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Agilent technologies polymerase chain reaction genomic dna
Polymerase Chain Reaction Genomic Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Ability to Generate Patient-Derived Breast Cancer Xenografts Is Enhanced in Chemoresistant Disease and Predicts Poor Patient Outcomes
Article Snippet: .. Polymerase chain reaction Genomic DNA was amplified using Herculase II DNA polymerase (Agilent). ..

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Article Title: Ability to Generate Patient-Derived Breast Cancer Xenografts Is Enhanced in Chemoresistant Disease and Predicts Poor Patient Outcomes
Article Snippet: .. Polymerase chain reaction Genomic DNA was amplified using Herculase II DNA polymerase (Agilent). ..

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    Agilent technologies bonobo genomic dna
    Five sequencing libraries were re-quantified in parallel to show the performance of qPCR quantification for libraries derived from different types of initial material. ( A ) Amplification plots of the standard and the libraries are drawn in black and red, respectively, with duplicates being treated collectively. The <t>bonobo</t> genomic <t>DNA</t> library was measured in a 1:30 dilution to obtain a signal within the range of the standard curve. The library from pooled PCR products was measured in the working dilution (1:100) used for emulsion PCR. ( B ) qPCR amplicons were size fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining in order to estimate the mean fragment size for each library.
    Bonobo Genomic Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bonobo genomic dna/product/Agilent technologies
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    92
    Agilent technologies genomic dna
    PRKCI amplification and overexpression in <t>CCOC</t> cells and patient specimens A. Copy number alterations in human chromosome 3 of five CCOC cell lines indicate amplification at 3q26 region, containing PRKCI gene. B. Correlation between PRKCI <t>DNA</t> copy number, mRNA expression and protein expression in 12 clear cell ovarian cancer cell lines. Relative PRKCI copy number, mRNA expression level and protein expression level of 12 clear cell ovarian cancer cell lines were determined by qPCR, qRT-PCR and Western blot respectively. The correlation was determined by Pearson correlation coefficient. C. PRKCI protein expression level of 12 CCOC cell lines and 2 normal ovarian surface epithelium cell lines (IOSE-80 and IOSE-120) as determined by Western blot analysis. D. Copy number variation qPCR on 27 CCOC tumor specimen. Relative PRKCI copy number was calculated by comparing normal and clear cell cancer specimen with pooled normal reference genomic DNA. E. Microarray and qRT-PCR data of PRKCI mRNA expression level by comparing 10 CCOC tumor specimens with 10 normal ovarian surface epithelium samples. F. Immunohistochemical staining of PRKCI protein expression in an additional 40 CCOC tumor tissues. Representative pictures are shown to demonstrate PRKCI intensity of (i) 0, (ii), 1+, (iii) 2+, (iv) 3+. Original magnification x200. G. Kaplan-Meier estimates of survival of the 40 CCOC patients according to the PRKCI expression level in cancer tissue with immunohistochemical staining. Analysis was done by median cut with the p-value of log-rank test. Blue line, samples with low PRKCI (
    Genomic Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies pex14 δc δc mice genomic dna
    BDNF expression in the cerebellum and brain stem region. (A) mRNA levels of Bdnf and Nt-4 relative to those of Rpl13a in the cerebellum from wild-type (+/+) and <t>Pex14</t> <t>ΔC/ΔC</t> ( ΔC/ΔC ) BL/ICR mice at P3 were determined by real-time PCR (n = 4). (B) Sagittal sections of the cerebellum at P7 were stained with antibodies to GFAP (green) and BDNF (red). Merged views of the two different proteins and staining with Hoechst 33242 (blue) are shown. Scale bar, 20 μm. (C) In situ hybridization analysis of Bdnf mRNA in the brain stem region of wild-type (upper panels) and Pex14 ΔC/ΔC (lower panels) mice at P0.5. Arrows indicate ION. Insets are higher magnification images of the dashed-line boxed regions. Scale bar, 100 μm. (D) Relative intensity of Bdnf mRNA staining by in situ hybridization shown in C was presented (n = 3). (E) Sagittal sections of the brain stem at P3 were stained with anti-BDNF antibody (red) and Hoechst 33242 (blue). Scale bar, 20 μm. (F) Fluorescent intensity of BDNF staining on the ION shown in E was quantified (n = 3). (G) The mRNA level of Bdnf in the brain stem region was quantified by real-time PCR (n = 3). (H) Brain stem lysates were analyzed by SDS–PAGE and immunoblotting using antibodies against BDNF and α-tubulin. The BDNF band was quantified (lower panel, n = 4). ns, not significant, * P
    Pex14 δc δc Mice Genomic Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Agilent technologies nsmce2 genomic dna
    Analysis of <t>NSMCE2</t> null cell growth characteristics: ( A ) Phase contrast images of WT and NSMCE2 null cells. The latter shows a large number of vacuoles in the cytoplasm (arrow). Bar = 20 µm; ( B ) Average cell cycle duration of WT and NSMCE2 null cells over multiple generations observed by live cell imaging. Data are presented as mean ± standard error of mean (SEM), n = 3; ( C ) Cell cycle phase distribution analysis of WT and NSMCE2 null cells by <t>DNA</t> histograms shows a 10% increase NSMCE2 null cells in G 0-1 ; ( D ) G 0-1 phase depletion by colcemid treatment. WT and NSMCE2 null cells were treated with colcemid for 0–96 h. In NSMCE2 null cells, a fraction of cells remained in G 0-1 even after 96 h; ( E ) Quantification for depletion of G 0-1 cells with the time of colcemid treatment. Data are presented as mean ± SEM, n = 3; ( F ) Western blot analysis of SMC5 and SMC6 proteins in WT and NSMCE2 null cells. β-Actin was used as a loading control.
    Nsmce2 Genomic Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Five sequencing libraries were re-quantified in parallel to show the performance of qPCR quantification for libraries derived from different types of initial material. ( A ) Amplification plots of the standard and the libraries are drawn in black and red, respectively, with duplicates being treated collectively. The bonobo genomic DNA library was measured in a 1:30 dilution to obtain a signal within the range of the standard curve. The library from pooled PCR products was measured in the working dilution (1:100) used for emulsion PCR. ( B ) qPCR amplicons were size fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining in order to estimate the mean fragment size for each library.

