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  • 94
    Vector Laboratories rhodamine conjugated lens culinaris agglutinin
    Rhodamine Conjugated Lens Culinaris Agglutinin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhodamine conjugated lens culinaris agglutinin/product/Vector Laboratories
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    93
    ATCC feeder mefs
    Validation <t>of</t> <t>Firre</t> knockout in <t>MEFs.</t> a qRT-PCR analysis of Firre expression in wild-type and knockout MEFs. Error bars: s.e.m. b Plot showing transcripts per million (TPM) values for wild-type and Firre KO MEF RNA-seq. Error bars: s.d. c CTCF ChIP-seq signal tracks showing the complete loss of CTCF binding at the Firre locus in Firre KO MEFs (mm9, chrX:47.8–49 Mb). d , e Hi-C reads per million (RPM) values for the Firre locus in ( e ) wild-type and ( f ) Firre KO MEFs
    Feeder Mefs, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    feeder mefs - by Bioz Stars, 2023-01
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    mef  (ATCC)
    95
    ATCC mef
    Validation <t>of</t> <t>Firre</t> knockout in <t>MEFs.</t> a qRT-PCR analysis of Firre expression in wild-type and knockout MEFs. Error bars: s.e.m. b Plot showing transcripts per million (TPM) values for wild-type and Firre KO MEF RNA-seq. Error bars: s.d. c CTCF ChIP-seq signal tracks showing the complete loss of CTCF binding at the Firre locus in Firre KO MEFs (mm9, chrX:47.8–49 Mb). d , e Hi-C reads per million (RPM) values for the Firre locus in ( e ) wild-type and ( f ) Firre KO MEFs
    Mef, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATCC mef cells
    (A) Structural model of the predicted extended structure <t>of</t> <t>Mfn2</t> based on the crystal structure of the structurally related protein BDLP with 5′-Guanylyl imidodiphosphate (GMPPNP) (protein data base [PDB] 2W6D ). The GTPase domain is green, HB1 is blue, HB2 is red, the transmembrane (TM) domain is grey, and Loops 1/2 are purple. Structural prediction performed by I-TASSER server ( ; ). (B) Enlarged view of hinge 1 showing the positions of relevant CMT2A-related amino acids in pink with side chains. (C) Structural model of the predicted closed structure of Mfn2 based on the crystal structure of the structurally related protein BDLP with GDP (PDB 2J69 ). (A) Domains are colored as described in (A). Structural prediction performed by I-TASSER server ( ; ). (D) Enlarged view of Loop 1 from hinge 1 showing the positions of the CMT2A-related amino acids in pink with side chains.
    Mef Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATCC c tropicalis
    Reproducibility of MICs of AMB against eight fungal strains, as determined by the PDA method
    C Tropicalis, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Validation of Firre knockout in MEFs. a qRT-PCR analysis of Firre expression in wild-type and knockout MEFs. Error bars: s.e.m. b Plot showing transcripts per million (TPM) values for wild-type and Firre KO MEF RNA-seq. Error bars: s.d. c CTCF ChIP-seq signal tracks showing the complete loss of CTCF binding at the Firre locus in Firre KO MEFs (mm9, chrX:47.8–49 Mb). d , e Hi-C reads per million (RPM) values for the Firre locus in ( e ) wild-type and ( f ) Firre KO MEFs

    Journal: Nature Communications

    Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

    doi: 10.1038/s41467-018-03614-0

    Figure Lengend Snippet: Validation of Firre knockout in MEFs. a qRT-PCR analysis of Firre expression in wild-type and knockout MEFs. Error bars: s.e.m. b Plot showing transcripts per million (TPM) values for wild-type and Firre KO MEF RNA-seq. Error bars: s.d. c CTCF ChIP-seq signal tracks showing the complete loss of CTCF binding at the Firre locus in Firre KO MEFs (mm9, chrX:47.8–49 Mb). d , e Hi-C reads per million (RPM) values for the Firre locus in ( e ) wild-type and ( f ) Firre KO MEFs

    Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

    Techniques: Knock-Out, Quantitative RT-PCR, Expressing, RNA Sequencing Assay, ChIP-sequencing, Binding Assay, Hi-C

