hygromycin b  (Millipore)

 
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    Name:
    Hygromycin B
    Description:
    Formula C20H37N3O13 LD50 6 mg kg in mouse i v 13 mg kg in guinea pig i p 63 mg kg in rat i p Molecular weight Mr 527 5 Working concentration 50 1 000 mug ml in cell culture A commonly used concentration for the selection of mammalian cells is 200 mug ml However the optimal concentration must be experimentally determined since it may vary depending on the type of cell used
    Catalog Number:
    10843555001
    Price:
    None
    Applications:
    Hygromycin B is an aminoglycosidic antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes. It is used to select and maintain the phenotype of eukaryotic cells that are stably transfected with the E. coli hygromycin-resistance gene (hyg or hph).
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    Structured Review

    Millipore hygromycin b
    Hygromycin B
    Formula C20H37N3O13 LD50 6 mg kg in mouse i v 13 mg kg in guinea pig i p 63 mg kg in rat i p Molecular weight Mr 527 5 Working concentration 50 1 000 mug ml in cell culture A commonly used concentration for the selection of mammalian cells is 200 mug ml However the optimal concentration must be experimentally determined since it may vary depending on the type of cell used
    https://www.bioz.com/result/hygromycin b/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hygromycin b - by Bioz Stars, 2021-04
    86/100 stars

    Images

    1) Product Images from "Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3?-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation"

    Article Title: Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3?-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq349

