48 h, cells were selected using 5 μg/ mL puromycin (Life Technologies, CA) as suggested by the manufacturer's instructions.
B7H3 shRNAs (Sigma Aldrich, St Louis, MI) and B7H3 siRNAs (Thermo Scientific Dharmacon; cat#J-007813-11, J-007813-12) were transfected into A172 and GBM2 cell lines (which have high expression of B7H3). After transfection for 48 h, cells were selected using 5 μg/ mL puromycin (Life Technologies, CA) as suggested by the manufacturer's instructions.
10
Evaluating KIM-1 Expression in Renal Cells
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Human renal adenocarcinoma cells 769‐P (CRL‐1933) purchased from ATCC (Manassas, VA, USA) were grown in RPMI 1640 Medium supplemented with 10% FBS (Gibco/Thermo Fisher Scientific, Waltham, MA, USA). Human embryonic kidney 293 cells (HEK‐293) purchased from Sigma‐Aldrich (St. Louis, MO, USA) were grown in MEM Medium supplemented with 2 mmol/L glutamine, 1% nonessential amino acids and 10%FBS (Gibco/Thermo Fisher Scientific). Cells were transfected with human UMOD construct expressed in the pcDNA3.1 vector (gift from Dr. L. Rampoldi, Dulbecco Telethon Institute, Milan, Italy) (Bernascone et al. 2006 ) using jetPRIME transfection agent (Polyplus Transfection, Illkirch, France), according to the manufacturer's instructions. For in vitro KIM‐1 expression experiments, 769‐P and 293 cells transfected with pcDNA3.1 vector alone (Control, pcDNA3.1‐C) and full length UMOD in pcDNA3.1 vector (pcDNA3.1‐UMOD) were reseeded 4 h after transfection into cell culture inserts with 1.0 μm pores (BD Biosciences, Mississauga, ON, Canada). Twenty‐four hours after transfection, the inserts were transferred to six‐well plate containing 293‐C and 293‐UMOD transfected cells, and incubated for an additional 24 h. Cells and conditioned medium were collected and analyzed for mRNA and protein levels of KIM‐1.
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