    Firre KO does not result in disruption of TAD boundaries. a-d Hi-C heatmaps showing ± 5 Mb of the Firre gene locus (mm9, chr.X: 45–51 Mb) in female wild-type and Firre KO MEFs, ( b ) male wild-type and Firre KO MEFs, ( c ) allele-specific haploid chromosomes for female Cast (wild type) and C57BL6 ( Firre KO), and ( d ) male C57BL6 ( Firre KO). The TAD boundaries and the insulation plot for each Hi-C dataset is depicted below. e Dot plots showing the boundary strength of the Firre -centered and the neighboring TAD boundaries in wildtype (gray) and Firre KO (red) samples. The TAD insulation scores of all TAD boundaries on chromosome X is shown as boxplots on the right panel. f Hi-C heatmaps from wild-type (grown on 2i) and Firre KO mouse embryonic stem cells (mESCs) (grown on feeders + 2i) showing ± 5 Mb of the Firre locus. g , h Boxplot showing the inter-TAD interaction frequency between the TAD domains neighboring the Firre locus in wild-type (gray) and Firre KO (red) cells ( g ) in female MEFs and ( h ) in mESCs. Error bars: s.d. (* p -value: Wilcoxon rank-sum test)

    Journal: Nature Communications

    Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

    doi: 10.1038/s41467-018-03614-0

    Figure Lengend Snippet: Firre KO does not result in disruption of TAD boundaries. a-d Hi-C heatmaps showing ± 5 Mb of the Firre gene locus (mm9, chr.X: 45–51 Mb) in female wild-type and Firre KO MEFs, ( b ) male wild-type and Firre KO MEFs, ( c ) allele-specific haploid chromosomes for female Cast (wild type) and C57BL6 ( Firre KO), and ( d ) male C57BL6 ( Firre KO). The TAD boundaries and the insulation plot for each Hi-C dataset is depicted below. e Dot plots showing the boundary strength of the Firre -centered and the neighboring TAD boundaries in wildtype (gray) and Firre KO (red) samples. The TAD insulation scores of all TAD boundaries on chromosome X is shown as boxplots on the right panel. f Hi-C heatmaps from wild-type (grown on 2i) and Firre KO mouse embryonic stem cells (mESCs) (grown on feeders + 2i) showing ± 5 Mb of the Firre locus. g , h Boxplot showing the inter-TAD interaction frequency between the TAD domains neighboring the Firre locus in wild-type (gray) and Firre KO (red) cells ( g ) in female MEFs and ( h ) in mESCs. Error bars: s.d. (* p -value: Wilcoxon rank-sum test)

    Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

    Techniques: Hi-C

    TAD boundaries are preserved upon ectopic Firre cDNA insertion and its induced expression at target sites. a Cartoon depicting the approach for the generation of the transgenic MEFs with endogeous Firre KO and ectopic Firre cDNA insertions. b qRT-PCR showing the induced expression of the Firre cDNA in wild type, DOX − , and DOX + conditions. Error bars: s.e.m. (* p -value: t -test). c Chromosome ideograms showing the Firre cDNA insertion sites on four different chromosomes. d CTCF ChIP-seq signal from DOX − transgenic MEFs for each of the exons of Firre cDNA at randomly inserted loci. As the transgenic MEFs harbor an endogenous Firre deletion, the intronic regions did not harbor any ChIP-seq signal. e-h Hi-C heatmaps showing the TAD organization, TAD boundary position, and the insulation plots for male Firre KO, DOX − , and DOX + samples ± ~ 5 Mb of Firre cDNA insertion sites on ( e ) chr 8, ( f ) chr 10, ( g ) chr 11, and ( h ) chr 15

    Journal: Nature Communications

    Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

    doi: 10.1038/s41467-018-03614-0

    Figure Lengend Snippet: TAD boundaries are preserved upon ectopic Firre cDNA insertion and its induced expression at target sites. a Cartoon depicting the approach for the generation of the transgenic MEFs with endogeous Firre KO and ectopic Firre cDNA insertions. b qRT-PCR showing the induced expression of the Firre cDNA in wild type, DOX − , and DOX + conditions. Error bars: s.e.m. (* p -value: t -test). c Chromosome ideograms showing the Firre cDNA insertion sites on four different chromosomes. d CTCF ChIP-seq signal from DOX − transgenic MEFs for each of the exons of Firre cDNA at randomly inserted loci. As the transgenic MEFs harbor an endogenous Firre deletion, the intronic regions did not harbor any ChIP-seq signal. e-h Hi-C heatmaps showing the TAD organization, TAD boundary position, and the insulation plots for male Firre KO, DOX − , and DOX + samples ± ~ 5 Mb of Firre cDNA insertion sites on ( e ) chr 8, ( f ) chr 10, ( g ) chr 11, and ( h ) chr 15

    Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, ChIP-sequencing, Hi-C

    Firre KO results in a loss of Firre -DXZ4, and changes in super-loop interactions in female MEFs. a Hi-C heatmaps at 40 kb resolution in male and female wildtype and Firre KO MEFs showing the interaction frequency between Firre and DXZ4 ± 1 Mb. b Forty-two windows of 40 kb bins were slid across the entire heatmap (2 Mb × 2 Mb in size) to compare the interaction frequency at each position of female and male WT versus KO conditions to the Firre -DXZ4 interaction by t -tests. The p -value distribution in the female or male wild-type samples indicate a drastically enriched significance of Firre -DXZ4 interactions when compared with either the female Firre KO or male samples (* p < 0.05, one-way ANOVA). c Hi-C heatmap at 100 kb resolution showing the zoomed-in interactions between the mouse Firre , DXZ4, x75, and ICCE regions ± 100 kb in female and male wild-type and Firre KO MEFs. d Boxplots showing the interactions among the super-loop regions in wild-type (gray) and Firre KO (red) female (top) and male (bottom) MEF samples. The sample sizes of the boxplots are n = 12 for Firre -DXZ4, DXZ4-x75, and DXZ4-ICCE interactions, and n = 9 for all other combinations. p -value: t -test. Error bars: s.d

    Journal: Nature Communications

    Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

    doi: 10.1038/s41467-018-03614-0

    Figure Lengend Snippet: Firre KO results in a loss of Firre -DXZ4, and changes in super-loop interactions in female MEFs. a Hi-C heatmaps at 40 kb resolution in male and female wildtype and Firre KO MEFs showing the interaction frequency between Firre and DXZ4 ± 1 Mb. b Forty-two windows of 40 kb bins were slid across the entire heatmap (2 Mb × 2 Mb in size) to compare the interaction frequency at each position of female and male WT versus KO conditions to the Firre -DXZ4 interaction by t -tests. The p -value distribution in the female or male wild-type samples indicate a drastically enriched significance of Firre -DXZ4 interactions when compared with either the female Firre KO or male samples (* p < 0.05, one-way ANOVA). c Hi-C heatmap at 100 kb resolution showing the zoomed-in interactions between the mouse Firre , DXZ4, x75, and ICCE regions ± 100 kb in female and male wild-type and Firre KO MEFs. d Boxplots showing the interactions among the super-loop regions in wild-type (gray) and Firre KO (red) female (top) and male (bottom) MEF samples. The sample sizes of the boxplots are n = 12 for Firre -DXZ4, DXZ4-x75, and DXZ4-ICCE interactions, and n = 9 for all other combinations. p -value: t -test. Error bars: s.d

    Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

    Techniques: Hi-C

    CRISPR live-cell imaging and 3C validates the changes in super-loop interactions. a Four-color CRISPR live-cell imaging (CLING) from female wild-type and Firre KO MEFs. Firre (red), DXZ4 (green), and x75 (white) loci were simultaneously visualized with Hoechst staining (blue). Pseudo-coloring was used for visual simplicity. Scale bar: 5 μm. b Quantification of the colocalization percentages between Firre -DXZ4, DXZ4-x75, and Firre -x75 between wild-type (black) and Firre KO (red) MEFs. (* p -value: χ 2 -test, n > 80 nuclei). Error bars: s.e.m. c Chromosome conformation capture (3C) analysis showing the interaction frequency ratios of Firre -DXZ4, DXZ4-x75, and Firre -x75 in female Firre KO vs. wild-type MEFs (* p -value: t -test, n = 3). The black arc indicates 3C enrichment in the wild-type samples, whereas the red arc represents enrichment in the Firre KO samples

    Journal: Nature Communications

    Article Title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

    doi: 10.1038/s41467-018-03614-0

    Figure Lengend Snippet: CRISPR live-cell imaging and 3C validates the changes in super-loop interactions. a Four-color CRISPR live-cell imaging (CLING) from female wild-type and Firre KO MEFs. Firre (red), DXZ4 (green), and x75 (white) loci were simultaneously visualized with Hoechst staining (blue). Pseudo-coloring was used for visual simplicity. Scale bar: 5 μm. b Quantification of the colocalization percentages between Firre -DXZ4, DXZ4-x75, and Firre -x75 between wild-type (black) and Firre KO (red) MEFs. (* p -value: χ 2 -test, n > 80 nuclei). Error bars: s.e.m. c Chromosome conformation capture (3C) analysis showing the interaction frequency ratios of Firre -DXZ4, DXZ4-x75, and Firre -x75 in female Firre KO vs. wild-type MEFs (* p -value: t -test, n = 3). The black arc indicates 3C enrichment in the wild-type samples, whereas the red arc represents enrichment in the Firre KO samples

    Article Snippet: The Firre KO mESCs were co-cultured with irradiated feeder MEFs (ATCC, SCRC-1040.1) with 2i media.