    Journal: Nucleic Acids Research

    Article Title: From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing

    doi: 10.1093/nar/gkm1095

    Figure Lengend Snippet: Five sequencing libraries were re-quantified in parallel to show the performance of qPCR quantification for libraries derived from different types of initial material. ( A ) Amplification plots of the standard and the libraries are drawn in black and red, respectively, with duplicates being treated collectively. The bonobo genomic DNA library was measured in a 1:30 dilution to obtain a signal within the range of the standard curve. The library from pooled PCR products was measured in the working dilution (1:100) used for emulsion PCR. ( B ) qPCR amplicons were size fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining in order to estimate the mean fragment size for each library.

    Article Snippet: Except for the bonobo genomic DNA, the sequencing libraries could not be detected by gel electrophoresis on an RNA 6000 pico chip (Agilent, data not shown).

    Techniques: Sequencing, Real-time Polymerase Chain Reaction, Derivative Assay, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    PRKCI amplification and overexpression in CCOC cells and patient specimens A. Copy number alterations in human chromosome 3 of five CCOC cell lines indicate amplification at 3q26 region, containing PRKCI gene. B. Correlation between PRKCI DNA copy number, mRNA expression and protein expression in 12 clear cell ovarian cancer cell lines. Relative PRKCI copy number, mRNA expression level and protein expression level of 12 clear cell ovarian cancer cell lines were determined by qPCR, qRT-PCR and Western blot respectively. The correlation was determined by Pearson correlation coefficient. C. PRKCI protein expression level of 12 CCOC cell lines and 2 normal ovarian surface epithelium cell lines (IOSE-80 and IOSE-120) as determined by Western blot analysis. D. Copy number variation qPCR on 27 CCOC tumor specimen. Relative PRKCI copy number was calculated by comparing normal and clear cell cancer specimen with pooled normal reference genomic DNA. E. Microarray and qRT-PCR data of PRKCI mRNA expression level by comparing 10 CCOC tumor specimens with 10 normal ovarian surface epithelium samples. F. Immunohistochemical staining of PRKCI protein expression in an additional 40 CCOC tumor tissues. Representative pictures are shown to demonstrate PRKCI intensity of (i) 0, (ii), 1+, (iii) 2+, (iv) 3+. Original magnification x200. G. Kaplan-Meier estimates of survival of the 40 CCOC patients according to the PRKCI expression level in cancer tissue with immunohistochemical staining. Analysis was done by median cut with the p-value of log-rank test. Blue line, samples with low PRKCI (

    Journal: Oncotarget

    Article Title: Integrated genomic analysis of clear cell ovarian cancers identified PRKCI as a potential therapeutic target

    doi: 10.18632/oncotarget.19946

    Figure Lengend Snippet: PRKCI amplification and overexpression in CCOC cells and patient specimens A. Copy number alterations in human chromosome 3 of five CCOC cell lines indicate amplification at 3q26 region, containing PRKCI gene. B. Correlation between PRKCI DNA copy number, mRNA expression and protein expression in 12 clear cell ovarian cancer cell lines. Relative PRKCI copy number, mRNA expression level and protein expression level of 12 clear cell ovarian cancer cell lines were determined by qPCR, qRT-PCR and Western blot respectively. The correlation was determined by Pearson correlation coefficient. C. PRKCI protein expression level of 12 CCOC cell lines and 2 normal ovarian surface epithelium cell lines (IOSE-80 and IOSE-120) as determined by Western blot analysis. D. Copy number variation qPCR on 27 CCOC tumor specimen. Relative PRKCI copy number was calculated by comparing normal and clear cell cancer specimen with pooled normal reference genomic DNA. E. Microarray and qRT-PCR data of PRKCI mRNA expression level by comparing 10 CCOC tumor specimens with 10 normal ovarian surface epithelium samples. F. Immunohistochemical staining of PRKCI protein expression in an additional 40 CCOC tumor tissues. Representative pictures are shown to demonstrate PRKCI intensity of (i) 0, (ii), 1+, (iii) 2+, (iv) 3+. Original magnification x200. G. Kaplan-Meier estimates of survival of the 40 CCOC patients according to the PRKCI expression level in cancer tissue with immunohistochemical staining. Analysis was done by median cut with the p-value of log-rank test. Blue line, samples with low PRKCI (

    Article Snippet: In brief, genomic DNA from 12 human CCOC cell lines were extracted and profiled on the Agilent 105k Human Genome CGH Microarray according to manufacturer’s instructions.

    Techniques: Amplification, Over Expression, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Microarray, Immunohistochemistry, Staining

