Mouse melanoma cell line B16-F10 (CRL-6475) was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). HEK293 cells stably expressing human LAT1 and human LAT2 (designated as HEK293-hLAT1 and HEK293-hLAT2, respectively) were established as described previously through transfection with plasmids encoding human LAT1 and human LAT226 (link). B16-F10 cells were cultured in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan)48 (link). The HEK293-hLAT1 and HEK293-hLAT2 cells were maintained in MEM medium (Nacalai Tesque, Kyoto, Japan) supplemented with 1% non-essential amino acids (Wako, Osaka, Japan) and G418 disulfate (0.9 g/L, Nacalai Tesque, Kyoto, Japan)26 (link). Both cell media were supplemented with 10% fetal bovine serum (FBS, Nichirei Biosciences Inc., Tokyo, Japan) and a combination antibiotic consisting of 100 units/mL penicillin and 100 μg/mL streptomycin (Nacalai Tesque, Kyoto, Japan). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
G418 disulfate
Manufactured by Nacalai Tesque
Sourced in Japan
G418 disulfate is a laboratory reagent used for the selection and maintenance of mammalian cell lines that have been transfected with a resistance gene. It is a broad-spectrum antibiotic that inhibits protein synthesis in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine ltx, by Thermo Fisher Scientific (3 mentions)
Nahco3, by Fujifilm (2 mentions)
L glutamine, by Fujifilm (2 mentions)
Amphotericin b, by Bristol-Myers Squibb (2 mentions)
Kanamycin, by Meiji Seika Pharma (2 mentions)
Dmem ham s f 12, by Fujifilm (2 mentions)
Polyethyleneimine, by Polysciences (2 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Mem medium, by Nacalai Tesque (1 mentions)
Non essential amino acids (neaa), by Fujifilm (1 mentions)
Rpmi 1640 medium, by Nacalai Tesque (1 mentions)
Hygromycin b, by Fujifilm (1 mentions)
Ac5 stable2 neo, by Addgene (1 mentions)
Hilymax, by Dojindo Laboratories (1 mentions)
Deltavision elite, by GE Healthcare (1 mentions)
Veroe6 tmprss2 cell, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
10 protocols using g418 disulfate
1
Establishment and Maintenance of B16-F10 and HEK293 Cell Lines
2
Fission Yeast Strain Maintenance
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All S. pombe strains used in this study are listed in Supplementary Data 1 . The minimum medium used was EMMG or EMMG5S (EMMG with five supplements); the rich medium used was YES55 (link). Cells were cultured in an appropriate medium at 30 °C for 3–6 days, depending on their growth. The strains including lem2Δ were maintained in EMMG medium as previously reported23 ,24 (link),56 (link). For selection, 100 μg/ml G418 disulfate (Nacalai Tesque, Kyoto, Japan), 200 μg/ml hygromycin B (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), 100 μg/ml nourseothricin sulfate (WERNER BioAgents, Jena, Germany), and 100 μg/ml blastcidin S (FUJIFILM Wako Pure Chemical Corp.) were added to the media, respectively.
3
SARS-CoV-2 Infection in VeroE6/TMPRSS2 Cells
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SARS-CoV-2 (JPN/TY/WK-521 strain) and transmembrane protease serine 2 (TMPRSS2)-expressing VeroE6 (VeroE6/TMPRSS2) cells [25 (link)] were obtained from the National Institute of Infectious Diseases (Tokyo, Japan). For passaging, VeroE6/TMPRSS2 cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum, 2 mM l-glutamine (FUJIFILM Wako Pure Chemical Co., Osaka, Japan), 0.15% NaHCO3 (FUJIFILM Wako Pure Chemical Co.), 2 µg/mL amphotericin B (Bristol-Myers Squibb Co., New York, NY, USA), 100 µg/mL kanamycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and 100 µg/mL G418 disulfate (Nacalai Tesque Inc., Kyoto, Japan). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2, cultured in viral growth medium (VGM) composed of DMEM supplemented with 1% fetal bovine serum, 20 mM l-glutamine, 0.15% NaHCO3, 2 µg/mL amphotericin B, and 100 µg/mL kanamycin for 3 d. Culture supernatants were stored at −80 °C as viral stock (viral titer: 7.25 log10 50% tissue culture infective dose (TCID50)/mL). SARS-CoV-2 was handled in a biosafety level 3 facility.
