The vectors of annexin C1-GFP and annexin C2-GFP were newly constructed. Each gene was amplified from cDNA library (a gift from Dr. D. Robinson) and inserted into the pA15GFP expression vector including a G418 resistance gene. Dd-GCaMP6s was used as a Ca2+ probe, in which the codon usage in the original GCaMP6s51 (link) was optimized for Dictyostelium. To generate the knockout construct of annexin C1, annexin C1 fragment was subcloned outside of two loxP sites in pLRBLP. The left arm (nucleotides 50–600) and right arm (nucleotides 619–1196) of annexin C1 gene were amplified from the cDNA by PCR using the following primer sets with restriction enzyme sites (underscore): 5′-ATGTCGAC CACAACAAGGTTATCCACCACAACAAGGCT-3′ (SalI) and 5′-ATATCGAT GTGAATTTGTTCAACATCGAAATGAGCTGG-3′ (ClaI) (for the left arm of the KO construct); and 5′-ATGGATCC GGTACCAACGAGAACACTATAATTGAAATTTTAG-3′ (BamHI) and 5′-ATACTAGT GCTAATGAATTCTTGAAGAGAGTTGAATAAGC-3′ (SpeI) (for the right arm of the KO construct). The amplified left arm fragments were subcloned between SalI and ClaI sites lying upstream of N-terminal loxP site in pLRBLP. The right arm fragments were subsequently subcloned between BamH1 and SpeI sites lying downstream of C-terminal loxP site. After transformation with the knockout construct, cells were selected in HL5 medium containing 10 µg/ml of blasticidin S hydrochloride (Wako).
Blasticidin s hydrochloride
Manufactured by Fujifilm
Sourced in Japan
Blasticidin S hydrochloride is a broad-spectrum antibiotic used as a selective agent for cell culture applications. It inhibits protein synthesis and is effective against a variety of eukaryotic and prokaryotic organisms.
Automatically generated - may contain errors
Lab products found in correlation
Virapower packaging mix, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dnase 1 dn25, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (1 mentions)
Gateway lr clonase 2 plus enzyme mix, by Thermo Fisher Scientific (1 mentions)
Pentr directional topo vector, by Thermo Fisher Scientific (1 mentions)
Pcdna6.2 v5 dest vector, by Thermo Fisher Scientific (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Gene pulser 2 electroporator, by Bio-Rad (1 mentions)
Ham s f 12 nutrient mixture, by Thermo Fisher Scientific (1 mentions)
Polyethylene glycol 1000, by Fujifilm (1 mentions)
Colcemid, by Merck Group (1 mentions)
Serum free dmem, by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Lr clonase 2 plus enzyme mix, by Thermo Fisher Scientific (1 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (1 mentions)
Etoposide, by Fujifilm (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Sch772984, by Thermo Fisher Scientific (1 mentions)
12 protocols using blasticidin s hydrochloride
1
Annexin C1/C2 Overexpression and Knockout in Dictyostelium
2
Antibody-based Protein Analysis Protocol
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The antibodies used were listed in Table S7 . The following reagents were used in the analyses: blasticidin S hydrochloride (029-18701, Wako Pure Chemical Industries, Ltd.), complete protease inhibitor cocktail (11697498001, Roche Applied Science, Basel, Switzerland), and DNase I (DN25, Sigma-Aldrich, St. Louis, MO).
3
Cloning and Expression of E3 Ligases
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Human E3 ligases were amplified from cDNA of Tm-treated HeLa cells using gene-specific primers (forward primers with the 5′-CACC overhang and reverse primers without stop codons; Supplementary Table S2 ) and cloned into the pENTR Directional TOPO vector (entry clone; Life Technologies, Carlsbad, CA). The cloned sequences were then transferred into the pcDNA6.2/V5-DEST vector, including the V5 epitope tag at the C-terminal of the insert sequences (Life Technologies), using Gateway LR Clonase II Enzyme mix (Life Technologies). The CS mutants were constructed by PCR using the overlapping method with the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA).