    BDNF expression in the cerebellum and brain stem region. (A) mRNA levels of Bdnf and Nt-4 relative to those of Rpl13a in the cerebellum from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice at P3 were determined by real-time PCR (n = 4). (B) Sagittal sections of the cerebellum at P7 were stained with antibodies to GFAP (green) and BDNF (red). Merged views of the two different proteins and staining with Hoechst 33242 (blue) are shown. Scale bar, 20 μm. (C) In situ hybridization analysis of Bdnf mRNA in the brain stem region of wild-type (upper panels) and Pex14 ΔC/ΔC (lower panels) mice at P0.5. Arrows indicate ION. Insets are higher magnification images of the dashed-line boxed regions. Scale bar, 100 μm. (D) Relative intensity of Bdnf mRNA staining by in situ hybridization shown in C was presented (n = 3). (E) Sagittal sections of the brain stem at P3 were stained with anti-BDNF antibody (red) and Hoechst 33242 (blue). Scale bar, 20 μm. (F) Fluorescent intensity of BDNF staining on the ION shown in E was quantified (n = 3). (G) The mRNA level of Bdnf in the brain stem region was quantified by real-time PCR (n = 3). (H) Brain stem lysates were analyzed by SDS–PAGE and immunoblotting using antibodies against BDNF and α-tubulin. The BDNF band was quantified (lower panel, n = 4). ns, not significant, * P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: BDNF expression in the cerebellum and brain stem region. (A) mRNA levels of Bdnf and Nt-4 relative to those of Rpl13a in the cerebellum from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice at P3 were determined by real-time PCR (n = 4). (B) Sagittal sections of the cerebellum at P7 were stained with antibodies to GFAP (green) and BDNF (red). Merged views of the two different proteins and staining with Hoechst 33242 (blue) are shown. Scale bar, 20 μm. (C) In situ hybridization analysis of Bdnf mRNA in the brain stem region of wild-type (upper panels) and Pex14 ΔC/ΔC (lower panels) mice at P0.5. Arrows indicate ION. Insets are higher magnification images of the dashed-line boxed regions. Scale bar, 100 μm. (D) Relative intensity of Bdnf mRNA staining by in situ hybridization shown in C was presented (n = 3). (E) Sagittal sections of the brain stem at P3 were stained with anti-BDNF antibody (red) and Hoechst 33242 (blue). Scale bar, 20 μm. (F) Fluorescent intensity of BDNF staining on the ION shown in E was quantified (n = 3). (G) The mRNA level of Bdnf in the brain stem region was quantified by real-time PCR (n = 3). (H) Brain stem lysates were analyzed by SDS–PAGE and immunoblotting using antibodies against BDNF and α-tubulin. The BDNF band was quantified (lower panel, n = 4). ns, not significant, * P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Staining, In Situ Hybridization, SDS Page

    Impaired peroxisomal biogenesis in the cortex of Pex14 ΔC/ΔC mice. Immunofluorescence labeling of the cortex in wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) mice. (A–D) The coronal sections of brains were stained with antibodies to PTS1 (green) and Pex14pC (red) (A), ADAPS (green) and Pex14pC (red) (B), PMP70 (green) and catalase (red) (C), or Pex14pN (green) and Pex14pC (red) (D). Staining with Hoechst 33242 (blue) and the merged view are also shown. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown (insets). Scale bar, 2 μm. (E) Brain lysates prepared from wild-type, heterozygous ( +/ΔC ), and homozygous Pex14 mutant mice were subjected to SDS–PAGE and immunoblotting analysis using antibodies against Pex14pN, Pex14pC, AOx, and α-tubulin. Arrowhead indicates C-terminally truncated Pex14p corresponding to Pex14p-Mut. AOx is synthesized as a 75-kD A-chain and converted to a 53-kD B-chain and a 22-kD C-chain in peroxisomes. AOx A and AOx B chains are shown in immunoblots. Dots, non-specific bands. (F–H) Total amounts of PlsEtn (F), VLCPC (G), and DHA-PLs (H) are represented relative to those in the wild-type mouse brain (n = 3). ** P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: Impaired peroxisomal biogenesis in the cortex of Pex14 ΔC/ΔC mice. Immunofluorescence labeling of the cortex in wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) mice. (A–D) The coronal sections of brains were stained with antibodies to PTS1 (green) and Pex14pC (red) (A), ADAPS (green) and Pex14pC (red) (B), PMP70 (green) and catalase (red) (C), or Pex14pN (green) and Pex14pC (red) (D). Staining with Hoechst 33242 (blue) and the merged view are also shown. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown (insets). Scale bar, 2 μm. (E) Brain lysates prepared from wild-type, heterozygous ( +/ΔC ), and homozygous Pex14 mutant mice were subjected to SDS–PAGE and immunoblotting analysis using antibodies against Pex14pN, Pex14pC, AOx, and α-tubulin. Arrowhead indicates C-terminally truncated Pex14p corresponding to Pex14p-Mut. AOx is synthesized as a 75-kD A-chain and converted to a 53-kD B-chain and a 22-kD C-chain in peroxisomes. AOx A and AOx B chains are shown in immunoblots. Dots, non-specific bands. (F–H) Total amounts of PlsEtn (F), VLCPC (G), and DHA-PLs (H) are represented relative to those in the wild-type mouse brain (n = 3). ** P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mouse Assay, Immunofluorescence, Labeling, Staining, Mutagenesis, SDS Page, Synthesized, Western Blot

    A schematic model of dysmorphogenesis of Purkinje cells in the Pex14 ΔC/ΔC mouse. In the wild-type cerebellum (left), BDNF targets to TrkB-TK+ on the surface of Purkinje cells. The cytosolic tyrosine kinase domain (TK) then undergoes autophosphorylation and activates MAPK/ERK and PI3K/AKT signaling, leading to the dendritic arborization of Purkinje cells. In the Pex14 ΔC/ΔC mouse, the BDNF level is increased around the Purkinje cells through the climbing fiber from ION neurons. On the Purkinje cells of the Pex14 ΔC/ΔC mouse (right), TrkB-T1 is up-regulated and dominant-negatively inhibits autophosphorylation of TrkB-TK+. Decrease of TrkB-TK+ phosphorylation deactivates the MAPK/ERK and PI3K/AKT signaling, resulting in the dysmorphogenesis of Purkinje cells.

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: A schematic model of dysmorphogenesis of Purkinje cells in the Pex14 ΔC/ΔC mouse. In the wild-type cerebellum (left), BDNF targets to TrkB-TK+ on the surface of Purkinje cells. The cytosolic tyrosine kinase domain (TK) then undergoes autophosphorylation and activates MAPK/ERK and PI3K/AKT signaling, leading to the dendritic arborization of Purkinje cells. In the Pex14 ΔC/ΔC mouse, the BDNF level is increased around the Purkinje cells through the climbing fiber from ION neurons. On the Purkinje cells of the Pex14 ΔC/ΔC mouse (right), TrkB-T1 is up-regulated and dominant-negatively inhibits autophosphorylation of TrkB-TK+. Decrease of TrkB-TK+ phosphorylation deactivates the MAPK/ERK and PI3K/AKT signaling, resulting in the dysmorphogenesis of Purkinje cells.

