The plasmids were transfected into cells using Lipofectamine LTX and Plus Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Control cells were mock transfected with the same amount of the pcDNA or pCMV empty vector. Experiments using the transiently transfected cells were performed 48 h after transfection. To establish stably transfected cells with these plasmids, the transfected cells were cultured in 400 μg/mL hygromycin B (Nacalai Tesque, Kyoto, Japan) medium for 14–20 days. After drug selection, we aligned HER2 expression to the same level using cell sorting on a FACSAria III flow cytometer (BD Biosciences, Franklin Lakes, NJ) using allophycocyanin-conjugated anti-human HER2 antibody (Biolegend, San Diego, CA). Seven days after cell sorting, the experiments using stably transfected cells were performed.
Hygromycin b
Manufactured by Nacalai Tesque
Sourced in Japan, United States
Hygromycin B is a broad-spectrum antibiotic derived from the bacterium Streptomyces hygroscopicus. It is commonly used as a selective agent in cell culture and genetic engineering applications to identify and maintain cells that have been successfully transfected or transformed with a gene of interest.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pgl4.50 luc2 cmv hygro vector, by Promega (3 mentions)
D luciferin, by Promega (3 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt reagent, by Merck Group (2 mentions)
L glutamine, by Nacalai Tesque (2 mentions)
Fetal bovine serum (fbs), by Equitech-Bio (2 mentions)
Antibiotic antimycotic cocktail solution, by Nacalai Tesque (2 mentions)
Geneticin g418, by Nacalai Tesque (2 mentions)
Lc ms grade acetonitrile, by Merck Group (2 mentions)
Geneticin disulfate, by Nacalai Tesque (2 mentions)
Hexane, by Kanto Chemical (2 mentions)
Toluene, by Kanto Chemical (2 mentions)
Facsaria 3, by BD (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Hygromycin, by Merck Group (1 mentions)
Hexadimethrine bromide, by Merck Group (1 mentions)
Agarose, by Nacalai Tesque (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
30 protocols using hygromycin b
1
Stable HER2 Expression in Cells
2
Construction of N-terminally FLAG-tagged GDE7
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cDNA of human or mouse GDE7 with a C-terminal FLAG tag was previously constructed16 (link). A cDNA sequence encoding a FLAG peptide was introduced by PCR using PrimeSTAR HS DNA Polymerase (TaKaRa Bio, Kusatsu, Japan) to construct cDNA for N-terminally FLAG-tagged GDE7. This was generated using a forward primer containing a XbaI site, Kozak sequence, FLAG, and 5×Gly linker, and a reverse primer with a BamHI site. Site-directed mutagenesis was performed by overlap extension PCR. Each DNA fragment was amplified using primer sets shown in Supplementary Table 1 and subcloned into the third generation lentiviral backbone vector containing a hygromycin resistant gene as previously described60 (link).
COS-7, MCF-7, and 3T3-L1 cells were transduced with each obtained lentivirus using 8 μg mL−1 hexadimethrine bromide (Sigma-Aldrich) and then selected with hygromycin B (Nacalai Tesque).
COS-7, MCF-7, and 3T3-L1 cells were transduced with each obtained lentivirus using 8 μg mL−1 hexadimethrine bromide (Sigma-Aldrich) and then selected with hygromycin B (Nacalai Tesque).
3
Cell Viability Assay with SN-38
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The following reagents and drugs were purchased from the commercial sources indicated in parentheses: antibiotic-antimycotic cocktail solution, l -glutamine, high-glucose Dulbecco’s modified Eagle medium (DMEM), hygromycin B (Nacalai Tesque, Inc., Kyoto, Japan); fetal bovine serum (FBS) (Equitech-Bio, Inc., Kerrville, TX, USA); and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT reagent) (Sigma-Aldrich Co., St. Louis, MO, USA). SN-38 was generously provided by Yakult Honsha Co., Ltd. (Tokyo, Japan). All other chemicals used were of analytical grade.