For stable cell lines, entry clone products and the pENTR5′/CMVp vector were recombined into the pLenti 6.4/R4R2/V5-DEST vector using Gateway LR Clonase II Plus enzyme mix (Life Technologies). To produce lentivirus carrying V5-tagged E3s, the pLenti-based expression vectors and the ViraPower Packaging mix (Life Technologies) were cotransfected into the 293FT cell line. The virus-containing supernatant was harvested and transduced into COS-1 and N2a cells. Cells stably expressing E3s were selected with 5 μg/ml blasticidin S hydrochloride (WAKO Pure Chemical Industries, Osaka, Japan).
For stable cell lines, entry clone products and the pENTR5′/CMVp vector were recombined into the pLenti 6.4/R4R2/V5-DEST vector using Gateway LR Clonase II Plus enzyme mix (Life Technologies). To produce lentivirus carrying V5-tagged E3s, the pLenti-based expression vectors and the ViraPower Packaging mix (Life Technologies) were cotransfected into the 293FT cell line. The virus-containing supernatant was harvested and transduced into COS-1 and N2a cells. Cells stably expressing E3s were selected with 5 μg/ml blasticidin S hydrochloride (WAKO Pure Chemical Industries, Osaka, Japan).
4
Lentiviral Transduction of ALDH1L1 in HuH-7 Cells
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Lentivirus and packaging plasmids CSII-CMV-MCS-IRES2-Bsd and pCAG-HIVgp and pCMV-VSV-G-RSV-Rev were provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan. Human ALDH1L1 cDNA was amplified by PCR (corresponding primers are shown in Supplementary Table 1 ) from Human BioBank cDNA (Primer Design, UK), cloned into pBluescriptII SK(+) plasmid, and then sub-cloned into CSII-CMV-MCS-IRES2-Bsd lentiviral vector. Lentivirus particles were produced by transfection of CSII-CMV-MCS-IRES2-Bsd or CSII-CMV-ALDH1L1-IRES2-Bsd, pCAG-HIVgp and pCMV-VSV-G-RSV-Rev into HEK293 cells, which were subsequently used to infect to HuH-7 cells. Transduced HuH-7 cells were selected with 5 mg/mL blasticidin S hydrochloride (Fujifilm Wako Pure Chemical).
5
Generating CENP-A-GFP expressing DT40 cells
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To generate DT40 cells expressing GFP-CENP-A or GFP-CENP-AQD, ggCENP-A_pEGFP-C2 or ggCENP-AQD_pEGFP-C2 plasmids containing a Blasticidin resistance gene were transfected into CENP-A conditional knockout cells [CENP-A (–/Flox), Mer-Cre-Mer]22 (link) with a Gene Pulser II electroporator (Bio-Rad). Cells transfected with the ggCENP-A_pEGFP-C2 plasmid were selected in medium containing 2 mg/ml G418 (Santa Cruz Biotechnology), and cells transfected with the ggCENP-AQD_pEGFP-C2 plasmid were selected in medium containing 25 μg/ml Blasticidin S hydrochloride (Wako). After 10 days of selection, the drug resistant and GFP-positive clones were isolated. To knockout the endogenous CENP-A gene in the isolated clones, 100 nM 4-hydroxytamoxifen (OHT, Sigma) was added to the culture medium to activate the Mer-fused Cre-recombinase (Mer-Cre-Mer), and then the 5′ portion of the CENP-A gene [from exon 1 to 3 (CENP-A 1–87)] flanked by the LoxP sequences was excised from the genome. After the OHT treatment, we further isolated the monoclonal lines by limited dilution in 96-well plates, and verified the depletion of endogenous CENP-A in the isolated clones by a southern blot analysis. CENP-A disruption was also confirmed by an immunoblot analysis.