    SIDER2 anti - sense RNA complementary to the cleavage region blocks degradation of unstable SIDER2-containing reporter transcripts. ( A ) Schematic representation of the chimeric LUC reporter constructs bearing either a sense (s) or anti-sense (as) SIDER2 retroposon from transcripts 1270 or 3810 . Pairs of sense/anti-sense vectors LUC-3′-UTR1270/SIDER1270as LUC-3′-UTR3810/SIDER3810as, LUC-3′-UTR3810/SIDER3810Δ79IIas and 3′-UTR1270/SIDER3810as were co-transfected into L. major. The plasmids expressing the full-length 3′-UTRs of 3810 and 1270 cloned downstream of the LUC gene harbor a neomycin phosphotransferase ( NEO ) gene as a selection marker. Anti-sense SIDER2 sequences were inserted downstream of a hygromycin B ( HYG ) selection marker. Arrows indicate the orientation of SIDER2 elements. Y corresponds to a 92-bp polypyrimidine stretch ( 34 ) and α-IR refers to the IR of α-tubulin, both necessary for NEO , HYG and SIDER2as RNA processing. ( B ) Northern blot analysis to compare LUC mRNA expression levels in single (−) and double (+) transfectants using a LUC-specific probe. Expression and correct processing of both fully complementary anti-sense SIDER2 transcripts (1270as and 3810as) was verified with riboprobes specific to each SIDER2 sequence. Their sizes of ∼1.2 kb indicate that the transcripts are spliced within the α-IR and do not contain HYG sequences. The LUC signal intensities were normalized with α-tubulin mRNA levels and calculated with respect to the LUC-plasmid copy numbers ( Supplementary Table S2 ). ( C ) Western blot analysis using L. major protein lysates and a LUC-specific antibody. Protein loading was controlled with an anti-α-tubulin antibody. ( D ) Deadenylation profile and decay kinetics in single (LUC-3′-UTR3810) and double L. major transfectants (LUC-3′-UTR3810/SIDER3810as) as determined by northern blotting (identical samples). The numbers below the blots represent the relative LUC mRNA levels with respect to time point 0 (before addition of ActD). Histone 4A was used as a loading control. The mRNA half-lives of the uncut LUC transcripts are shown below the blots. ( E ) Northern blot hybridization to evaluate LUC- 3′-UTR3810 mRNA accumulation in the absence or presence of a truncated SIDER3810 anti-sense RNA-lacking signature II (SIDER3810Δ79IIas) (left panel). Northern blot analysis to compare LUC -3′-UTR1270 mRNA abundance in the presence or absence of a heterologous SIDER3810 anti-sense RNA (right panel). SIDER3810 and SIDER1270 retroposons share a 76.5% sequence identity at the level of signature II and > 80% identity within the cleavage site ( Figure 1 A). LUC signal intensities were normalized as indicated in (B).
    Figure Legend Snippet: SIDER2 anti - sense RNA complementary to the cleavage region blocks degradation of unstable SIDER2-containing reporter transcripts. ( A ) Schematic representation of the chimeric LUC reporter constructs bearing either a sense (s) or anti-sense (as) SIDER2 retroposon from transcripts 1270 or 3810 . Pairs of sense/anti-sense vectors LUC-3′-UTR1270/SIDER1270as LUC-3′-UTR3810/SIDER3810as, LUC-3′-UTR3810/SIDER3810Δ79IIas and 3′-UTR1270/SIDER3810as were co-transfected into L. major. The plasmids expressing the full-length 3′-UTRs of 3810 and 1270 cloned downstream of the LUC gene harbor a neomycin phosphotransferase ( NEO ) gene as a selection marker. Anti-sense SIDER2 sequences were inserted downstream of a hygromycin B ( HYG ) selection marker. Arrows indicate the orientation of SIDER2 elements. Y corresponds to a 92-bp polypyrimidine stretch ( 34 ) and α-IR refers to the IR of α-tubulin, both necessary for NEO , HYG and SIDER2as RNA processing. ( B ) Northern blot analysis to compare LUC mRNA expression levels in single (−) and double (+) transfectants using a LUC-specific probe. Expression and correct processing of both fully complementary anti-sense SIDER2 transcripts (1270as and 3810as) was verified with riboprobes specific to each SIDER2 sequence. Their sizes of ∼1.2 kb indicate that the transcripts are spliced within the α-IR and do not contain HYG sequences. The LUC signal intensities were normalized with α-tubulin mRNA levels and calculated with respect to the LUC-plasmid copy numbers ( Supplementary Table S2 ). ( C ) Western blot analysis using L. major protein lysates and a LUC-specific antibody. Protein loading was controlled with an anti-α-tubulin antibody. ( D ) Deadenylation profile and decay kinetics in single (LUC-3′-UTR3810) and double L. major transfectants (LUC-3′-UTR3810/SIDER3810as) as determined by northern blotting (identical samples). The numbers below the blots represent the relative LUC mRNA levels with respect to time point 0 (before addition of ActD). Histone 4A was used as a loading control. The mRNA half-lives of the uncut LUC transcripts are shown below the blots. ( E ) Northern blot hybridization to evaluate LUC- 3′-UTR3810 mRNA accumulation in the absence or presence of a truncated SIDER3810 anti-sense RNA-lacking signature II (SIDER3810Δ79IIas) (left panel). Northern blot analysis to compare LUC -3′-UTR1270 mRNA abundance in the presence or absence of a heterologous SIDER3810 anti-sense RNA (right panel). SIDER3810 and SIDER1270 retroposons share a 76.5% sequence identity at the level of signature II and > 80% identity within the cleavage site ( Figure 1 A). LUC signal intensities were normalized as indicated in (B).

    Techniques Used: Construct, Transfection, Expressing, Clone Assay, Selection, Marker, Northern Blot, Sequencing, Plasmid Preparation, Western Blot, Hybridization

    2) Product Images from "RRE-deleting self-inactivating and self-activating HIV-1 vectors for improved safety"

    Article Title: RRE-deleting self-inactivating and self-activating HIV-1 vectors for improved safety

