Hygromicin b

Manufactured by Thermo Fisher Scientific

Sourced in Belgium, United States

Hygromicin B is a broad-spectrum antibiotic produced by the bacterium Streptomyces hygroscopicus. It functions as a protein synthesis inhibitor, preventing the formation of the 70S ribosomal initiation complex.

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Hygromicin (3 citations)

8 protocols using hygromicin b

Optimization of AC Inhibitor Assays

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For FD, FD/AC, HEK293, HeLa T-REX, and ASAH2^(−/−) mouse embryonic fibroblasts (MEFs) and melanoma cell lines, Dulbecco's modified Eagle's medium, FBS, penicillin/streptomycin solution, nonessential amino acids, tetracycline, and puromycin were from Sigma. Zeocin was from Genaxxon Bioscience, lasticidin was from CalBiochem, and hygromicin B was from Invitrogen. For AML cell lines, RPMI-1640 medium and FBS were obtained from Corning (ref. 10-040) and VWR (ref. 97068-085), respectively. Recombinant human NC (rhNC) was obtained from R&D Systems. URB597 was from Sigma-Aldrich (ref. 341249), and ARN726 was from Tocris (ref. 5861). Primary antibodies used were AC (BD Biosciences, ref. 612302; Research Resource Identifier [RRID]: AB_399617) and β-actin (Cell Signaling Technology, ref. 3700; RRID: AB_2242334). Secondary antibody used was HRP-linked horse anti-mouse (Cell Signaling Technology, ref. 7076; RRID: AB_330924). All antibodies were prepared as per the recommended manufacturer’s protocol. SACLAC and SOCLAC were synthesized in our laboratories. Since both are irreversible AC inhibitors with similar activities (supplemental Fig. S1), they were used interchangeably in this work.

Casasampere M., Ung J., Iñáñez A., Dufau C., Tsuboi K., Casas J., Tan S.F., Feith D.J., Andrieu-Abadie N., Segui B., Loughran TP J.r., Abad J.L, & Fabrias G. (2024). A fluorogenic substrate for the detection of lipid amidases in intact cells. Journal of Lipid Research, 65(3), 100520.

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Cell Culture Conditions for HIV Research

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MT-4, U373-CD4-CXCR4, U373-CD4-CCR5, HeLa and TZM-Bl cell lines were obtained through the NIH AIDS Reagent Program. HEK 293, Vero, MDCK, and CRFK cells were purchased from ATCC (Manassas, VA, USA). HeLa-P4-CXCR4-LTRLacZ and Hela-ENV-Lai cells [26 (link)] were kindly given by Dr. Marc Alizon, Institute Pasteur, Paris. MT-4 cells were cultured in RPMI 1640 (Lonza, Wijchen, The Netherlands) containing 10% heat-inactivated fetal bovine serum (FBS) (Lonza, Netherlands) and 2mM L-glutamine (Invitrogen, Gosselies, Belgium). U373-CD4-CXCR4 and U373-CD4-CCR5 were cultured in RPMI 1640 containing 10% FBS, 2mM L-glutamine, 200 μg/mL geneticin (Invitrogen, Belgium), and 100 μg/mL hygromicin B (Invitrogen). HEK 293, Vero, Hela, and TZM-Bl were cultured in dulbecco’s modified eagle medium (DMEM) (Lonza) containing 10% FBS and 2mM L-glutamine. MDCK and CRFK cells were in eagle’s minimum essential medium (Lonza) containing 10% FBS. HeLa-P4-CXCR4-LTRLacZ cells were cultured in DMEM containing 10% FBS, 2mM L-glutamine, and 500 μg/mL geneticin. Hela-ENV-Lai cells were cultured in DMEM containing 10% FBS, 2mM L-glutamine, and 2 μM methotrexate (Sigma-Aldrich, Liège, Belgium).

Zheng Y., Yang X.W., Schols D., Mori M., Botta B., Chevigné A., Mulinge M., Steinmetz A., Schmit J.C, & Seguin-Devaux C. (2021). Active Components from Cassia abbreviata Prevent HIV-1 Entry by Distinct Mechanisms of Action. International Journal of Molecular Sciences, 22(9), 5052.