4
Genetic Manipulation of Fission Yeast
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The fission yeast strains used in this study are detailed in Supplementary Table S1 . Standard genetic procedures were used as described previously (35 (link)). Antibiotic marker genes were obtained from pFA6a-kanMX6 and pFA6a-hphMX6 (44 (link)). Yeast transformation was carried out using lithium acetate and transformants were selected on yeast extract (YE) medium supplemented with G418 (Nacalai Tesque) or hygromycin B (Nacalai Tesque) at a final concentration of 100 μg/ml. Correct integration was confirmed by polymerase chain reaction (PCR). Cells were grown on Yeast extract (YE) medium or Edinburgh minimal medium (EMM) supplemented with appropriate amino acids at a final concentration of 225 μg/ml. 5-fluoroorotic acid (5FOA; 1 mg/ml) (Apollo Science) and uracil (56 μg/ml) were added to Yeast Nitrogen Base (YNB) media containing 1.7 g/l YNB (Difco 233520, BD Biosciences), 5 g/l of ammonium sulphate and 2% glucose. Solid medium contained 1.5% agarose (Nacalai Tesque). To generate the mhf1-L78R mutant strain, the ura4+ gene was introduced at 303 bp upstream of the mhf1 coding region, after which the Ura+ cells were transformed with a 1.9 kb PCR fragment that contains the mhf1-L78R mutation and the ura4+ integration site and were selected on 5FOA plates. The integration of the mhf1-L78R mutation was confirmed by DNA sequencing. Cells were grown at 33°C, unless otherwise indicated.
5
Transfection of P2X2 in HEK293 Cells
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HEK293T cells (RIKEN Bio‐Resource Center, Tsukuba, Japan) were cultured in HEK medium consisting of Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Taufkirchen, Germany) containing 10% FBS (Biowest, Nuaillé, France), penicillin (100 U·mL−1), and streptomycin (100 µg·mL−1; P/S; Nacalai Tesque Inc., Kyoto, Japan). HEK293 cells stably expressing murine choline acetyltransferase (ChAT; ChAT‐HEK293 cells) [29 (link)] were cultured in ChAT‐HEK medium consisting of DMEM containing 10% FBS and hygromycin B (500 µg·mL−1; Nacalai Tesque, Inc., Kyoto, Japan). HEK293T or ChAT‐HEK293 cells were plated on 10‐cm dishes (2 × 106 cells per dish) maintained at 37 °C in a 5% CO2/air atmosphere. HEK293T or ChAT‐HEK293 cells were transfected with the pP2X2‐IRES2‐AcGFP1‐Nuc vector (10 μg plasmid DNA per dish) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and transfected cells were incubated for 2 days.
6
Isoleucine Biosynthesis Pathway
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Isoleucine, CoA, acetoacetyl-CoA, acetyl-CoA, malonyl-CoA, ATP and TeA were purchased from Sigma-Aldrich (St Louis, MO, USA). Kanamycin (Km), carbenicillin and hygromycin B were purchased from Nacalai (Kyoto, Japan). Blasticidin S was purchased from Funakoshi (Tokyo, Japan). All other reagents were of analytical grade.
7
CRISPR-Mediated Knockout of Autophagy Genes
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The CRISPR/Cas9 system (Mali et al., 2013 (link)) was used to knockout NLRX1, Beclin 1, or UVRAG as described previously (Oda et al., 2016 (link)). We selected the CRISPR guide RNA (gRNAs) that targeted an exon common to all splicing variants of the gene of interest (target sequence: NLRX1, 5′-GAGCTGTGCTAGCTCAGCT-3′, Beclin 1, 5′-TCCAACAACAGCACCATGC-3′, UVRAG, 5′-GAGATGAGCGCCTCCGCGT-3′, Rubicon, 5′-CAGTTCAGCTCACGTGATT-3′). For CRISPR/Cas9 gene editing, HeLa cells were transfected with a gRNA-hyg vector containing the CRISPR target sequence and hCAS9 vector (41815; Addgene). Two days after transfection, the untransfected cells were removed by selection with 300 μg/mL hygromycin B (Nacalai Tesque) and 750 μg/mL geneticin (G418; Nacalai Tesque). Single colonies were expanded in 24-well plates, and depletion of the target gene was confirmed by immunoblotting. As a secondary screen for KO lines, genomic DNA was isolated from cells and the genomic regions were amplified by PCR. These PCR products were sequenced to confirm the presence of the desired frameshift insertions and deletions.