    Journal: PeerJ

    doi: 10.7717/peerj.84

    Reconstitution of functional Hyg gene during transduction of HeLa cells with RRE-deleting HIV-1 vectors. Vector stocks were produced by transient transfection of 293T cells with a packaging plasmid (pgp3virin), a VSV-G envelope expression construct and the indicated vector, as described in Materials and Methods. Transfections were done in duplicate. Each individual vector stock was used for infection of HeLa cells in 6-well plates using the indicated amounts (200 µl, 20 µl or 2 µl). The cells were then subjected to selection using hygromycin B as described in in the text for 12–14 days. The resultant colonies were fixed and stained with crystal violet in 50% methanol and enumerated. The results of two independent experiments (Expt 1 and Expt 2) are shown. Each experiment consisted of two parallel transfections (biological replicates) done on the same day. The calculated titers in colony forming units (CFU)/ml as well as the p24 levels in ng/ml are shown to the right of each titration.
    Figure Legend Snippet: Reconstitution of functional Hyg gene during transduction of HeLa cells with RRE-deleting HIV-1 vectors. Vector stocks were produced by transient transfection of 293T cells with a packaging plasmid (pgp3virin), a VSV-G envelope expression construct and the indicated vector, as described in Materials and Methods. Transfections were done in duplicate. Each individual vector stock was used for infection of HeLa cells in 6-well plates using the indicated amounts (200 µl, 20 µl or 2 µl). The cells were then subjected to selection using hygromycin B as described in in the text for 12–14 days. The resultant colonies were fixed and stained with crystal violet in 50% methanol and enumerated. The results of two independent experiments (Expt 1 and Expt 2) are shown. Each experiment consisted of two parallel transfections (biological replicates) done on the same day. The calculated titers in colony forming units (CFU)/ml as well as the p24 levels in ng/ml are shown to the right of each titration.

    Techniques Used: Functional Assay, Transduction, Plasmid Preparation, Produced, Transfection, Expressing, Construct, Infection, Selection, Staining, Titration

    Related Articles

    Selection:

    Article Title: RRE-deleting self-inactivating and self-activating HIV-1 vectors for improved safety
    Article Snippet: To this end, we compared efficiency of gene delivery by the wild-type vector containing an RRE with a vector lacking RRE (ΔRRE) ( ). .. The wild-type and ΔRRE vectors were packaged in 293 T cells, as previously described, and the resultant titers (not normalized to p24 levels) were determined on HeLa cells following selection with hygromycin B. .. The wild-type vector titer (1.3 ± 0.1 × 104 cfu/ml) was approximately 61-fold higher than that of the ΔRRE vector (2.2 ± 0.1 × 102 cfu/ml) that exhibited colonies only at the highest volumes tested.

    Modification:

    Article Title: Sequences in pol Are Required for Transfer of Human Foamy Virus-Based Vectors
    Article Snippet: Stably transfected vector cell lines were generated with Lipofectin (GIBCO BRL) from BHK/Bel-1 cells, which constitutively express the viral transactivator Bel-1 ( ). .. They were selected in Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum, 0.5 mg of G-418 (GIBCO BRL) per ml, and 400 μg of hygromycin (Sigma) per ml. .. The selection medium was changed daily until colonies of hygromycin-resistant cells were observed.

    Infection:

    Article Title: Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition
    Article Snippet: Five days post-transfection the cells were assayed using Dual-glo luciferase reagents (Promega) following the manufacturer's protocol and quantitated using an Analyst GT plate reader (Molecular Devices). .. Groups of 25 flies were placed on standard media plus serial dilutions of Hygromycin B (17.5–280 μM; Calbiochem) 1 d prior to infection. .. HeLa cells were cultured in antibiotic-free DMEM medium, supplemented with 10% FBS and L-glutamine.

    other:

    Article Title: Overexpression of YPT6 restores invasive filamentous growth and secretory vesicle clustering in a Candida albicans arl1 mutant
    Article Snippet: Congo red and Hygromycin B were from Fluka, Sigma-Aldrich, Saint Quentin Fallavier, France.

    Article Title: Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition
    Article Snippet: The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit.