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques:

    The BDNF-TrkB signaling pathway is impaired in the cerebellum of Pex14 ΔC/ΔC BL/ICR mice at P3. (A) Cerebellum lysates from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice were analyzed by SDS–PAGE and immunoblotting with antibodies against TrkB, phosphorylated Trk (p-TrkB-TK+, Y496), PLCγ1, phosphorylated PLCγ1 (p-PLCγ1, Y783), ERK, phosphorylated ERK (p-ERK1 and 2, T202 and Y204, respectively), AKT, and phosphorylated AKT (p-AKT, S473). Dots, non-specific bands. (B–E) The amount of phosphorylated TrkB-TK+ to total TrkB-TK+ (B), phosphorylated ERK1/2 relative to total ERK1/2 (C), phosphorylated AKT to total AKT (D), and phosphorylated PLCγ1 to total PLCγ1 (E) were represented (n = 3). ns, not significant, ** P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: The BDNF-TrkB signaling pathway is impaired in the cerebellum of Pex14 ΔC/ΔC BL/ICR mice at P3. (A) Cerebellum lysates from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice were analyzed by SDS–PAGE and immunoblotting with antibodies against TrkB, phosphorylated Trk (p-TrkB-TK+, Y496), PLCγ1, phosphorylated PLCγ1 (p-PLCγ1, Y783), ERK, phosphorylated ERK (p-ERK1 and 2, T202 and Y204, respectively), AKT, and phosphorylated AKT (p-AKT, S473). Dots, non-specific bands. (B–E) The amount of phosphorylated TrkB-TK+ to total TrkB-TK+ (B), phosphorylated ERK1/2 relative to total ERK1/2 (C), phosphorylated AKT to total AKT (D), and phosphorylated PLCγ1 to total PLCγ1 (E) were represented (n = 3). ns, not significant, ** P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mouse Assay, SDS Page

    Impaired TrkB signaling in the cerebellum of Pex14 ΔC/ΔC BL/ICR mice. (A) Sagittal sections of the cerebellum of wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice at P7 were stained with anti-TrkB (green) and calbindin-D 28k (red) antibodies. TrkB-positive punctate structures that were observed at P3 ( Fig 6A ) were not detected in the wild-type mouse cerebellum at P7, suggesting that climbing fiber terminals shifted to the Purkinje cell dendritic compartment. Scale bar, 10 μm. (B) Sagittal sections of the cerebellum at P7 were stained with antibodies against vGlut2 (red) and calbindin-D 28k (white). The number of dots stained with anti-vGlut2 antibody was quantified (right panel, n = 4). Scale bar, 10 μm. (C, D) Primary cerebellar neurons from a wild-type mouse at P0.5 were cultured for five DIV and then treated with 50 ng/ml BDNF for two DIV. Levels of TrkB-TK+ (C) and TrkB-T1 (D) mRNAs were determined by real-time PCR (n = 5). (E) Relative mRNA levels of c-fos and c-jun in the cerebellum at P3 were analyzed by real-time PCR (n = 3). (F) Cerebellar lysates of wild-type and Pex14 ΔC/ΔC BL/ICR mice at P0.5, P3, P5, and P7 were analyzed as in Fig 6E . TrkB-TK+, full-length TrkB; TrkB-T1, truncated isoform of TrkB; Dot, a non-specific band. (G) Amounts of TrkB-TK+ and TrkB-T1 were normalized by α-tubulin level and presented relative to those of TrkB-TK+ in control mice at P0.5 (n = 3). (H) Cerebellum lysates from wild-type and Pex14 ΔC/ΔC BL/ICR mice at P5 and P7 were analyzed by SDS–PAGE and immunoblotting with antibodies against TrkB (lower panels) and phosphorylated Trk (p-TrkB-TK+, Y496, upper panels). (I) Total ERK1/2 (lower panels) and phosphorylated ERK1/2 (p-ERK1 and 2, T202, and Y204, respectively, upper panels) at P5 and P7 were analyzed as in Fig 7C . The amount of phosphorylated ERK1/2 relative to total ERK1/2 is represented (P5; n = 3, P7; n = 4). (J, K) Sagittal sections of the cerebellum from wild-type (upper panels) and Pex14 ΔC/ΔC (lower panels) BL/ICR mice at P3 (J) and P7 (K) were stained with anti-calbindin-D 28k antibody (green) and phalloidin-TRITC (red). Scale bar, 10 μm. Higher magnification images of the boxed regions are shown (inset). Scale bar, 5 μm. Actin-positive structures located on the dendrites (arrowheads) were decreased in Pex14 ΔC/ΔC BL/ICR mice. ns, not significant, * P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: Impaired TrkB signaling in the cerebellum of Pex14 ΔC/ΔC BL/ICR mice. (A) Sagittal sections of the cerebellum of wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice at P7 were stained with anti-TrkB (green) and calbindin-D 28k (red) antibodies. TrkB-positive punctate structures that were observed at P3 ( Fig 6A ) were not detected in the wild-type mouse cerebellum at P7, suggesting that climbing fiber terminals shifted to the Purkinje cell dendritic compartment. Scale bar, 10 μm. (B) Sagittal sections of the cerebellum at P7 were stained with antibodies against vGlut2 (red) and calbindin-D 28k (white). The number of dots stained with anti-vGlut2 antibody was quantified (right panel, n = 4). Scale bar, 10 μm. (C, D) Primary cerebellar neurons from a wild-type mouse at P0.5 were cultured for five DIV and then treated with 50 ng/ml BDNF for two DIV. Levels of TrkB-TK+ (C) and TrkB-T1 (D) mRNAs were determined by real-time PCR (n = 5). (E) Relative mRNA levels of c-fos and c-jun in the cerebellum at P3 were analyzed by real-time PCR (n = 3). (F) Cerebellar lysates of wild-type and Pex14 ΔC/ΔC BL/ICR mice at P0.5, P3, P5, and P7 were analyzed as in Fig 6E . TrkB-TK+, full-length TrkB; TrkB-T1, truncated isoform of TrkB; Dot, a non-specific band. (G) Amounts of TrkB-TK+ and TrkB-T1 were normalized by α-tubulin level and presented relative to those of TrkB-TK+ in control mice at P0.5 (n = 3). (H) Cerebellum lysates from wild-type and Pex14 ΔC/ΔC BL/ICR mice at P5 and P7 were analyzed by SDS–PAGE and immunoblotting with antibodies against TrkB (lower panels) and phosphorylated Trk (p-TrkB-TK+, Y496, upper panels). (I) Total ERK1/2 (lower panels) and phosphorylated ERK1/2 (p-ERK1 and 2, T202, and Y204, respectively, upper panels) at P5 and P7 were analyzed as in Fig 7C . The amount of phosphorylated ERK1/2 relative to total ERK1/2 is represented (P5; n = 3, P7; n = 4). (J, K) Sagittal sections of the cerebellum from wild-type (upper panels) and Pex14 ΔC/ΔC (lower panels) BL/ICR mice at P3 (J) and P7 (K) were stained with anti-calbindin-D 28k antibody (green) and phalloidin-TRITC (red). Scale bar, 10 μm. Higher magnification images of the boxed regions are shown (inset). Scale bar, 5 μm. Actin-positive structures located on the dendrites (arrowheads) were decreased in Pex14 ΔC/ΔC BL/ICR mice. ns, not significant, * P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mouse Assay, Staining, Cell Culture, Real-time Polymerase Chain Reaction, SDS Page