6
Lentiviral Transduction of RLMVECs for eGFP Expression
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Viral particles were produced and used to infect RLMVECs. Lentiviral vectors were generated by lipofectamine-mediated virus infection of HEK 293T cells. HEK 293T cells (8 × 106) were cultured in 3 mL of Opti-MEM and 5 mL of DMEM/10% fetal bovine serum (FBS) in 10-cm petri dishes and infected the following day. HEK cells were infected with 3 μg of transfer vector plasmid carrying the eGFP gene under the CMV promoter, 9 μg of package mix, and 36 μL of Lipofectamine 2000 (Thermo Fisher Scientific). The medium was exchanged for 10 mL DMEM/10% FBS 8 h after infection. Supernatant (10 mL) containing infectious particles was collected after 48 h. Viral supernatants were concentrated for preservation and were diluted (1:10) with EGM-2MV medium prior to infection of RLMVECs for 24 h. Three days after the initial viral infection, 10 μg/mL blasticidin S hydrochloride (Wako Pure Chemical, Osaka, Japan) was administered for 3 days to enrich the successfully labeled cells. The term eGFP-ECs refers to eGFP-RLMVECs in this study. Preservation of vascular phenotype and function in lentiviral vector-transfected RLMVECs was verified by flow cytometric analysis in comparison with untransfected RLMVECs (Supplementary Fig. 5 ).
7
CHO Cell Culture and Maintenance
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CHO K1 cells were obtained from ATCC (Manassas, VA, USA), and CHO DG44 cells were obtained from Dr. Lawrence Chasin of Columbia University and Dr. Motonobu Katoh of our laboratory. These cells were maintained in Ham’s F-12 nutrient mixture (Invitrogen, Carlsbad, CA, USA) plus 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA). CHO K1 hybrids carrying the HAC (21HAC4) were cultured with 8 μg/ml blasticidin S hydrochloride (Wako, Tokyo, Japan) as previously described24 (link). In antibody purification analysis, CHO K1 and CHO DG44 cells were maintained in Ham’s F-12 nutrient mixture (Invitrogen) 10% plus IgG free fetal bovine serum (FBS) (GE Healthcare, Piscataway, NJ, USA).
8
Micronuclei Isolation and Chromosome Transfer
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MMCT was performed as previously described (Ohira et al. 2019a (link), b (link)). Briefly, donor cells were incubated with 0.05 μg/ml colcemid (Sigma) in F12 (Invitrogen) containing 20% FBS (JRH Biosciences) for 72 h. Micronuclei were harvested by treatment with 10 μg/ml cytochalasin B (Sigma) and centrifugation and sequentially filtered through 8, 5, and 3 μm polycarbonate filters (Whatman Nuclepore, Kent, UK). The fusion was mediated by 47% polyethylene glycol 1000 (Wako), followed by extensive washing with serum-free DMEM (Sigma). After incubation for 24 h in the culture medium, cells with chromosome transfer were selected with G418 (Sigma) or blasticidin S hydrochloride (Wako).
9
NIH-H358 Cell Transfection Protocol
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NIH-H358 cells were cultured to 25% confluence, the medium was then removed, and equivalent amounts of vector-containing medium and RPMI-1640 were added. Cells stably expressing each gene were obtained by selection with 10 μg/ml blasticidin S hydrochloride (Wako Pure Chemical Industries, Ltd) for 1week.
10
Heterologous Expression of Cytoskeletal Proteins
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Descriptions of the plasmid sources and null mutant cells used in the present study are listed in Supplementary Table S1 . Cells were transformed with expression vectors of GFP-tagged proteins were transformed into cells by electroporation or laserporation, as described previously [46 (link),47 (link)]. The transformed cells were selected in HL5 medium containing 10 µg/mL G418 (Wako Pure Chemical Corporation, Osaka, Japan) or 10 µg/mL blasticidin S hydrochloride (Wako Pure Chemical Corporation, Osaka, Japan) in plastic dishes.
We constructed expression vectors for some GFP-tagged proteins, including RacA, severin, filamin, ABD34, CAP32, fimbrin, α-actinin, and CARMIL. The individual genes were amplified from a cDNA library by PCR and subcloned between the BamH1 and Sac1 sites downstream of the C-terminal GFP site in the pA15GFP expression vector, as described previously [16 (link)].
We constructed expression vectors for some GFP-tagged proteins, including RacA, severin, filamin, ABD34, CAP32, fimbrin, α-actinin, and CARMIL. The individual genes were amplified from a cDNA library by PCR and subcloned between the BamH1 and Sac1 sites downstream of the C-terminal GFP site in the pA15GFP expression vector, as described previously [16 (link)].
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