    Sampling:

    Article Title: Asymmetric positive feedback loops reliably control biological responses
    Article Snippet: .. For sampling, 10 technical and two biological replicates of equal volumes of dilutions were plated onto YPD, which is nonselective and allows both strains to grow equally, and YPD with 400 ug/ml Hygromycin B (HPH; Sigma) medium, to select for the engineered strain. ..

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    Millipore hygromycin b
    Inhibition of ribosomal function protects from infection in vivo and from poliovirus infection. ( A ) Adult wild-type flies (male, Canton-S) fed serial dilutions of <t>Hygromycin</t> B (280–17.5 μM) and then challenged with DCV were monitored daily
    Hygromycin B, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hygromycin b/product/Millipore
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    Inhibition of ribosomal function protects from infection in vivo and from poliovirus infection. ( A ) Adult wild-type flies (male, Canton-S) fed serial dilutions of Hygromycin B (280–17.5 μM) and then challenged with DCV were monitored daily

    Journal: Genes & Development

    Article Title: Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition

    doi: 10.1101/gad.1267905

    Figure Lengend Snippet: Inhibition of ribosomal function protects from infection in vivo and from poliovirus infection. ( A ) Adult wild-type flies (male, Canton-S) fed serial dilutions of Hygromycin B (280–17.5 μM) and then challenged with DCV were monitored daily

    Article Snippet: The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit.

    Techniques: Inhibition, Infection, In Vivo

    The degree of paramutation in tetraploids depends on growth temperature in F1. (A) Experimental setup: Diploid or tetraploid plants with R epialleles were crossed with those containing S or W, and F1 hybrids from reciprocal crossings were grown at 19°C for three weeks before transfer to either 10°C, 19°C, or 24°C until seed maturity. F2 seeds were germinated at 19°C on GM plates containing 20 mg/L hygromycin B and resistance ratios determined after 14 days for tetraploids (B) and diploids (C). Data for reciprocal crosses were combined, as no parent-of-origin difference was observed. Number in parentheses: different F2 populations / technical repetitions for each population. N = number of tested seedlings in each group. Bars represent the mean from two biological with three technical replicates each (B) and one biological with three technical replicates (C), with 100 plated seeds (B) each, or 50 plated seeds (C). Error bars indicate standard deviation. Dashed lines represent the expected segregation for tetraploid (B, 35:1 ≙ 97.2%) and diploid F2 populations (C, 3:1 ≙ 75%). F1 growth temperature is indicated by colour: 10°C (blue), 19°C (green), 24°C (red); light colours: F2 of R/S hybrids, dark colours: F2 of R/W hybrids. Statistical analysis was performed by summed Chi-square goodness-of-fit test with the indicated values.

    Journal: PLoS Genetics

    Article Title: Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure

    doi: 10.1371/journal.pgen.1009444

    Figure Lengend Snippet: The degree of paramutation in tetraploids depends on growth temperature in F1. (A) Experimental setup: Diploid or tetraploid plants with R epialleles were crossed with those containing S or W, and F1 hybrids from reciprocal crossings were grown at 19°C for three weeks before transfer to either 10°C, 19°C, or 24°C until seed maturity. F2 seeds were germinated at 19°C on GM plates containing 20 mg/L hygromycin B and resistance ratios determined after 14 days for tetraploids (B) and diploids (C). Data for reciprocal crosses were combined, as no parent-of-origin difference was observed. Number in parentheses: different F2 populations / technical repetitions for each population. N = number of tested seedlings in each group. Bars represent the mean from two biological with three technical replicates each (B) and one biological with three technical replicates (C), with 100 plated seeds (B) each, or 50 plated seeds (C). Error bars indicate standard deviation. Dashed lines represent the expected segregation for tetraploid (B, 35:1 ≙ 97.2%) and diploid F2 populations (C, 3:1 ≙ 75%). F1 growth temperature is indicated by colour: 10°C (blue), 19°C (green), 24°C (red); light colours: F2 of R/S hybrids, dark colours: F2 of R/W hybrids. Statistical analysis was performed by summed Chi-square goodness-of-fit test with the indicated values.