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Lentiviral Overexpression of H3.3 and Knockdown of PRC2

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H3.3 wild-type and H3.3K27M cDNA full length fused to a short sequence codifying 6 histidine residues were cloned into the pINDUCER lentiviral vector (Addgene plasmid # 44012, ^{39 (link)}) using gateway technology (Thermo Fisher). Lentiviral particle assembly and infection procedure were performed as previously described. HCT116 cells were transduced with H3.3 wild-type and H3.3K27M expressing lentiviral vectors. Neomycin [1mg/mL] was added to select infected cells for 5 days. Transgene expression was induced for 4 days by adding doxycyline [200ng/mL] every two days. For the experiment in Fig. S5 doxycycline was initially added for 7 days and then withdrawn for 21 days. SF8628 and SU-DIPG-IV were transduced with SUZ12 shRNA, EED shRNA and control vectors as previously described. Briefly, 1e6 cells were plated the day before the infection. The following day 0.45μL filtered medium enriched with viral particles was applied to the cells and 10μg/ml polybrene (Sigma) was added. After 6–8 hrs, cells were washed in PBS 1X and fresh medium was applied overnight. The entire procedure was repeated the following day. After that the cells were selected with 150μg/mL hygromicin B (Invitrogen) for 3–4 days (SUZ12 shRNA and control) or with Puromycin 2μg/mL (EED shRNA and control).

Piunti A., Hashizume R., Morgan M.A., Bartom E.T., Horbinski C.M., Marshall S.A., Rendleman E.J., Ma Q., Takahashi Y.H., Woodfin A.R., Misharin A.V., Abshiru N.A., Lulla R.R., Saratsis A.M., Kelleher N.L., James C.D, & Shilatifard A. (2017). Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas. Nature medicine, 23(4), 493-500.

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Isolation and Culture of Mammary Cells

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Cells were isolated from collected mammary glands, and mammosphere cultures were established as described previously [6 (link)] (see also Supplemental Materials). Comma-Dβ cells were grown in DMEM:F12 medium (Gibco BRL) supplemented with 2% FBS, 2 mM L-glutamine (Gibco BRL), 10 μg ml^–1 bovine insulin (Sigma), and 5 ng ml^–1 murine EGF (Sigma) [25 (link)]. The SUM159pt cell line (Asterand, Detroit, MI) was cultured in Ham’sF12 medium supplemented with 5% FBS, 2 mM L-glutamine, 5 µg ml^–1 insulin, 1 µg ml^–1 hydrocortisone, 10 mM HEPES, and incubated at 37 °C with 10% CO₂ in a humidified cell-culture incubator. Doxycycline (Sigma-Aldrich) was dissolved in water (10 mg ml^–1 stock solution) and used at final concentration of 2 µg ml^–1. Hygromicin B (Invitrogen; 50 mg ml^–1 stock solution) was used at final concentration of 200 µg ml^–1. 4-hydroxytamoxifen (4-OHT; Sigma-Aldrich) was used at final concentrations of 200 or 500 nM.

Bonetti P., Climent M., Panebianco F., Tordonato C., Santoro A., Marzi M.J., Pelicci P.G., Ventura A, & Nicassio F. (2018). Dual role for miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer. Oncogene, 38(3), 360-374.

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Agrobacterium-mediated Transformation of Marchantia

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Agrobacterium-mediated transformation using regenerating thalli of M. polymorpha was done by co-cultivation 15-day old plant fragments with transformed Agrobacterium tumefaciens (GV3101) cells [94] , [95] . After three days of incubation, positive transformants were selected on Gamborg's B5 plates supplemented with 10 μg/ml Hygromicin B (Invitrogen), 0.5 μM Chlorosulfuron or Kanamycin, and 100 μg/ml of Cefotaxime sodium. Isogenic lines were obtained by using plants derived from gemmae of the T1 generation (G1 generation). G1 or subsequent gemmae generations were used for all experiments, as they are derived from single cells [96] , [97] . Transformation of A. thaliana via A. tumefaciens (GV3101) was performed by floral dipping according to [98] .
Primers used for amplification of promoter fragments of MpFER are listed in Table S2.