    Defect of cerebellar development and malformation of Purkinje cells in Pex14 ΔC/ΔC BL/ICR mice. (A) Percentage of pups surviving at postnatal days. The survival days were based on the pups of 22 wild-type (+/+) and 25 Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice. (B) Body weights of pups at postnatal days were plotted. (C) Hematoxylin and eosin staining of the sagittal sections of the cerebellum (P7). Arrowheads indicate the shallow cerebellar folia in the cerebellum of the Pex14 ΔC/ΔC BL/ICR mouse (lower panel). Scale bar, 500 μm. (D) The sagittal section of the cerebellum at P7 was stained with hematoxylin and eosin. Scale bar, 20 μm. (E) Thickness of EGL was quantified (n = 3). (F, G) Confocal microscopy images of the sagittal sections of the cerebellum at P3 labeled with an antibody to calbindin-D 28k , a Purkinje cell marker. Arrows indicate axons of wild-type Purkinje cells and arrowheads indicate swollen axons of Pex14 mutant Purkinje cells. Scale bar, 10 μm. (H) Percentage of swollen axons was quantified (+/+, n = 54; ΔC/ΔC , n = 52). (I) Confocal microscopy images of sagittal sections of the cerebellum at P7 labeled with an antibody to calbindin-D 28k are shown. Arrows indicate axons of wild-type Purkinje cells and arrowheads indicate axonal reticular structures of Pex14 mutant Purkinje cells. Scale bar, 10 μm. *** P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: Defect of cerebellar development and malformation of Purkinje cells in Pex14 ΔC/ΔC BL/ICR mice. (A) Percentage of pups surviving at postnatal days. The survival days were based on the pups of 22 wild-type (+/+) and 25 Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice. (B) Body weights of pups at postnatal days were plotted. (C) Hematoxylin and eosin staining of the sagittal sections of the cerebellum (P7). Arrowheads indicate the shallow cerebellar folia in the cerebellum of the Pex14 ΔC/ΔC BL/ICR mouse (lower panel). Scale bar, 500 μm. (D) The sagittal section of the cerebellum at P7 was stained with hematoxylin and eosin. Scale bar, 20 μm. (E) Thickness of EGL was quantified (n = 3). (F, G) Confocal microscopy images of the sagittal sections of the cerebellum at P3 labeled with an antibody to calbindin-D 28k , a Purkinje cell marker. Arrows indicate axons of wild-type Purkinje cells and arrowheads indicate swollen axons of Pex14 mutant Purkinje cells. Scale bar, 10 μm. (H) Percentage of swollen axons was quantified (+/+, n = 54; ΔC/ΔC , n = 52). (I) Confocal microscopy images of sagittal sections of the cerebellum at P7 labeled with an antibody to calbindin-D 28k are shown. Arrows indicate axons of wild-type Purkinje cells and arrowheads indicate axonal reticular structures of Pex14 mutant Purkinje cells. Scale bar, 10 μm. *** P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mouse Assay, Staining, Confocal Microscopy, Labeling, Marker, Mutagenesis

    Brain morphology of Pex14 mutant mice. Cresyl violet staining of coronal sections of the brains from wild-type (+/+, left panel) and Pex14 ΔC/ΔC ( ΔC/ΔC , right panel) mice (P0.5). Scale bar, 1 mm.

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: Brain morphology of Pex14 mutant mice. Cresyl violet staining of coronal sections of the brains from wild-type (+/+, left panel) and Pex14 ΔC/ΔC ( ΔC/ΔC , right panel) mice (P0.5). Scale bar, 1 mm.