    Article Snippet: Seeds were sown on plates containing germination medium (GM, https://www.oeaw.ac.at/gmi/research/research-groups/ortrun-mittelsten-scheid/resources/ ) with or without 20 μg/ml hygromycin B (Calbiochem) and grown for 14 d under long day (LD) conditions (16 h light, 8 h dark 21°C).

    Techniques: Standard Deviation

    Lack of paramutation in the background of RdDM mutants. (A) F2 seeds obtained by selfing tetraploid F1 hybrids were germinated on GM plates containing 20 mg/L hygromycin B and resistance ratios determined after 14 days. RRWW control (white), paramutation test hybrid RRSS in wild type (grey), nrpd1 mutant (blue) or rdr2 mutant (red). Data for reciprocal crosses were combined. Number in parentheses: different F2 populations / technical repetitions for each population. N = number of tested seedlings in each group. The dashed line represents the expected F2 segregation for tetraploids (97.2%), statistical analysis is based on a summed Chi-square goodness-of-fit test with indicated values. (B) Representative images for resistance assays quantified in (A). Orange asterisks indicate sensitive seedlings, scale bar = 3 cm.

    Journal: PLoS Genetics

    Article Title: Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure

    doi: 10.1371/journal.pgen.1009444

    Figure Lengend Snippet: Lack of paramutation in the background of RdDM mutants. (A) F2 seeds obtained by selfing tetraploid F1 hybrids were germinated on GM plates containing 20 mg/L hygromycin B and resistance ratios determined after 14 days. RRWW control (white), paramutation test hybrid RRSS in wild type (grey), nrpd1 mutant (blue) or rdr2 mutant (red). Data for reciprocal crosses were combined. Number in parentheses: different F2 populations / technical repetitions for each population. N = number of tested seedlings in each group. The dashed line represents the expected F2 segregation for tetraploids (97.2%), statistical analysis is based on a summed Chi-square goodness-of-fit test with indicated values. (B) Representative images for resistance assays quantified in (A). Orange asterisks indicate sensitive seedlings, scale bar = 3 cm.

    Article Snippet: Seeds were sown on plates containing germination medium (GM, https://www.oeaw.ac.at/gmi/research/research-groups/ortrun-mittelsten-scheid/resources/ ) with or without 20 μg/ml hygromycin B (Calbiochem) and grown for 14 d under long day (LD) conditions (16 h light, 8 h dark 21°C).

    Techniques: Mutagenesis

    Paramutation depends on epiallele structure, and paramutated epialleles can exert secondary paramutation. (A) Tetraploid F2 seeds obtained by selfing F1 hybrids of crosses between S/W/R and RΔ2 (light grey) or RΔ4 (dark grey) were germinated on GM plates containing 20 mg/L hygromycin B and resistance ratios determined after 14 days. Data for reciprocal crosses were combined. Number in parentheses: different F2 populations / technical repetitions for each population. N = number of tested seedlings in each group. The dashed line represents the expected F2 segregation for tetraploids (97.2%), statistical analysis is based on a summed Chi-square goodness-of-fit test with indicated values. (B) RΔ4 deletion alleles were combined with either W or S partners (all tetraploid). Individual plants in F2 progeny were genotyped for RΔ4, sorted into groups heterozygous (+/-) or homozygous (+/+) for the deletion and material from14 d-old seedlings subjected to HPT expression analysis relative to homozygous RRRR and the housekeeping gene EIF4A1 (At3g13920) . Number in parentheses: biological / technical replicates. Error bars indicate standard deviation of biological replicates. Statistical analysis was performed by one-way ANOVA followed by a post-hoc Tuckey HSD test (α = 0.05). The black star indicates individuals homozygous for the R allele with the deletion and lacking any S copy, nevertheless with substantially reduced HPT expression. (C) Plants from primary paramutation crosses, homozygous for RΔ4 alleles (excluding the presence of the old S allele) and only residual HPT expression (black star in B, now called SΔ4SΔ4SΔ4SΔ4, Fig 1 ) were crossed to plants with the active, full length R alleles and analysed for hygromycin as described before in F2, in parallel to controls. The dashed line represents expected F2 segregation for tetraploid populations at 97.2%; statistical analysis is based on a summed Chi-square goodness-of-fit test with indicated values. N = number of tested seedlings in each group. Number in brackets: different F2 populations / technical repetitions of each population.