Mecchia M.A., Rövekamp M., Giraldo-Fonseca A., Meier D., Gadient P., Vogler H., Limacher D., Bowman J.L, & Grossniklaus U. (2022). The single Marchantia polymorpha FERONIA homolog reveals an ancestral role in regulating cellular expansion and integrity. Development (Cambridge, England), 149(19).

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Plasmid Creation for Stable Cell Lines

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Reagents for creating plasmids for stable cell lines and the T-Rex™-293 cell line were purchased from Invitrogen (T-Rex™-293 System; Carlsbad, CA). Tetracycline-free fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA). Doxycycline hyclate and resveratrol compounds were purchased from Sigma (St. Louis, MO). Hygromicin B and blasticidin S HCl were obtained from Invitrogen (Carlsbad, CA). Hamaelis crude extracts were obtained from Dr. Frank Döring, University of Kiel [15 ].

Teixeira D.C., Cordonier E.L., Wijeratne S.S., Huebbe P., Jamin A., Jarecke S., Wiebe M, & Zempleni J. (2018). A cell death assay for assessing the mitochondrial targeting of proteins. The Journal of nutritional biochemistry, 56, 48-54.

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Selecting Antibodies for Osteopontin Isoforms

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OPN full-length was used as a target for selecting antibodies from a phage display Ab library as previously described (28 (link)). The selection was performed in immuno tubes coated with recombinant protein incubated overnight at 4°C in PBS. The panning procedure was repeated twice. In total, 96 random clones were selected, and a specific positive clone to OPN-C was identified using phage ELISA. The positive scFv clone was converted into the human scFv–Fc format by subcloning into the pMB-SV5 (29 (link)) vector containing the human hinge–CH2–CH3 domain. For antibody production, CHOs cell lines were transfected and stable clones obtained through selection with hygromicin B (500 μg/ml, Invitrogen). The scFv–Fc molecules were purified from cell culture supernatant using a HiTrap protein G column (GE Healthcare). After elution, the preparations of purified scFv–Fc were dialyzed in BupHTM phosphate buffer (Thermo Fisher Scientific, Waltham, MA, USA), aliquoted, and stored at −80°C. The selected mAb was tested by ELISA for its capacity to bind both human and mouse OPN-C and OPN-FL. The mAb was also tested during EAE in a passive immunization protocol.

Clemente N., Comi C., Raineri D., Cappellano G., Vecchio D., Orilieri E., Gigliotti C.L., Boggio E., Dianzani C., Sorosina M., Martinelli-Boneschi F., Caldano M., Bertolotto A., Ambrogio L., Sblattero D., Cena T., Leone M., Dianzani U, & Chiocchetti A. (2017). Role of Anti-Osteopontin Antibodies in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology, 8, 321.

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Recombinant IgG Antibody Production

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The reagents: dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), phenyl vinyl sulfone, RNAse I, propidium iodide, Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), laemmli buffer, glycine, iodoacetic acid (IAA); were supplied by Sigma-Aldrich (St. Louis, Missouri, USA). OptiCHO TM free serum media, L-glutamine were supplied by Thermo Fisher Scientific (Ulm, Germany). Hygromicin B was supplied by Invitrogen (Carlsbad, California, USA). Tris was supplied by SLS (Nottingham, UK).
The cell line used was a stable recombinant DG44 CHO cell line that expressed an IgG monoclonal antibody, provided by Fujifilm Diosynth Biotechnologies (Billingham, UK). The cell line was grown in suspension in an OptiCHO TM free serum media supplemented with 8mM L-glutamine and 100 mg/L Hygromicin B. An initial culture was maintained in a shaking incubator with 5% CO2 modified atmosphere at 37 o C. Cells were then seeded into new media at 0.2x10 6 viable cells/ml every 3-4 days.

Toronjo-Urquiza L., Acosta-Martin A.E., James D.C., Nagy T, & Falconer R.J. (2020). Resveratrol addition to Chinese hamster ovary cell culture media: The effect on cell growth, monoclonal antibody synthesis, and its chemical modification. Biotechnology progress, 36(3).

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