    Techniques: CRISPR, Live Cell Imaging, Staining

    (A) Structural model of the predicted extended structure of Mfn2 based on the crystal structure of the structurally related protein BDLP with 5′-Guanylyl imidodiphosphate (GMPPNP) (protein data base [PDB] 2W6D ). The GTPase domain is green, HB1 is blue, HB2 is red, the transmembrane (TM) domain is grey, and Loops 1/2 are purple. Structural prediction performed by I-TASSER server ( ; ). (B) Enlarged view of hinge 1 showing the positions of relevant CMT2A-related amino acids in pink with side chains. (C) Structural model of the predicted closed structure of Mfn2 based on the crystal structure of the structurally related protein BDLP with GDP (PDB 2J69 ). (A) Domains are colored as described in (A). Structural prediction performed by I-TASSER server ( ; ). (D) Enlarged view of Loop 1 from hinge 1 showing the positions of the CMT2A-related amino acids in pink with side chains.

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: (A) Structural model of the predicted extended structure of Mfn2 based on the crystal structure of the structurally related protein BDLP with 5′-Guanylyl imidodiphosphate (GMPPNP) (protein data base [PDB] 2W6D ). The GTPase domain is green, HB1 is blue, HB2 is red, the transmembrane (TM) domain is grey, and Loops 1/2 are purple. Structural prediction performed by I-TASSER server ( ; ). (B) Enlarged view of hinge 1 showing the positions of relevant CMT2A-related amino acids in pink with side chains. (C) Structural model of the predicted closed structure of Mfn2 based on the crystal structure of the structurally related protein BDLP with GDP (PDB 2J69 ). (A) Domains are colored as described in (A). Structural prediction performed by I-TASSER server ( ; ). (D) Enlarged view of Loop 1 from hinge 1 showing the positions of the CMT2A-related amino acids in pink with side chains.

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques:

    The Mfn2 IM crystal structure with the GTPase domain in green, HB1 in blue and S378, A383, Q386, and C390F in dark pink. The positions of the N-terminal truncation (R400) and the start of the C-terminal helix (R707) are shown with arrows (PDB 6JFK ).

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: The Mfn2 IM crystal structure with the GTPase domain in green, HB1 in blue and S378, A383, Q386, and C390F in dark pink. The positions of the N-terminal truncation (R400) and the start of the C-terminal helix (R707) are shown with arrows (PDB 6JFK ).

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques:

    Whole-cell lysates prepared from the indicated cell lines were subject to SDS–PAGE and immunoblotting with α-Mfn2 and α-tubulin. Molecular weight markers are indicated in kD on the left.

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: Whole-cell lysates prepared from the indicated cell lines were subject to SDS–PAGE and immunoblotting with α-Mfn2 and α-tubulin. Molecular weight markers are indicated in kD on the left.

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: SDS Page, Western Blot, Molecular Weight

    (A) Representative images of mitochondrial networks in wild-type (Mfn1 +/+ Mfn2 +/+ ) or Mfn2-null (Mfn1 +/+ Mfn2 −/− ) MEFs expressing the indicated Mfn2 variant. Mitochondria were stained with MitoTracker Red CMXRos and visualized by fluorescence microscopy. Images represent a maximum intensity projection. Scale bars = 5 μm. (A, B) Quantification of mitochondrial morphology in cells represented in (A). Error bars indicate mean + SD from three independent, blinded experiments (n ≧ 100 cells per population per experiment).

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: (A) Representative images of mitochondrial networks in wild-type (Mfn1 +/+ Mfn2 +/+ ) or Mfn2-null (Mfn1 +/+ Mfn2 −/− ) MEFs expressing the indicated Mfn2 variant. Mitochondria were stained with MitoTracker Red CMXRos and visualized by fluorescence microscopy. Images represent a maximum intensity projection. Scale bars = 5 μm. (A, B) Quantification of mitochondrial morphology in cells represented in (A). Error bars indicate mean + SD from three independent, blinded experiments (n ≧ 100 cells per population per experiment).