    Journal: PLoS Genetics

    Article Title: Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure

    doi: 10.1371/journal.pgen.1009444

    Figure Lengend Snippet: Paramutation depends on epiallele structure, and paramutated epialleles can exert secondary paramutation. (A) Tetraploid F2 seeds obtained by selfing F1 hybrids of crosses between S/W/R and RΔ2 (light grey) or RΔ4 (dark grey) were germinated on GM plates containing 20 mg/L hygromycin B and resistance ratios determined after 14 days. Data for reciprocal crosses were combined. Number in parentheses: different F2 populations / technical repetitions for each population. N = number of tested seedlings in each group. The dashed line represents the expected F2 segregation for tetraploids (97.2%), statistical analysis is based on a summed Chi-square goodness-of-fit test with indicated values. (B) RΔ4 deletion alleles were combined with either W or S partners (all tetraploid). Individual plants in F2 progeny were genotyped for RΔ4, sorted into groups heterozygous (+/-) or homozygous (+/+) for the deletion and material from14 d-old seedlings subjected to HPT expression analysis relative to homozygous RRRR and the housekeeping gene EIF4A1 (At3g13920) . Number in parentheses: biological / technical replicates. Error bars indicate standard deviation of biological replicates. Statistical analysis was performed by one-way ANOVA followed by a post-hoc Tuckey HSD test (α = 0.05). The black star indicates individuals homozygous for the R allele with the deletion and lacking any S copy, nevertheless with substantially reduced HPT expression. (C) Plants from primary paramutation crosses, homozygous for RΔ4 alleles (excluding the presence of the old S allele) and only residual HPT expression (black star in B, now called SΔ4SΔ4SΔ4SΔ4, Fig 1 ) were crossed to plants with the active, full length R alleles and analysed for hygromycin as described before in F2, in parallel to controls. The dashed line represents expected F2 segregation for tetraploid populations at 97.2%; statistical analysis is based on a summed Chi-square goodness-of-fit test with indicated values. N = number of tested seedlings in each group. Number in brackets: different F2 populations / technical repetitions of each population.

    Article Snippet: Seeds were sown on plates containing germination medium (GM, https://www.oeaw.ac.at/gmi/research/research-groups/ortrun-mittelsten-scheid/resources/ ) with or without 20 μg/ml hygromycin B (Calbiochem) and grown for 14 d under long day (LD) conditions (16 h light, 8 h dark 21°C).