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mutagenesis, Mouse Assay, Staining

    Targeted disruption of the mouse Pex14 gene. (A) Schematic representation of the Pex14 genome locus (top), targeting vector (pMC-KO, middle), and targeted allele of the mutated locus following the homologous recombination (bottom). Exon sequences are indicated by black bars and boxes. (B) PCR-based genotyping using tail-derived DNA of wild-type (+/+), heterozygous ( +/ΔC ), and homozygous ( ΔC/ΔC ) Pex14 mutant mice. Arrows indicate the sequences used for primers described in the Materials and Methods section. Primers P14F (F) and P14R (R) amplify a 599-bp fragment, and primers F and KN52-2 (K) amplify a 169-bp fragment specific for the recombined Pex14 gene. (C) Schematic structure of predicted Pex14p proteins in wild-type mice, Pex14 mutant mice (Pex14p-Mut), and patients with a Pex14 nonsense mutation, C553T (Pex14p-Q185X). Gray bar, Pex5p-binding domain; brown bar, transmembrane domain (TM); yellow bar, coiled-coil domain (CC); shaded area, altered amino acid sequence caused by a frameshift mutation (129–163). (D) Phenotypic appearance of Pex14 mutant mice ∼12 h after birth. Scale bar, 1 cm. (E) Postnatal body weights were determined at P0.5. The number of pups with each genotype is indicated. *** P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: Targeted disruption of the mouse Pex14 gene. (A) Schematic representation of the Pex14 genome locus (top), targeting vector (pMC-KO, middle), and targeted allele of the mutated locus following the homologous recombination (bottom). Exon sequences are indicated by black bars and boxes. (B) PCR-based genotyping using tail-derived DNA of wild-type (+/+), heterozygous ( +/ΔC ), and homozygous ( ΔC/ΔC ) Pex14 mutant mice. Arrows indicate the sequences used for primers described in the Materials and Methods section. Primers P14F (F) and P14R (R) amplify a 599-bp fragment, and primers F and KN52-2 (K) amplify a 169-bp fragment specific for the recombined Pex14 gene. (C) Schematic structure of predicted Pex14p proteins in wild-type mice, Pex14 mutant mice (Pex14p-Mut), and patients with a Pex14 nonsense mutation, C553T (Pex14p-Q185X). Gray bar, Pex5p-binding domain; brown bar, transmembrane domain (TM); yellow bar, coiled-coil domain (CC); shaded area, altered amino acid sequence caused by a frameshift mutation (129–163). (D) Phenotypic appearance of Pex14 mutant mice ∼12 h after birth. Scale bar, 1 cm. (E) Postnatal body weights were determined at P0.5. The number of pups with each genotype is indicated. *** P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Plasmid Preparation, Homologous Recombination, Polymerase Chain Reaction, Derivative Assay, Mutagenesis, Mouse Assay, Binding Assay, Sequencing

    Axonal swelling and impairment of dendritic development in Purkinje cells from Pex14 ΔC/ΔC BL/ICR mouse upon treatment with BDNF. (A) Sagittal sections of the cerebellum from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice were labeled with anti-calbindin-D 28k (left panels, green) and BDNF (middle panels, red) antibodies. Merged views of the two different proteins and staining with Hoechst 33242 (blue) are shown on the right. Scale bar, 50 μm. (B) Relative fluorescent intensity of BDNF was quantified (n = 4). (C) Primary cerebellar neurons were cultured in the absence or presence of 50 ng/ml BDNF for 14 DIV. The cells were fixed and stained with antibody against calbindin-D 28K . Reverse images of Purkinje cells are shown. Scale bar, 100 μm. Higher magnification image of the boxed region is shown in the inset. Arrows indicate swollen axons of Purkinje cell. Scale bar, 20 μm. (D, E) Statistical analyses were performed for neuronal axon length (D) and the number of collaterals per 100-μm axon (E). Data represent means ± SEM. (F) The percentage of swollen axons ( > 2 μm diameter) was quantified. (G) Enlarged view of the reverse images of Purkinje cell bodies are shown (upper and lower panels). Scale bar, 20 μm. (H) Areas of the cell body and dendrites of Purkinje cells were measured (+/+: n = 64; +/+, rBDNF: n = 42; ΔC/ΔC : n = 110; ΔC/ΔC , rBDNF: n = 109). Data represent means ± SEM. ns, not significant, * P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: Axonal swelling and impairment of dendritic development in Purkinje cells from Pex14 ΔC/ΔC BL/ICR mouse upon treatment with BDNF. (A) Sagittal sections of the cerebellum from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice were labeled with anti-calbindin-D 28k (left panels, green) and BDNF (middle panels, red) antibodies. Merged views of the two different proteins and staining with Hoechst 33242 (blue) are shown on the right. Scale bar, 50 μm. (B) Relative fluorescent intensity of BDNF was quantified (n = 4). (C) Primary cerebellar neurons were cultured in the absence or presence of 50 ng/ml BDNF for 14 DIV. The cells were fixed and stained with antibody against calbindin-D 28K . Reverse images of Purkinje cells are shown. Scale bar, 100 μm. Higher magnification image of the boxed region is shown in the inset. Arrows indicate swollen axons of Purkinje cell. Scale bar, 20 μm. (D, E) Statistical analyses were performed for neuronal axon length (D) and the number of collaterals per 100-μm axon (E). Data represent means ± SEM. (F) The percentage of swollen axons ( > 2 μm diameter) was quantified. (G) Enlarged view of the reverse images of Purkinje cell bodies are shown (upper and lower panels). Scale bar, 20 μm. (H) Areas of the cell body and dendrites of Purkinje cells were measured (+/+: n = 64; +/+, rBDNF: n = 42; ΔC/ΔC : n = 110; ΔC/ΔC , rBDNF: n = 109). Data represent means ± SEM. ns, not significant, * P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mouse Assay, Labeling, Staining, Cell Culture