8
Generating IFN-β Responsive Reporter Cell Line
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Briefly, 293T cells (ATCC, Cat# CRL-3216) were transfected with 1 µg pGL4.45[luc2P/ISRE/Hygro] Vector with TransIT-LT1 Transfection Reagent (TaKaRa, Cat# V2304T) in Opti-MEM (Thermo Fisher Scientific, Minoto-Ku, Japan, Cat# 31985062). After 3 d, the cells were cultured in 250 µg/mL of hygromycin B (Nacalai Tesque, Cat# 09287-84) for 10 d. Single-cell cloning was then performed. After cell growth, we evaluated the induction of luciferase activity upon treatment with recombinant human IFN-β (PeproTech, Cranbury, NJ, USA, Cat# 300-02BC) in each clone.
9
CRISPR-Mediated Knockout of STING, TBC1D9, and TBK1
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CRISPR/Cas9 was used to KO STING, TBC1D9, and TBK1. CRISPR guide (g)RNAs were designed to target an exon common to all splicing variants of the gene of interest (5′-GAGAGTGTGCTCTGGTGGC-3′ for STING, 5′-AACCCGGAGGAGGTGTTGC-3′ for TBC1D9, and 5′-GAGCACTTCTAATCATCTG-3′ for TBK1). HeLa cells were transfected with the vector hCAS9 (Addgene #41815) and a gRNA-hyg vector containing the CRISPR target sequence. Untransfected cells were removed by selection on plates containing 300 μg mL−1 hygromycin B (Nacalai Tesque) and 750 μg mL−1 geneticin (G418; Nacalai Tesque). Single colonies were expanded, and depletion of the target gene was confirmed by immunoblot. As a secondary screen for some KO lines, genomic DNA was isolated, and target regions were amplified by PCR and sequenced to confirm the presence of the desired frameshift insertions and deletions.
10
Genetic Engineering of Chlamydomonas Strains
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Chlamydomonas reinhardtii cells were grown in Tris-acetate-phosphate (TAP) medium. To screen transformants, cells were grown on TAP agar supplemented with hygromycin B (20 μg/ml; Nacalai Tesque, Kyoto, Japan). The fap44 (LMJ.SG0182.019151), fap43 (LMJ.SG0182.005221), and fap244 (LMJ.RY0402.124246) mutant strains were obtained from the CLiP Library (Li et al., 2016 (link)). The CliP strains were back-crossed with the wild-type strain at least five times before the experiments. Chlamydomonas reinhardtii strains used in this study are listed in Table 2 . For generation of anti-FAP44 and FAP43 antibodies, cDNA sequence encoding the amino acids 1-271 of FAP44 and 689-1312 of FAP43 were each inserted into the pGEX-6p-2 plasmid and polypeptides were expressed in Escherichia coli cells. Anti-FAP44 and FAP43 rabbit polyclonal antibodies were then raised against the purified proteins. Anti-DRC3 and anti-IC140 antibodies were generated in the previous studies (Oda et al., 2014 , 2015 (link)). Anti–human influenza hemagglutinin (HA) antibody 4B2 was purchased from Wako Pure Chemical Industries (Tokyo, Japan). Anti-IC138 antisera was a kind gift from W. S. Sale (Emory University, Atlanta, GA).
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