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Expressing, Variant Assay, Staining, Fluorescence, Microscopy

    Wild-type (Mfn1 +/+ Mfn2 +/+ ) or clonal populations of Mfn2-null MEFs expressing the indicated Mfn2 variant were transduced with the mitochondrial matrix-targeted photoactivatable-GFP (mito-PAGFP). These cells were labeled with MitoTracker Red CMXRos before imaging. A small region of the cells that contained mitochondria (∼1 μm 2 ) was activated using a 405-nm laser, and the cell was imaged immediately after the activation and 50 min later. The area of green pixels and red pixels was determined for each image, where red pixels represent the area of the mitochondrial network. The proportion of the mitochondrial network that possessed green pixels was determined at time = 0 and time = 50 min. The increase in this proportion of t = 50 relative to t = 0 is shown. Paired t test analysis indicated that the decreased overlap observed was not statistically significant.

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: Wild-type (Mfn1 +/+ Mfn2 +/+ ) or clonal populations of Mfn2-null MEFs expressing the indicated Mfn2 variant were transduced with the mitochondrial matrix-targeted photoactivatable-GFP (mito-PAGFP). These cells were labeled with MitoTracker Red CMXRos before imaging. A small region of the cells that contained mitochondria (∼1 μm 2 ) was activated using a 405-nm laser, and the cell was imaged immediately after the activation and 50 min later. The area of green pixels and red pixels was determined for each image, where red pixels represent the area of the mitochondrial network. The proportion of the mitochondrial network that possessed green pixels was determined at time = 0 and time = 50 min. The increase in this proportion of t = 50 relative to t = 0 is shown. Paired t test analysis indicated that the decreased overlap observed was not statistically significant.

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Expressing, Variant Assay, Transduction, Labeling, Imaging, Activation Assay

    Wild-type (Mfn1 +/+ Mfn2 +/+ ) or Mfn2-null MEFs expressing the indicated Mfn2 variant were treated with 100 μM diamide for 1 h. Mitochondria were stained with MitoTracker Red CMXRos and visualized by fluorescence microscopy. The graph represents the mitochondrial morphology as scored in three independent experiments. Error bars indicate mean + SD (n ≧ 100 cells per population per experiment).

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: Wild-type (Mfn1 +/+ Mfn2 +/+ ) or Mfn2-null MEFs expressing the indicated Mfn2 variant were treated with 100 μM diamide for 1 h. Mitochondria were stained with MitoTracker Red CMXRos and visualized by fluorescence microscopy. The graph represents the mitochondrial morphology as scored in three independent experiments. Error bars indicate mean + SD (n ≧ 100 cells per population per experiment).

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Expressing, Variant Assay, Staining, Fluorescence, Microscopy

    (A) Mitochondria isolated from wild-type cells or clonal populations of Mfn2-null MEFs either transduced with empty vector or expressing the indicated Mfn2 variant were subject to in vitro fusion conditions at 37°C for 60 min. The data are represented as relative to wild-type controls performed in parallel. Error bars indicate mean + SD from at least three independent experiments. The average values of each is indicated in white within the individual bars. (B) Mitochondrial in vitro fusion assay performed as in (A) except with the addition of cytosol-enriched fraction to the reaction buffer. Data are represented as proportion of control reactions performed in parallel without cytosol. Error bars indicate mean + SD from at least three independent experiments. The average values of each is indicated in white within the individual bars. (C) Mitochondrial in vitro fusion assay performed as in (A) except with the addition of 300 nM purified Bax protein to the reaction buffer. Data are represented as proportion of control reactions performed in parallel without Bax. Error bars indicate mean + SD from at least three independent experiments. The average values of each is indicated in white within the individual bars.

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: (A) Mitochondria isolated from wild-type cells or clonal populations of Mfn2-null MEFs either transduced with empty vector or expressing the indicated Mfn2 variant were subject to in vitro fusion conditions at 37°C for 60 min. The data are represented as relative to wild-type controls performed in parallel. Error bars indicate mean + SD from at least three independent experiments. The average values of each is indicated in white within the individual bars. (B) Mitochondrial in vitro fusion assay performed as in (A) except with the addition of cytosol-enriched fraction to the reaction buffer. Data are represented as proportion of control reactions performed in parallel without cytosol. Error bars indicate mean + SD from at least three independent experiments. The average values of each is indicated in white within the individual bars. (C) Mitochondrial in vitro fusion assay performed as in (A) except with the addition of 300 nM purified Bax protein to the reaction buffer. Data are represented as proportion of control reactions performed in parallel without Bax. Error bars indicate mean + SD from at least three independent experiments. The average values of each is indicated in white within the individual bars.