    Expressions of TrkB and BDNF were not altered in the cortex of Pex14 ΔC/ΔC mice. (A) Lysates of the cortex from wild-type and Pex14 ΔC/ΔC mice at P0.5 were analyzed by SDS–PAGE and immunoblotting with antibodies against TrkB, α-tubulin, and BDNF. (B) Amounts of TrkB-TK+ and TrkB-T1 were normalized by α-tubulin level and presented relative to those of TrkB-TK+ in wild-type mice at P0.5 (n = 4). (C) Amount of BDNF was normalized by α-tubulin level and presented relative to that in wild-type mice at P0.5 (n = 4). (D, E) Lysates of the cortex from wild-type and Pex14 ΔC/ΔC mice at P0.5 were analyzed as in (A) with antibodies against ERK (D), phosphorylated ERK (p-ERK1 and 2, T202, and Y204, respectively) (D), AKT (E), and phosphorylated AKT (p-AKT, S473) (E). Amounts of phosphorylated ERK1/2 relative to total ERK1/2 (D) and phosphorylated AKT to total AKT (E) were shown on the respective lower panels (n = 4). Dot, a non-specific band. ns, not significant, by t test (B–E). Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: Expressions of TrkB and BDNF were not altered in the cortex of Pex14 ΔC/ΔC mice. (A) Lysates of the cortex from wild-type and Pex14 ΔC/ΔC mice at P0.5 were analyzed by SDS–PAGE and immunoblotting with antibodies against TrkB, α-tubulin, and BDNF. (B) Amounts of TrkB-TK+ and TrkB-T1 were normalized by α-tubulin level and presented relative to those of TrkB-TK+ in wild-type mice at P0.5 (n = 4). (C) Amount of BDNF was normalized by α-tubulin level and presented relative to that in wild-type mice at P0.5 (n = 4). (D, E) Lysates of the cortex from wild-type and Pex14 ΔC/ΔC mice at P0.5 were analyzed as in (A) with antibodies against ERK (D), phosphorylated ERK (p-ERK1 and 2, T202, and Y204, respectively) (D), AKT (E), and phosphorylated AKT (p-AKT, S473) (E). Amounts of phosphorylated ERK1/2 relative to total ERK1/2 (D) and phosphorylated AKT to total AKT (E) were shown on the respective lower panels (n = 4). Dot, a non-specific band. ns, not significant, by t test (B–E). Source data are available for this figure.

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mouse Assay, SDS Page

    TrkB-T1 is upregulated in cerebellum of Pex14 ΔC/ΔC BL/ICR mouse at P3. (A) Sagittal sections of the cerebellum from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice were labeled with anti-TrkB (green) and calbindin-D 28k (red) antibodies. Scale bar, 20 μm. Higher magnification images of the boxed regions are shown and cell boundaries were indicated as a dashed line (inset). Scale bar, 5 μm. (B) TrkB-positive dots (arrowheads) were quantified (n = 13). (C) Sagittal sections of the cerebellum at P3 were stained with antibodies against vGlut2 (red) and calbindin-D 28k (white). The number of dots stained with anti-vGlut2 antibody was quantified (lower panel, n = 4). Scale bar, 10 μm. ( D) Sagittal sections of the cerebellum at P3 were stained with antibodies against TrkB (green), vGlut2 (red), and calbindin-D 28k (blue). Scale bar, 10 μm. Higher magnification images of the boxed regions are shown (inset). Scale bar, 5 μm. Arrowheads indicate TrkB-positive punctate structures that partly coincided with or are located adjacent to vGlut2-positive punctate structures. (E) Cerebellum lysates were analyzed by SDS-PAGE and immunoblotting with antibodies against TrkB, Pex14pC, and α-tubulin (left panel). TrkB-TK+, full-length TrkB; TrkB-T1, a truncated isoform of TrkB. Amounts of TrkB-TK+ and TrkB-T1 are presented relative to those of TrkB-TK+ in the control mice (middle panel, n = 6). A schematic view of TrkB variants is shown on the right (right panel). TK, tyrosine kinase domain. (F, G) mRNA levels of TrkB-TK+ (F) and TrkB-T1 (G) were quantified by real-time PCR (n = 6). ns, not significant, ** P

    Journal: Life Science Alliance

    Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum

    doi: 10.26508/lsa.201800062

    Figure Lengend Snippet: TrkB-T1 is upregulated in cerebellum of Pex14 ΔC/ΔC BL/ICR mouse at P3. (A) Sagittal sections of the cerebellum from wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice were labeled with anti-TrkB (green) and calbindin-D 28k (red) antibodies. Scale bar, 20 μm. Higher magnification images of the boxed regions are shown and cell boundaries were indicated as a dashed line (inset). Scale bar, 5 μm. (B) TrkB-positive dots (arrowheads) were quantified (n = 13). (C) Sagittal sections of the cerebellum at P3 were stained with antibodies against vGlut2 (red) and calbindin-D 28k (white). The number of dots stained with anti-vGlut2 antibody was quantified (lower panel, n = 4). Scale bar, 10 μm. ( D) Sagittal sections of the cerebellum at P3 were stained with antibodies against TrkB (green), vGlut2 (red), and calbindin-D 28k (blue). Scale bar, 10 μm. Higher magnification images of the boxed regions are shown (inset). Scale bar, 5 μm. Arrowheads indicate TrkB-positive punctate structures that partly coincided with or are located adjacent to vGlut2-positive punctate structures. (E) Cerebellum lysates were analyzed by SDS-PAGE and immunoblotting with antibodies against TrkB, Pex14pC, and α-tubulin (left panel). TrkB-TK+, full-length TrkB; TrkB-T1, a truncated isoform of TrkB. Amounts of TrkB-TK+ and TrkB-T1 are presented relative to those of TrkB-TK+ in the control mice (middle panel, n = 6). A schematic view of TrkB variants is shown on the right (right panel). TK, tyrosine kinase domain. (F, G) mRNA levels of TrkB-TK+ (F) and TrkB-T1 (G) were quantified by real-time PCR (n = 6). ns, not significant, ** P

    Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).

    Techniques: Mouse Assay, Labeling, Staining, SDS Page, Real-time Polymerase Chain Reaction

    Analysis of NSMCE2 null cell growth characteristics: ( A ) Phase contrast images of WT and NSMCE2 null cells. The latter shows a large number of vacuoles in the cytoplasm (arrow). Bar = 20 µm; ( B ) Average cell cycle duration of WT and NSMCE2 null cells over multiple generations observed by live cell imaging. Data are presented as mean ± standard error of mean (SEM), n = 3; ( C ) Cell cycle phase distribution analysis of WT and NSMCE2 null cells by DNA histograms shows a 10% increase NSMCE2 null cells in G 0-1 ; ( D ) G 0-1 phase depletion by colcemid treatment. WT and NSMCE2 null cells were treated with colcemid for 0–96 h. In NSMCE2 null cells, a fraction of cells remained in G 0-1 even after 96 h; ( E ) Quantification for depletion of G 0-1 cells with the time of colcemid treatment. Data are presented as mean ± SEM, n = 3; ( F ) Western blot analysis of SMC5 and SMC6 proteins in WT and NSMCE2 null cells. β-Actin was used as a loading control.