4
Tetracycline-Inducible Neurogenin2 Differentiation
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To establish a robust, quick differentiation method, we utilized direct conversion technology. Human neurogenin2 (NGN2) cDNA, under tetracycline-inducible promoter (tetO), was transfected into iPSCs by a piggyBac transposon system and Lipofectamine LTX (Thermo Fisher Scientific Inc., Waltham, MA). We used the vector containing tetO::NGN2 After antibiotic selection of G418 disulfate (Nacalai-Tesque, Kyoto, Japan), we picked out colonies and selected subclones that could efficiently differentiate into neurons by inducing the temporal expression of NGN2, with MAP2/DAPI 96% < purity21 (link) as iN-iPSCs.
5
Stable Expression of CAHS3 in Drosophila S2 Cells
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The expression vector Ac5-STABLE2-neo was obtained from Addgene (#32426) [62 (link)], and then the coding
sequence of FLAG-mCherry was replaced with the codon-optimized CAHS3 coding
sequence (Gene Universal) to express CAHS3-T2A-EGFP-T2A-neoR under the control
of Ac5 promoter. The empty vector was constructed by deleting FLAG-mCherry from
Ac5-STABLE2-neo, which was designed to express T2A-EGFP-T2A-neoR driven by the
same Ac5 promoter. Drosophila S2 cells were transfected using a
cationic liposome reagent Hilymax (Dojindo) with the expression construct or the
empty vector above. We established stably transfected cells by culturing for 6
weeks under the drug selection with G418 disulfate (2,000 μg/mL, Nacalai
Tesque).
sequence of FLAG-mCherry was replaced with the codon-optimized CAHS3 coding
sequence (Gene Universal) to express CAHS3-T2A-EGFP-T2A-neoR under the control
of Ac5 promoter. The empty vector was constructed by deleting FLAG-mCherry from
Ac5-STABLE2-neo, which was designed to express T2A-EGFP-T2A-neoR driven by the
same Ac5 promoter. Drosophila S2 cells were transfected using a
cationic liposome reagent Hilymax (Dojindo) with the expression construct or the
empty vector above. We established stably transfected cells by culturing for 6
weeks under the drug selection with G418 disulfate (2,000 μg/mL, Nacalai
Tesque).
6
Germination of Spores with G418
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Spores were prepared essentially as described in (28 (link)) with minor modifications. Spores were germinated in YES media containing 10 μg/ml G418 disulfate (Nacalai Tesque). We have confirmed that yeast cells lacking the kanMX6 gene do not proliferate in the presence of 10 μg/ml G418 (data not shown). Cell lengths were measured on images captured with DeltaVision Elite (GE Healthcare).
7
Comparative analysis of SARS-CoV-2 strain growth
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Multiple SARS-CoV-2 strains: 2019-nCoV/Japan/TY/WK-521/2020 (ancestral strain, Pango lineage: A, GISAID ID: EPI_ISL_408667), hCoV-19/Japan/QHN001/2020 (Alpha strain, B.1.1.7, EPI_ISL_804007), hCoV-19/Japan/TY8-612-P1/2021 (Beta strain, B.1.351, EPI_ISL_1123289), hCoV-19/Japan/TY7-501/2021 (Gamma strain, P.1, EPI_ISL_877769), hCoV-19/Japan/TY11-927-P1/2021 (Delta strain, AY.122, EPI_ISL_2158617), and hCoV-19/Japan/TY38-873P0/2021 (Omicron strain, BA.1.18, EPI_ISL_7418017) were obtained from the National Institute of Infectious Diseases (Tokyo, Japan). Vero E6/TMPRSS2 cells were obtained from the Japanese Collection of Research Bioresources (No. JCRB1819, Osaka, Japan) and passaged in a Dulbecco’s modified Eagle’s minimal essential medium (DMEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum, 2 mM L-glutamine (FUJIFILM Wako Pure Chemical Co., Osaka, Japan), 0.15% NaHCO3 (FUJIFILM Wako Pure Chemical Co.), 2 µg/mL amphotericin B (Bristol-Myers Squibb Co., New York, NY, USA), 100 µg/mL kanamycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and 100 µg/mL G418 disulfate (Nacalai Tesque Inc., Kyoto, Japan). After inoculation with SARS-CoV-2, the cells were cultured for three days in a viral growth medium composed of DMEM supplemented with 1% fetal bovine serum, 2 mM L-glutamine, 0.15% NaHCO3, 2 µg/mL amphotericin B, and 100 µg/mL kanamycin.