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Isolation, Transduction, Plasmid Preparation, Expressing, Variant Assay, In Vitro, Single Vesicle Fusion Assay, Purification

    (A) Schematic of the differential epitope labeling used in the co-immunoprecipitation assay. Interactions tested are Mfn2-FLAG with Mfn1 in cis (top left), Mfn2-FLAG with Mfn1-eGFP in trans (top center), and Mfn2-FLAG with Mfn2 in trans (bottom center). (B) Mitochondria were isolated from a clonal population of Mfn1-null cells expressing Mfn1 WT -eGFP at endogenous levels and clonal populations of Mfn2-null MEFs expressing the indicated Mfn2-FLAG variant. Mitochondria that possess Mfn1-eGFP and Mfn2 were combined with mitochondria that possess Mfn1 and Mfn2-FLAG; these mixtures were incubated with BeF 3 in the absence or presence of GDP. After lysis, immunoprecipitation was performed with α-FLAG magnetic beads. Proteins eluted from the beads were subjected to SDS–PAGE and immunoblotting with α-Mfn1 and α-Mfn2, as indicated. Arrows indicate endogenous Mfn1 (black) and Mfn2 (white); arrowheads indicate Mfn1-eGFP (white) and Mfn2-FLAG (black). Input represents 3% of the input and elution represents 37.5% of the immunoprecipitated protein. (C) Quantification of the percentage of Mfn1-eGFP in the elution compared with Mfn2-FLAG is shown as the mean + SD of three independent experiments.

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: (A) Schematic of the differential epitope labeling used in the co-immunoprecipitation assay. Interactions tested are Mfn2-FLAG with Mfn1 in cis (top left), Mfn2-FLAG with Mfn1-eGFP in trans (top center), and Mfn2-FLAG with Mfn2 in trans (bottom center). (B) Mitochondria were isolated from a clonal population of Mfn1-null cells expressing Mfn1 WT -eGFP at endogenous levels and clonal populations of Mfn2-null MEFs expressing the indicated Mfn2-FLAG variant. Mitochondria that possess Mfn1-eGFP and Mfn2 were combined with mitochondria that possess Mfn1 and Mfn2-FLAG; these mixtures were incubated with BeF 3 in the absence or presence of GDP. After lysis, immunoprecipitation was performed with α-FLAG magnetic beads. Proteins eluted from the beads were subjected to SDS–PAGE and immunoblotting with α-Mfn1 and α-Mfn2, as indicated. Arrows indicate endogenous Mfn1 (black) and Mfn2 (white); arrowheads indicate Mfn1-eGFP (white) and Mfn2-FLAG (black). Input represents 3% of the input and elution represents 37.5% of the immunoprecipitated protein. (C) Quantification of the percentage of Mfn1-eGFP in the elution compared with Mfn2-FLAG is shown as the mean + SD of three independent experiments.

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Labeling, Co-Immunoprecipitation Assay, Isolation, Expressing, Variant Assay, Incubation, Lysis, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot

    Mitochondria were isolated from a clonal population of Mfn1-null cells expressing Mfn1 WT -eGFP at endogenous levels (Mfn1-eGFP) or a clonal population of Mfn2-null cells expressing Mfn2-FLAG, or a clonal population of Mfn1-null cells expressing Mfn1-FLAG. Mitochondria were combined in the indicated combinations and incubated with BeF 3 in the presence or absence of GDP. After lysis, immunoprecipitation was performed with α-FLAG magnetic beads. Proteins eluted from the beads were subjected to SDS–PAGE and immunoblotting with α-Mfn1, α-FLAG, α-VDAC, or α-Hsp60, as indicated. Black arrow indicates endogenous Mfn1, white arrowhead indicates Mfn1-Egfp, black arrowhead indicates Mfn2-FLAG, and line indicates Mfn1-FLAG. Input represents 3% of the input and elution represents 37.5% of the immunoprecipitated protein. The molecular weight markers are shown in kD on the left.

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: Mitochondria were isolated from a clonal population of Mfn1-null cells expressing Mfn1 WT -eGFP at endogenous levels (Mfn1-eGFP) or a clonal population of Mfn2-null cells expressing Mfn2-FLAG, or a clonal population of Mfn1-null cells expressing Mfn1-FLAG. Mitochondria were combined in the indicated combinations and incubated with BeF 3 in the presence or absence of GDP. After lysis, immunoprecipitation was performed with α-FLAG magnetic beads. Proteins eluted from the beads were subjected to SDS–PAGE and immunoblotting with α-Mfn1, α-FLAG, α-VDAC, or α-Hsp60, as indicated. Black arrow indicates endogenous Mfn1, white arrowhead indicates Mfn1-Egfp, black arrowhead indicates Mfn2-FLAG, and line indicates Mfn1-FLAG. Input represents 3% of the input and elution represents 37.5% of the immunoprecipitated protein. The molecular weight markers are shown in kD on the left.