    Journal: International Journal of Molecular Sciences

    Article Title: Non-SMC Element 2 (NSMCE2) of the SMC5/6 Complex Helps to Resolve Topological Stress

    doi: 10.3390/ijms17111782

    Figure Lengend Snippet: Analysis of NSMCE2 null cell growth characteristics: ( A ) Phase contrast images of WT and NSMCE2 null cells. The latter shows a large number of vacuoles in the cytoplasm (arrow). Bar = 20 µm; ( B ) Average cell cycle duration of WT and NSMCE2 null cells over multiple generations observed by live cell imaging. Data are presented as mean ± standard error of mean (SEM), n = 3; ( C ) Cell cycle phase distribution analysis of WT and NSMCE2 null cells by DNA histograms shows a 10% increase NSMCE2 null cells in G 0-1 ; ( D ) G 0-1 phase depletion by colcemid treatment. WT and NSMCE2 null cells were treated with colcemid for 0–96 h. In NSMCE2 null cells, a fraction of cells remained in G 0-1 even after 96 h; ( E ) Quantification for depletion of G 0-1 cells with the time of colcemid treatment. Data are presented as mean ± SEM, n = 3; ( F ) Western blot analysis of SMC5 and SMC6 proteins in WT and NSMCE2 null cells. β-Actin was used as a loading control.

    Article Snippet: Sequencing Analysis of the Targeting Site in NSMCE2 Genomic DNA was extracted from each single cell-derived colonies, and the region flanking the targeting site was amplified by PCR with Easy-A High-Fidelity PCR Cloning Enzyme (Agilent Technologies).

    Techniques: Live Cell Imaging, Western Blot

    Etoposide-induced DSB foci formation in the absence of NSMCE2. ( A ) WT and NSMCE2 null cells were subjected to 3 µM etoposide for 1 h, fixed at different time points post treatment (0 min: immediately after etoposide treatment) and stained for γH2AX (a marker for DNA damage, red) and SMC6 (green). SMC6 localized to sites of DNA damage in both WT and NSMCE2 null cells. Bar = 5 μm; ( B ) Quantification of the average number of γH2AX foci in each cell. Significantly more γH2AX foci were formed in NSMCE2 null cells. Data are presented as mean ± SEM, n = 3; ( C ) Quantification of the average number of γH2AX foci that overlap with a SMC6 focus. Data are presented as mean ± SEM, n = 3.

    Journal: International Journal of Molecular Sciences

    Article Title: Non-SMC Element 2 (NSMCE2) of the SMC5/6 Complex Helps to Resolve Topological Stress

    doi: 10.3390/ijms17111782

    Figure Lengend Snippet: Etoposide-induced DSB foci formation in the absence of NSMCE2. ( A ) WT and NSMCE2 null cells were subjected to 3 µM etoposide for 1 h, fixed at different time points post treatment (0 min: immediately after etoposide treatment) and stained for γH2AX (a marker for DNA damage, red) and SMC6 (green). SMC6 localized to sites of DNA damage in both WT and NSMCE2 null cells. Bar = 5 μm; ( B ) Quantification of the average number of γH2AX foci in each cell. Significantly more γH2AX foci were formed in NSMCE2 null cells. Data are presented as mean ± SEM, n = 3; ( C ) Quantification of the average number of γH2AX foci that overlap with a SMC6 focus. Data are presented as mean ± SEM, n = 3.

    Article Snippet: Sequencing Analysis of the Targeting Site in NSMCE2 Genomic DNA was extracted from each single cell-derived colonies, and the region flanking the targeting site was amplified by PCR with Easy-A High-Fidelity PCR Cloning Enzyme (Agilent Technologies).

    Techniques: Staining, Marker

    Ionizing irradiation (IR)-induced double-strand break (DSB) foci formation in the absence of NSMCE2. ( A ) WT and NSMCE2 null cells were subjected to 1 Gy of IR, fixed at different time points post IR (0 min: immediately after IR) and stained for γH2AX (a marker for DNA damage, red) and SMC6 (green). SMC6 localized to sites of DNA damage in both WT and NSMCE2 null cells. Bar = 5 μm; ( B ) Quantification of the average number of γH2AX foci in each cell. Data are presented as mean ± SEM, n = 3; ( C ) Quantification of the average number of γH2AX foci that overlap with a SMC6 focus. Data are presented as mean ± SEM, n = 3.

    Journal: International Journal of Molecular Sciences

    Article Title: Non-SMC Element 2 (NSMCE2) of the SMC5/6 Complex Helps to Resolve Topological Stress

    doi: 10.3390/ijms17111782

    Figure Lengend Snippet: Ionizing irradiation (IR)-induced double-strand break (DSB) foci formation in the absence of NSMCE2. ( A ) WT and NSMCE2 null cells were subjected to 1 Gy of IR, fixed at different time points post IR (0 min: immediately after IR) and stained for γH2AX (a marker for DNA damage, red) and SMC6 (green). SMC6 localized to sites of DNA damage in both WT and NSMCE2 null cells. Bar = 5 μm; ( B ) Quantification of the average number of γH2AX foci in each cell. Data are presented as mean ± SEM, n = 3; ( C ) Quantification of the average number of γH2AX foci that overlap with a SMC6 focus. Data are presented as mean ± SEM, n = 3.

    Article Snippet: Sequencing Analysis of the Targeting Site in NSMCE2 Genomic DNA was extracted from each single cell-derived colonies, and the region flanking the targeting site was amplified by PCR with Easy-A High-Fidelity PCR Cloning Enzyme (Agilent Technologies).

    Techniques: Irradiation, Staining, Marker