    Techniques: Expressing, Standard Deviation

    Overexpression of YPT6 rescues the hyphal invasive growth defect in an arl1 deletion mutant. (A-D) Overexpression of YPT6 specifically rescues invasive growth in arl1/arl1 cells. Indicated strains were grown on agar-containing YEPD with 50% FCS (A, C, D) or on Spider media (B) and images were taken after 5–6 days. Similar results were observed in 2 independent experiments. (C) Rescue of invasive growth in arl1/arl1 cells depends on Ypt6 activity. Indicated strains were grown on agar-containing YEPD with FCS as in A. (D) Overexpression of ARL1 does not rescue invasive growth in ypt6/ypt6 cells. Indicated strains were grown on agar-containing YEPD with FCS as in A. (E) Overexpression of YPT6 partially rescues arl1/arl1 hyphal length defect. Cells from the indicated strains were incubated with FCS for 90 min and the graph shows the average hyphal length (mean of 200–400 cells each strain, from 3 experiments); error bars indicate SD. Student t test was used to calculate the p values: arl1 + ARL1 vs arl1 : 0.0028 and arl1 + YPT6 vs arl1 : 0.0042. (F) YPT6 and ARL1 transcripts in the overexpression mutants. mRNA and cDNA were prepared from the indicated strains and the transcripts were quantified by RT-PCR; actin ( ACT1 ) was used for normalization. (G) Overexpression of YPT6 does not rescue cell wall defects in arl1/arl1 cells. Serial dilutions of the indicated strains were spotted on YEPD media (Ctrl) containing 400 μg/ml Congo red (CR) or 1 mg/ml hygromycin B (HygB). Images were taken after 2 days.

    Journal: Small GTPases

    Article Title: Overexpression of YPT6 restores invasive filamentous growth and secretory vesicle clustering in a Candida albicans arl1 mutant

    doi: 10.1080/21541248.2017.1378157

    Figure Lengend Snippet: Overexpression of YPT6 rescues the hyphal invasive growth defect in an arl1 deletion mutant. (A-D) Overexpression of YPT6 specifically rescues invasive growth in arl1/arl1 cells. Indicated strains were grown on agar-containing YEPD with 50% FCS (A, C, D) or on Spider media (B) and images were taken after 5–6 days. Similar results were observed in 2 independent experiments. (C) Rescue of invasive growth in arl1/arl1 cells depends on Ypt6 activity. Indicated strains were grown on agar-containing YEPD with FCS as in A. (D) Overexpression of ARL1 does not rescue invasive growth in ypt6/ypt6 cells. Indicated strains were grown on agar-containing YEPD with FCS as in A. (E) Overexpression of YPT6 partially rescues arl1/arl1 hyphal length defect. Cells from the indicated strains were incubated with FCS for 90 min and the graph shows the average hyphal length (mean of 200–400 cells each strain, from 3 experiments); error bars indicate SD. Student t test was used to calculate the p values: arl1 + ARL1 vs arl1 : 0.0028 and arl1 + YPT6 vs arl1 : 0.0042. (F) YPT6 and ARL1 transcripts in the overexpression mutants. mRNA and cDNA were prepared from the indicated strains and the transcripts were quantified by RT-PCR; actin ( ACT1 ) was used for normalization. (G) Overexpression of YPT6 does not rescue cell wall defects in arl1/arl1 cells. Serial dilutions of the indicated strains were spotted on YEPD media (Ctrl) containing 400 μg/ml Congo red (CR) or 1 mg/ml hygromycin B (HygB). Images were taken after 2 days.

    Article Snippet: Congo red and Hygromycin B were from Fluka, Sigma-Aldrich, Saint Quentin Fallavier, France.

    Techniques: Over Expression, Mutagenesis, Activity Assay, Incubation, Reverse Transcription Polymerase Chain Reaction

    Construction of the targeted replacement construct for mbtB deletion. A 10-kb portion of the mbt locus that has been proposed to be involved in mycobactin synthesis. Below this is the 5,281-nt Sph I fragment, from which an internal, 1.3-kb fragment was removed and replaced with the hygromycin resistance cassette as shown at the bottom.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages

    doi:

    Figure Lengend Snippet: Construction of the targeted replacement construct for mbtB deletion. A 10-kb portion of the mbt locus that has been proposed to be involved in mycobactin synthesis. Below this is the 5,281-nt Sph I fragment, from which an internal, 1.3-kb fragment was removed and replaced with the hygromycin resistance cassette as shown at the bottom.

    Article Snippet: Where indicated, antibiotics were included at the following concentrations: kanamycin (Sigma), 25 μg/ml; and hygromycin (Calbiochem), 50 μg/ml.

    Techniques: Construct