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Isolation, Expressing, Incubation, Lysis, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot, Molecular Weight

    (A) Mitochondria were isolated from clonal populations of Mfn2-null MEFs expressing the indicated Mfn2 variant. Mitochondria were either untreated or incubated with 2 mM GTP or 2 mM GMPPNP as indicated before lysis and separation by BN-PAGE followed by immunoblotting with anti-FLAG antibody. Arrow indicates predicted dimer, closed arrowhead indicates ∼320-kD species, and open arrowhead indicates ∼450-kD species. Asterisk (*) indicates nonspecific signal. Approximate molecular weights are in kD on the left. (B) Quantification of proportion of the total protein observed in the ~320 or ~450-kD band after treatment with GMPPNP (filled arrowhead). Error bars indicate mean + SD from at least three independent experiments and the statistical significance were determined by paired t test analysis between the indicated data and wild type (* P < 0.05). (C) Mitochondria were prepared as in (A) and incubated with 2 mM GMPPNP with or without 1 μM recombinant purified Bax. Arrow indicates predicted dimer, closed arrowhead indicates ∼320-kD species, and open arrowhead indicates ∼450-kD species. Asterisk (*) indicates nonspecific signal. Approximate molecular weights are in kD on the left. (D) Quantification of proportion of the total protein observed in the ∼320-kD band. Error bars indicate mean + SD from at least three independent experiments and the statistical significance were determined by paired t test analysis between the indicated data and wild type (* P < 0.05).

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: (A) Mitochondria were isolated from clonal populations of Mfn2-null MEFs expressing the indicated Mfn2 variant. Mitochondria were either untreated or incubated with 2 mM GTP or 2 mM GMPPNP as indicated before lysis and separation by BN-PAGE followed by immunoblotting with anti-FLAG antibody. Arrow indicates predicted dimer, closed arrowhead indicates ∼320-kD species, and open arrowhead indicates ∼450-kD species. Asterisk (*) indicates nonspecific signal. Approximate molecular weights are in kD on the left. (B) Quantification of proportion of the total protein observed in the ~320 or ~450-kD band after treatment with GMPPNP (filled arrowhead). Error bars indicate mean + SD from at least three independent experiments and the statistical significance were determined by paired t test analysis between the indicated data and wild type (* P < 0.05). (C) Mitochondria were prepared as in (A) and incubated with 2 mM GMPPNP with or without 1 μM recombinant purified Bax. Arrow indicates predicted dimer, closed arrowhead indicates ∼320-kD species, and open arrowhead indicates ∼450-kD species. Asterisk (*) indicates nonspecific signal. Approximate molecular weights are in kD on the left. (D) Quantification of proportion of the total protein observed in the ∼320-kD band. Error bars indicate mean + SD from at least three independent experiments and the statistical significance were determined by paired t test analysis between the indicated data and wild type (* P < 0.05).

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Isolation, Expressing, Variant Assay, Incubation, Lysis, Western Blot, Recombinant, Purification

    Structural models of the predicted closed structure of Mfn2 based on the crystal structure of the structurally related protein BDLP with GDP (PDB 2J69 ). The GTPase domain is green, HB1 is blue, HB2 is red, the transmembrane (TM) domain is grey, and Loops 1/2 are purple. (A) The positions of C390 and L710 are indicated in orange and labeled. (B) The positions of S378 and L710 are indicated in orange and labeled. Structural prediction performed by I-TASSER server ( ; ).

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: Structural models of the predicted closed structure of Mfn2 based on the crystal structure of the structurally related protein BDLP with GDP (PDB 2J69 ). The GTPase domain is green, HB1 is blue, HB2 is red, the transmembrane (TM) domain is grey, and Loops 1/2 are purple. (A) The positions of C390 and L710 are indicated in orange and labeled. (B) The positions of S378 and L710 are indicated in orange and labeled. Structural prediction performed by I-TASSER server ( ; ).

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Labeling

    (A) Representative images of mitochondrial networks in Mfn2-null MEFs expressing the indicated Mfn2-mNeonGreen variant. Mitochondria were stained with MitoTracker Red CMXRos and visualized by fluorescence microscopy. Images represent a maximum intensity projection. Scale bars = 5 μm. (B) Quantification of mitochondrial morphology of cells represented in (A). Error bars indicate mean + SD from at least three independent experiments and the statistical significance was determined by paired t test analysis between the indicated data and wild type (* P < 0.05) or between the indicated data and Mfn2 S378P/L710P († P < 0.05).