8
Establishment of Doxycycline-Inducible MTCL2 Cell Lines
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HeLa-Kyoto (HeLa-K), HEK293T, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, low glucose; Nissui, Japan) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM glutamine at 37°C in 5% CO2. The hTERT-immortalized human retinal pigment epithelial cells (RPE1 cells) were maintained in a 1:1 mixture of DMEM/Ham's F12 (FUJIFILM Wako Pure Chemical Corporation, Japan) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL hygromycin B, and 1 mM glutamine at 37°C in 5% CO2. For immunofluorescence analysis, cells were seeded on coverslips in 24-well plates and coated with atelocollagen (0.5 mg/mL IPC-50; KOKEN, Japan).
Plasmid transfections were performed using polyethyleneimine (Polysciences, Inc.) for HEK293T cells or Lipofectamine LTX (Life Technologies Corporation) for HeLa-K cells according to the manufacturer's instructions. To establish heterogenous stable HeLa-K cells expressing mMTCL2 or its mutants in a doxycycline-dependent manner, cells were transfected with pOSTet15.1 expression vector encoding the appropriate MTCL2 cDNA. The following day, cells were reseeded at one-twentieth of the cell density and subjected to selection using a medium containing 800 μg/mL G418 disulfate (Nacalai Tesque, Japan) for more than six days. Surviving cells were used in subsequent
Plasmid transfections were performed using polyethyleneimine (Polysciences, Inc.) for HEK293T cells or Lipofectamine LTX (Life Technologies Corporation) for HeLa-K cells according to the manufacturer's instructions. To establish heterogenous stable HeLa-K cells expressing mMTCL2 or its mutants in a doxycycline-dependent manner, cells were transfected with pOSTet15.1 expression vector encoding the appropriate MTCL2 cDNA. The following day, cells were reseeded at one-twentieth of the cell density and subjected to selection using a medium containing 800 μg/mL G418 disulfate (Nacalai Tesque, Japan) for more than six days. Surviving cells were used in subsequent
9
Overexpressing FABP5 in HCC Cells
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We used the expression vector pcDNA3.1 (+) (Invitrogen) with the whole exon sequence of FABP5 containing the Kozak sequence, GCCACC, to make cells with high FABP5 expression. The actual cloned sequence is presented in Supplementary Table 1. We transfected this expression vector into HCC cell lines using Lipofectamine 3000 (Invitrogen) and established stable clones under the selection pressure of G-418 Disulfate (Nacalai) for 14 days. Finally, we checked the FABP5 expression status of the stable clones using western blotting.
10
Establishment of Doxycycline-Inducible MTCL2 Cell Lines
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HeLa-Kyoto (HeLa-K), HEK293T, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, low glucose; Nissui, Japan) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM glutamine at 37°C in 5% CO2. The hTERT-immortalized human retinal pigment epithelial cells (RPE1 cells) were maintained in a 1:1 mixture of DMEM/Ham's F12 (FUJIFILM Wako Pure Chemical Corporation, Japan) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL hygromycin B, and 1 mM glutamine at 37°C in 5% CO2. For immunofluorescence analysis, cells were seeded on coverslips in 24-well plates and coated with atelocollagen (0.5 mg/mL IPC-50; KOKEN, Japan).
Plasmid transfections were performed using polyethyleneimine (Polysciences, Inc.) for HEK293T cells or Lipofectamine LTX (Life Technologies Corporation) for HeLa-K cells according to the manufacturer's instructions. To establish heterogenous stable HeLa-K cells expressing mMTCL2 or its mutants in a doxycycline-dependent manner, cells were transfected with pOSTet15.1 expression vector encoding the appropriate MTCL2 cDNA. The following day, cells were reseeded at one-twentieth of the cell density and subjected to selection using a medium containing 800 μg/mL G418 disulfate (Nacalai Tesque, Japan) for more than six days. Surviving cells were used in subsequent
Plasmid transfections were performed using polyethyleneimine (Polysciences, Inc.) for HEK293T cells or Lipofectamine LTX (Life Technologies Corporation) for HeLa-K cells according to the manufacturer's instructions. To establish heterogenous stable HeLa-K cells expressing mMTCL2 or its mutants in a doxycycline-dependent manner, cells were transfected with pOSTet15.1 expression vector encoding the appropriate MTCL2 cDNA. The following day, cells were reseeded at one-twentieth of the cell density and subjected to selection using a medium containing 800 μg/mL G418 disulfate (Nacalai Tesque, Japan) for more than six days. Surviving cells were used in subsequent
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