    Journal: Life Science Alliance

    Article Title: Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

    doi: 10.26508/lsa.201900527

    Figure Lengend Snippet: (A) Representative images of mitochondrial networks in Mfn2-null MEFs expressing the indicated Mfn2-mNeonGreen variant. Mitochondria were stained with MitoTracker Red CMXRos and visualized by fluorescence microscopy. Images represent a maximum intensity projection. Scale bars = 5 μm. (B) Quantification of mitochondrial morphology of cells represented in (A). Error bars indicate mean + SD from at least three independent experiments and the statistical significance was determined by paired t test analysis between the indicated data and wild type (* P < 0.05) or between the indicated data and Mfn2 S378P/L710P († P < 0.05).

    Article Snippet: MEF cells (Mfn wild-type and Mfn2-null) were purchased from American Type Culture Collection.

    Techniques: Expressing, Variant Assay, Staining, Fluorescence, Microscopy

    Reproducibility of MICs of AMB against eight fungal strains, as determined by the PDA method

    Journal:

    Article Title: Potato Dextrose Agar Antifungal Susceptibility Testing for Yeasts and Molds: Evaluation of Phosphate Effect on Antifungal Activity of CMT-3

    doi: 10.1128/AAC.46.5.1455-1461.2002

    Figure Lengend Snippet: Reproducibility of MICs of AMB against eight fungal strains, as determined by the PDA method

    Article Snippet: Overall, the reproducibility of the MICs of both drugs in the PDA method was satisfactory (97%). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Organism a No. of occurrences at an MIC (μg/ml) of: Tested range b (μg/ml) % Reproducibility c 0.12 0.25 0.5 1.0 2.0 4.0 8.0 C. parapsilosis (ATCC 22019) 0 0 1 9 5 0 0 0.5-2.0 100 C. krusei (ATCC 6258) 0 0 0 3 11 0 1 1.0-4.0 93 C. tropicalis (ATCC 750) 0 0 1 11 2 1 0 0.5-2.0 93 C. albicans (ATCC 24433) 0 1 2 10 2 0 0 0.5-2.0 93 A. flavus (CI) 0 0 1 12 2 0 0 0.5-2.0 100 A. fumigatus (ATCC 1022) 0 0 1 8 5 0 0 0.5-2.0 100 Penicillium sp. (CI) 1 11 3 0 0 0 0 0.12-0.5 100 Rhizopus sp. (CI) 0 1 8 6 0 0 0 0.25-1.0 100 Open in a separate window a CI, clinical isolate. b The range was determined as the peak and 1 log 2 dilution. c Percentage of MICs within the tested range.

    Techniques:

    Comparison of the MICs of AMB and itraconazole determined by the PDA method and the BMM

    Journal:

    Article Title: Potato Dextrose Agar Antifungal Susceptibility Testing for Yeasts and Molds: Evaluation of Phosphate Effect on Antifungal Activity of CMT-3

    doi: 10.1128/AAC.46.5.1455-1461.2002

    Figure Lengend Snippet: Comparison of the MICs of AMB and itraconazole determined by the PDA method and the BMM

    Article Snippet: Overall, the reproducibility of the MICs of both drugs in the PDA method was satisfactory (97%). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Organism a No. of occurrences at an MIC (μg/ml) of: Tested range b (μg/ml) % Reproducibility c 0.12 0.25 0.5 1.0 2.0 4.0 8.0 C. parapsilosis (ATCC 22019) 0 0 1 9 5 0 0 0.5-2.0 100 C. krusei (ATCC 6258) 0 0 0 3 11 0 1 1.0-4.0 93 C. tropicalis (ATCC 750) 0 0 1 11 2 1 0 0.5-2.0 93 C. albicans (ATCC 24433) 0 1 2 10 2 0 0 0.5-2.0 93 A. flavus (CI) 0 0 1 12 2 0 0 0.5-2.0 100 A. fumigatus (ATCC 1022) 0 0 1 8 5 0 0 0.5-2.0 100 Penicillium sp. (CI) 1 11 3 0 0 0 0 0.12-0.5 100 Rhizopus sp. (CI) 0 1 8 6 0 0 0 0.25-1.0 100 Open in a separate window a CI, clinical isolate. b The range was determined as the peak and 1 log 2 dilution. c Percentage of MICs within the tested range.

    Techniques: