nucleosome structures  (Worthington Biochemical)


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    Name:
    Deoxyribonuclease I
    Description:
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    Catalog Number:
    ls002004
    Price:
    33
    Size:
    5 mg
    Source:
    Bovine Pancreas
    Cas Number:
    9003.98.9
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    Structured Review

    Worthington Biochemical nucleosome structures
    Remodeling of <t>dinucleosomes</t> by ISWI complexes stimulates histone methylation by SET domain proteins. (A) The SET domain of SET7 binds histones, but not <t>nucleosomes.</t> GST pulldown experiments were conducted with immobilized GST-SET7 polypeptides (residues
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
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    Images

    1) Product Images from "SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿"

    Article Title: SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00775-09

    Remodeling of dinucleosomes by ISWI complexes stimulates histone methylation by SET domain proteins. (A) The SET domain of SET7 binds histones, but not nucleosomes. GST pulldown experiments were conducted with immobilized GST-SET7 polypeptides (residues
    Figure Legend Snippet: Remodeling of dinucleosomes by ISWI complexes stimulates histone methylation by SET domain proteins. (A) The SET domain of SET7 binds histones, but not nucleosomes. GST pulldown experiments were conducted with immobilized GST-SET7 polypeptides (residues

    Techniques Used: Methylation

    Analysis of ISW2 and SWI/SNF remodeled dinucleosomes. 32 P-end-labeled dinucleosomes were remodeled by ISW2 (A and B) or SWI/SNF (C and D) as described in the legend for Fig. . Remodeling was terminated by apyrase treatment, and nucleosomes
    Figure Legend Snippet: Analysis of ISW2 and SWI/SNF remodeled dinucleosomes. 32 P-end-labeled dinucleosomes were remodeled by ISW2 (A and B) or SWI/SNF (C and D) as described in the legend for Fig. . Remodeling was terminated by apyrase treatment, and nucleosomes

    Techniques Used: Labeling

    The SET domain of trithorax binds core histones and altered nucleosomal structures but not intact nucleosomes. (A) Schematic of the domain structure of trithorax. Positions of highly conserved blocks of homology with methyltransferases, a C-terminal cysteine-rich
    Figure Legend Snippet: The SET domain of trithorax binds core histones and altered nucleosomal structures but not intact nucleosomes. (A) Schematic of the domain structure of trithorax. Positions of highly conserved blocks of homology with methyltransferases, a C-terminal cysteine-rich

    Techniques Used:

    The SET domain of ALL1 does not bind remodeled mononucleosomes. (A, top) Analysis of remodeled mononucleosomes by native PAGE. Remodeling assay mixtures (50 μl) contained 1.2 μg of nucleosomes and 2.5 ng of ISWI or 5 ng of Swi-Snf complexes
    Figure Legend Snippet: The SET domain of ALL1 does not bind remodeled mononucleosomes. (A, top) Analysis of remodeled mononucleosomes by native PAGE. Remodeling assay mixtures (50 μl) contained 1.2 μg of nucleosomes and 2.5 ng of ISWI or 5 ng of Swi-Snf complexes

    Techniques Used: Clear Native PAGE

    The SET domain of ALL1 binds dinucleosomes remodeled by the ISWI class of chromatin remodeling enzymes. (A) Example of dinucleosome assembly. Dinucleosomes were reconstituted onto DNA containing two 601 minimal nucleosome positioning sequences (the orientation
    Figure Legend Snippet: The SET domain of ALL1 binds dinucleosomes remodeled by the ISWI class of chromatin remodeling enzymes. (A) Example of dinucleosome assembly. Dinucleosomes were reconstituted onto DNA containing two 601 minimal nucleosome positioning sequences (the orientation

    Techniques Used:

    2) Product Images from "Kinetics of rate-dependent shortening of action potential duration in guinea-pig ventricle; effects of IK1 and IKr blockade"

    Article Title: Kinetics of rate-dependent shortening of action potential duration in guinea-pig ventricle; effects of IK1 and IKr blockade

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0702443

    The time course of APD 90 shortening following a cycle length change from 450 ms to 300 ms in control for a Langendorff-perfused ventricular preparation, showing the two types of exponential fit. Zero on the time axis is the time at which the cycle length was changed. In the main plot, the data was fitted with a mono-exponential (solid line) and, in the inset plot, the same data was fitted with a bi-exponential. The first 50 s of shortening are clearly better fitted with a bi-exponential. Of nine experiments, all could be fit with a mono-exponential, although in five experiments, the first 30–50 s of data were better fit with a bi-exponential.
    Figure Legend Snippet: The time course of APD 90 shortening following a cycle length change from 450 ms to 300 ms in control for a Langendorff-perfused ventricular preparation, showing the two types of exponential fit. Zero on the time axis is the time at which the cycle length was changed. In the main plot, the data was fitted with a mono-exponential (solid line) and, in the inset plot, the same data was fitted with a bi-exponential. The first 50 s of shortening are clearly better fitted with a bi-exponential. Of nine experiments, all could be fit with a mono-exponential, although in five experiments, the first 30–50 s of data were better fit with a bi-exponential.

    Techniques Used: Mass Spectrometry

    3) Product Images from "Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1"

    Article Title: Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1

    Journal: Genes & Development

    doi: 10.1101/gad.310995.117

    Nucleosomes are excluded from a > 1-kb region surrounding a mismatch. ( A ) The DNA substrate used in this study. The 3011-base-pair (bp) DNA carries an A:T base pair (pMM1 homo ) or an A:C mispair (pMM1 AC ) at position 1. Positions of restriction enzyme sites used in this study, the site of biotin modification, and amplicons for quantitative PCR (qPCR) (P1: 2950–61, P2: 253–383, P3: 476–602, P4: 728–860, P5: 1498–1628, P6: 2266–2397, and P7: 2413–2537) are indicated. ( B ) Supercoiling assay in NPE. Covalently closed pMM1 homo (lanes 2 – 8 ) or pMM1 AC (lanes 9 – 15 ) was incubated in NPE and sampled at the indicated times. (Lane 1 ) Supercoiled pMM1 homo purified from Escherichia coli was used as a size standard. (oc/r) Open circular or relaxed DNA; (sc) supercoiled DNA. ( C ) pMM1 homo (lanes 1 – 4 ) or pMM1 AC (lanes 5 – 8 ) was incubated in NPE for 60 min and digested by micrococcal nuclease (MNase). DNA samples stained with SYBR Gold ( top ) and Southern blotting with the PvuII–PvuII probe ( middle ) and the DraI–DraI probe ( bottom ) are shown. ( D – F ) The MNase assay described in C was repeated in the presence of a control plasmid (pControl), and undigested DNA was quantified by qPCR. The amount of DNA relative to the input ( D ) and normalized to pControl ( E ) and pMM1 homo ( F ) is presented. Mean ± one standard deviation (SD) is shown. n = 3.
    Figure Legend Snippet: Nucleosomes are excluded from a > 1-kb region surrounding a mismatch. ( A ) The DNA substrate used in this study. The 3011-base-pair (bp) DNA carries an A:T base pair (pMM1 homo ) or an A:C mispair (pMM1 AC ) at position 1. Positions of restriction enzyme sites used in this study, the site of biotin modification, and amplicons for quantitative PCR (qPCR) (P1: 2950–61, P2: 253–383, P3: 476–602, P4: 728–860, P5: 1498–1628, P6: 2266–2397, and P7: 2413–2537) are indicated. ( B ) Supercoiling assay in NPE. Covalently closed pMM1 homo (lanes 2 – 8 ) or pMM1 AC (lanes 9 – 15 ) was incubated in NPE and sampled at the indicated times. (Lane 1 ) Supercoiled pMM1 homo purified from Escherichia coli was used as a size standard. (oc/r) Open circular or relaxed DNA; (sc) supercoiled DNA. ( C ) pMM1 homo (lanes 1 – 4 ) or pMM1 AC (lanes 5 – 8 ) was incubated in NPE for 60 min and digested by micrococcal nuclease (MNase). DNA samples stained with SYBR Gold ( top ) and Southern blotting with the PvuII–PvuII probe ( middle ) and the DraI–DraI probe ( bottom ) are shown. ( D – F ) The MNase assay described in C was repeated in the presence of a control plasmid (pControl), and undigested DNA was quantified by qPCR. The amount of DNA relative to the input ( D ) and normalized to pControl ( E ) and pMM1 homo ( F ) is presented. Mean ± one standard deviation (SD) is shown. n = 3.

    Techniques Used: Modification, Real-time Polymerase Chain Reaction, Incubation, Purification, Staining, Southern Blot, Plasmid Preparation, Standard Deviation

    4) Product Images from "Innate lymphotoxin receptor mediated signaling promotes HSV-1 associated neuroinflammation and viral replication"

    Article Title: Innate lymphotoxin receptor mediated signaling promotes HSV-1 associated neuroinflammation and viral replication

    Journal: Scientific Reports

    doi: 10.1038/srep10406

    Blockade of LT/LIGHT signaling inhibits viral replication in nervous tissue. ( a ) Rag1 –/– mice (n = 4 to 7/group) were infected with 2 × 10 6 pfu of HSV-1 and treated with either LTβR-Ig or control protein on day -1 and day 5 p.i.. At the indicated time points of figures, mice were euthanized. Footpad, DRG (L3, L4 and L5) and spinal cord were collected. Viral loads in different tissue homogenates were determined by plaque assay. ( b ) Rag1 –/– mice (n = 4 /group) were infected with 2 × 10 6 pfu of HSV-1 via footpad injection. For group with sciatic nerve (SN) resection, one segment of the SN was removed on day 2 (d2) p.i., For the group with SN resection and blockade of LT/LIGHT, 100 ug/mice of LTβR-Ig was administrated on day 2 p.i. (after sciatic nerve resection) and day 8 p.i. Data are representative of two independent experiments. Statistical analysis for a. unpaired t test, Error bar represents SEM, *p
    Figure Legend Snippet: Blockade of LT/LIGHT signaling inhibits viral replication in nervous tissue. ( a ) Rag1 –/– mice (n = 4 to 7/group) were infected with 2 × 10 6 pfu of HSV-1 and treated with either LTβR-Ig or control protein on day -1 and day 5 p.i.. At the indicated time points of figures, mice were euthanized. Footpad, DRG (L3, L4 and L5) and spinal cord were collected. Viral loads in different tissue homogenates were determined by plaque assay. ( b ) Rag1 –/– mice (n = 4 /group) were infected with 2 × 10 6 pfu of HSV-1 via footpad injection. For group with sciatic nerve (SN) resection, one segment of the SN was removed on day 2 (d2) p.i., For the group with SN resection and blockade of LT/LIGHT, 100 ug/mice of LTβR-Ig was administrated on day 2 p.i. (after sciatic nerve resection) and day 8 p.i. Data are representative of two independent experiments. Statistical analysis for a. unpaired t test, Error bar represents SEM, *p

    Techniques Used: Mouse Assay, Infection, Plaque Assay, Injection

    Blockade of LT/LIGHT inhibits chemokine expression and inflammatory cell infiltration into the DRG of infected Rag1 –/– mice. Rag1 –/– mice (n = 3 to 5/group) were infected with HSV-1 and treated with LTβR-Ig as Fig. 1a . Uninfected mice were chosen as the control group. On day 6 p.i., DRGs (L3, L4 and L5) were collected from euthanized mice. The mRNA level of various chemokines (CCL2, CXCL10 and CXCL1/2) ( a ) and viral ICP0 gene ( b ) were measured by real-time PCR. Data are representative of two independent experiments. ( c ) On day 8 p.i., innate immune cell subsets in DRG were determined by flow cytometry assay. Gate strategy: monocytes (CD45 + CD11b + Ly6C hi Ly6G middle ), macrophage (CD45 + F4/80 + ). Data are pooled from two independent experiments, n = 5 to 6/group. Statistical analysis for a , b , c was by unpaired t test. Error bar represents SEM, *p
    Figure Legend Snippet: Blockade of LT/LIGHT inhibits chemokine expression and inflammatory cell infiltration into the DRG of infected Rag1 –/– mice. Rag1 –/– mice (n = 3 to 5/group) were infected with HSV-1 and treated with LTβR-Ig as Fig. 1a . Uninfected mice were chosen as the control group. On day 6 p.i., DRGs (L3, L4 and L5) were collected from euthanized mice. The mRNA level of various chemokines (CCL2, CXCL10 and CXCL1/2) ( a ) and viral ICP0 gene ( b ) were measured by real-time PCR. Data are representative of two independent experiments. ( c ) On day 8 p.i., innate immune cell subsets in DRG were determined by flow cytometry assay. Gate strategy: monocytes (CD45 + CD11b + Ly6C hi Ly6G middle ), macrophage (CD45 + F4/80 + ). Data are pooled from two independent experiments, n = 5 to 6/group. Statistical analysis for a , b , c was by unpaired t test. Error bar represents SEM, *p

    Techniques Used: Expressing, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    5) Product Images from "Conditional Dicer1 depletion using Chrnb4-Cre leads to cone cell death and impaired photopic vision"

    Article Title: Conditional Dicer1 depletion using Chrnb4-Cre leads to cone cell death and impaired photopic vision

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-38294-9

    Chrnb4-GFP and Chrnb4-cre expression in developing and adult cone photoreceptor cells. (A) Immunostaining of adult (6 week old) Chrnb4-GFP retinas. Chrnb4-GFP expression co-labels with the expression of cone marker CA in the ONL. (B–D) Immunostaining of P8 Chrnb4-GFP retinas. Chrnb4-GFP expression co-labels with the expression of cone markers RxRγ (B) and CA (C) but not with the expression of rod marker RHO (D) . See Supplementary Figs 3 and 4 for the separate channels of the data shown in the merged images in A B. (E , F) Immunostaining on E17 Chrnb4-cre; R26 YFP retinas using an anti-GFP antibody to amplify the YFP signal intensity. YFP expression is detected in some RxRγ +ve cones (white arrows), while most cones remain YFP -ve ( E , yellow arrows). YFP expression is also seen in some OTX2-expressing photoreceptor progenitors ( F , white arrows). Yellow arrows indicate OTX2 +ve cells that are not YFP +ve . OTX2 +ve RPE cells do not express YFP (blue arrows). (G – I) Immunostaining on adult (6 weeks old) Chrnb4-cre; R26 YFP retinas. YFP expression was detected in CA +ve cones ( G , white arrows) and in a few inner retinal cells including some PAX6 +ve cells ( H , white arrowheads). Most Pax6 +ve cells in the inner retina remained YFP -ve ( H , yellow arrowheads). CHX10 +ve bipolar cells were mainly YFP -ve ( I , asterisks). (J) Immunostaining on E12 Chrnb4-cre; R26 YFP retinas was also performed. YFP expression was detected in a subset of CHX10 +ve RPCs (white arrows). CA: cone arrestin. RxRγ: retinoid x receptor gamma. RHO: rhodopsin. RPE: retinal pigment epithelium. ONL: outer nuclear layer. INL: inner nuclear layer. GCL: ganglion cell layer. RPC: retinal progenitor cells. Scale bars: 30 μm. Scale bars of insets: 10 μm.
    Figure Legend Snippet: Chrnb4-GFP and Chrnb4-cre expression in developing and adult cone photoreceptor cells. (A) Immunostaining of adult (6 week old) Chrnb4-GFP retinas. Chrnb4-GFP expression co-labels with the expression of cone marker CA in the ONL. (B–D) Immunostaining of P8 Chrnb4-GFP retinas. Chrnb4-GFP expression co-labels with the expression of cone markers RxRγ (B) and CA (C) but not with the expression of rod marker RHO (D) . See Supplementary Figs 3 and 4 for the separate channels of the data shown in the merged images in A B. (E , F) Immunostaining on E17 Chrnb4-cre; R26 YFP retinas using an anti-GFP antibody to amplify the YFP signal intensity. YFP expression is detected in some RxRγ +ve cones (white arrows), while most cones remain YFP -ve ( E , yellow arrows). YFP expression is also seen in some OTX2-expressing photoreceptor progenitors ( F , white arrows). Yellow arrows indicate OTX2 +ve cells that are not YFP +ve . OTX2 +ve RPE cells do not express YFP (blue arrows). (G – I) Immunostaining on adult (6 weeks old) Chrnb4-cre; R26 YFP retinas. YFP expression was detected in CA +ve cones ( G , white arrows) and in a few inner retinal cells including some PAX6 +ve cells ( H , white arrowheads). Most Pax6 +ve cells in the inner retina remained YFP -ve ( H , yellow arrowheads). CHX10 +ve bipolar cells were mainly YFP -ve ( I , asterisks). (J) Immunostaining on E12 Chrnb4-cre; R26 YFP retinas was also performed. YFP expression was detected in a subset of CHX10 +ve RPCs (white arrows). CA: cone arrestin. RxRγ: retinoid x receptor gamma. RHO: rhodopsin. RPE: retinal pigment epithelium. ONL: outer nuclear layer. INL: inner nuclear layer. GCL: ganglion cell layer. RPC: retinal progenitor cells. Scale bars: 30 μm. Scale bars of insets: 10 μm.

    Techniques Used: Expressing, Immunostaining, Marker

    6) Product Images from "Long disordered regions of the C-terminal domain of Abelson tyrosine kinase have specific and additive functions in regulation and axon localization"

    Article Title: Long disordered regions of the C-terminal domain of Abelson tyrosine kinase have specific and additive functions in regulation and axon localization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189338

    Protease susceptibility assay reveals disorder in the Abl CTD. (A) Predicted intrinsic disorder (IUPred) and protein binding regions (ANCHOR) within Abl. (B) Predicted clostripain (endoproteinase Arg-C) digestion sites within GST-1Q, GST-3Q and full-length Abl for protease susceptibility assay. Sites are well distributed over both the globular domains and predicted disordered regions, and sites within disordered regions should be preferentially utilized. Clostripain digest of purified GST-1Q (C) and GST-3Q (D) with 6 lanes of increasing doses, and two final lanes with 10x and 100x protease as indicated. All FLAG-tagged fragments are more than ~25kD smaller than the full-length protein, and no GST-tagged fragments are smaller than the ~25kD GST domain (▻) except at the 100x protease concentration for GST-3Q. Both indicate that protease sites within the CTD fragments are preferentially utilized by clostripain, compared to the GST domain. (E) Clostripain digest of full-length transgenic Abl in total cell lysates of 3 rd instar CNS. FLAG-tagged fragments are present at the size of the full-length CTD (~108 kD) or smaller, consistent with preferential digestion within the CTD. Fragments labeled with a polyclonal antibody against the Abl N-terminus show either fragments at or above the size of full-length AblN (~69 kD, ▻) or at the size of the SH3-SH2-domain (~49 kD, ►). In the last lane, AblN (N) alone was also expressed in 3 rd instar CNS, but the lysate was not subject to digestion.
    Figure Legend Snippet: Protease susceptibility assay reveals disorder in the Abl CTD. (A) Predicted intrinsic disorder (IUPred) and protein binding regions (ANCHOR) within Abl. (B) Predicted clostripain (endoproteinase Arg-C) digestion sites within GST-1Q, GST-3Q and full-length Abl for protease susceptibility assay. Sites are well distributed over both the globular domains and predicted disordered regions, and sites within disordered regions should be preferentially utilized. Clostripain digest of purified GST-1Q (C) and GST-3Q (D) with 6 lanes of increasing doses, and two final lanes with 10x and 100x protease as indicated. All FLAG-tagged fragments are more than ~25kD smaller than the full-length protein, and no GST-tagged fragments are smaller than the ~25kD GST domain (▻) except at the 100x protease concentration for GST-3Q. Both indicate that protease sites within the CTD fragments are preferentially utilized by clostripain, compared to the GST domain. (E) Clostripain digest of full-length transgenic Abl in total cell lysates of 3 rd instar CNS. FLAG-tagged fragments are present at the size of the full-length CTD (~108 kD) or smaller, consistent with preferential digestion within the CTD. Fragments labeled with a polyclonal antibody against the Abl N-terminus show either fragments at or above the size of full-length AblN (~69 kD, ▻) or at the size of the SH3-SH2-domain (~49 kD, ►). In the last lane, AblN (N) alone was also expressed in 3 rd instar CNS, but the lysate was not subject to digestion.

    Techniques Used: Drug Susceptibility Assay, Protein Binding, Purification, Concentration Assay, Transgenic Assay, Labeling

    7) Product Images from "Transcription regulation of the type II restriction-modification system AhdI"

    Article Title: Transcription regulation of the type II restriction-modification system AhdI

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1116

    DNase I footprinting of C.AhdI complexes on wild-type and mutant ahdICR promoters. 32 P-end labelled (bottom strand) ahdICR promoter-containing fragments (22.5 nM) were combined with increasing concentrations (0–188 nM) of C.AhdI and treated with DNase I. Two sets of inverted repeats are indicated at the left of each panel.
    Figure Legend Snippet: DNase I footprinting of C.AhdI complexes on wild-type and mutant ahdICR promoters. 32 P-end labelled (bottom strand) ahdICR promoter-containing fragments (22.5 nM) were combined with increasing concentrations (0–188 nM) of C.AhdI and treated with DNase I. Two sets of inverted repeats are indicated at the left of each panel.

    Techniques Used: Footprinting, Mutagenesis

    Footprinting of RNA polymerase complexes P ahdICR . The indicated proteins were combined with the wild-type P ahdICR DNA fragment, complexes were allowed to form and footprinted with DNase I ( A ) or probed with KMnO 4 ( B ). C.AhdI binding sites and the −10 element of the promoter are indicated.
    Figure Legend Snippet: Footprinting of RNA polymerase complexes P ahdICR . The indicated proteins were combined with the wild-type P ahdICR DNA fragment, complexes were allowed to form and footprinted with DNase I ( A ) or probed with KMnO 4 ( B ). C.AhdI binding sites and the −10 element of the promoter are indicated.

    Techniques Used: Footprinting, Binding Assay

    8) Product Images from "Involvement of unconventional myosin VI in myoblast function and myotube formation"

    Article Title: Involvement of unconventional myosin VI in myoblast function and myotube formation

    Journal: Histochemistry and Cell Biology

    doi: 10.1007/s00418-015-1322-6

    MVI localization to different compartments of undifferentiated myoblasts. MVI is visualized in green with anti-MVI antibody ( a – d ) or as GFP-associated fluorescence ( e – h ), and nuclei were stained with DAPI ( a – h ). a MVI is present in the regions next to cortical actin (in red , stained with Alexa Flour 536-conjugated phalloidin). b , c MVI is in the close proximity to calreticulin (in red ), an endoplasmic reticulum marker, and to GM130 (in red ), a Golgi apparatus marker, respectively. d MVI is present next to vinculin-containing (in red ) adhesive structures. Regions indicated in the merged images are shown at higher magnification in the right panel . e Overexpression of H246R MVI mutant (in green ) myoblasts with MVI knockdown; right panel , merged with DAPI staining. f Overexpression of H246R MVI mutant (in green ) in 2-day rat neonatal cardiomyocytes. In red , staining for α-actinin, the marker of Z lines. Arrows on e and f indicate vacuole-like structures. g , h , Day-0 myoblasts overexpressing the GFP-fused wild-type MVI (GFP-MVI) or H246R mutant, respectively. Golgi cisternae (in yellow ) were stained with anti-GM130 monoclonal antibody. Arrows in ( g ), Golgi cisternae in cells overexpressing GFP-MVI, and arrowhead in ( h ), Golgi cisternae in the cell overexpressing the MVI mutant. Bars 20 μm
    Figure Legend Snippet: MVI localization to different compartments of undifferentiated myoblasts. MVI is visualized in green with anti-MVI antibody ( a – d ) or as GFP-associated fluorescence ( e – h ), and nuclei were stained with DAPI ( a – h ). a MVI is present in the regions next to cortical actin (in red , stained with Alexa Flour 536-conjugated phalloidin). b , c MVI is in the close proximity to calreticulin (in red ), an endoplasmic reticulum marker, and to GM130 (in red ), a Golgi apparatus marker, respectively. d MVI is present next to vinculin-containing (in red ) adhesive structures. Regions indicated in the merged images are shown at higher magnification in the right panel . e Overexpression of H246R MVI mutant (in green ) myoblasts with MVI knockdown; right panel , merged with DAPI staining. f Overexpression of H246R MVI mutant (in green ) in 2-day rat neonatal cardiomyocytes. In red , staining for α-actinin, the marker of Z lines. Arrows on e and f indicate vacuole-like structures. g , h , Day-0 myoblasts overexpressing the GFP-fused wild-type MVI (GFP-MVI) or H246R mutant, respectively. Golgi cisternae (in yellow ) were stained with anti-GM130 monoclonal antibody. Arrows in ( g ), Golgi cisternae in cells overexpressing GFP-MVI, and arrowhead in ( h ), Golgi cisternae in the cell overexpressing the MVI mutant. Bars 20 μm

    Techniques Used: Fluorescence, Staining, Marker, Over Expression, Mutagenesis

    MVI colocalization with sarcoplasmic reticulum in neonatal rat cardiomyocytes. a and b , Localization of MVI (in green ) and SERCA2 (in red ) in day-2 and day-8 cardiomyocytes, respectively. Arrows point to the nuclear MVI presence. The far right panels , magnification (as marked in the figure) of the regions indicated in the merged panels . Nuclei were stained with TO-PRO ® -3 (in blue ). Bars 10 μm
    Figure Legend Snippet: MVI colocalization with sarcoplasmic reticulum in neonatal rat cardiomyocytes. a and b , Localization of MVI (in green ) and SERCA2 (in red ) in day-2 and day-8 cardiomyocytes, respectively. Arrows point to the nuclear MVI presence. The far right panels , magnification (as marked in the figure) of the regions indicated in the merged panels . Nuclei were stained with TO-PRO ® -3 (in blue ). Bars 10 μm

    Techniques Used: Staining

    9) Product Images from "All-in-One Bacmids: an Efficient Reverse Genetics Strategy for Influenza A Virus Vaccines"

    Article Title: All-in-One Bacmids: an Efficient Reverse Genetics Strategy for Influenza A Virus Vaccines

    Journal: Journal of Virology

    doi: 10.1128/JVI.01468-14

    Transfection-based inoculation of all-in-one bacmids leads to lethal PR8 virus replication in DBA mice. Five-week-old DBA/2J mice ( n = 5 [A to D] or n = 15 [E to G]) were intranasally inoculated with 100 μl of inoculum containing 5 × 10 6 293T cells transfected 6 h earlier with bcmd-P8PR8 (H1N1 PR8 ) or the corresponding 8-plasmid RG DNAs. Negative control mice received 5 × 10 6 mock-transfected 293T cells or cells transfected with either bcmd-P6PR8 without HA and NA cassettes or bcmd-DH10 empty DNA. Positive control mice were intranasally inoculated with wt PR8 virus (10 4 TCID 50 /100 μl). Mice were monitored for clinical signs, including body weight changes (A and E) and survival (B and F). At the time of death (C) or at 5 dpi (G), mice were necropsied or euthanized and lungs and brain were collected and analyzed for levels of virus in tissues by a TCID 50 assay in MDCK cells. *, P
    Figure Legend Snippet: Transfection-based inoculation of all-in-one bacmids leads to lethal PR8 virus replication in DBA mice. Five-week-old DBA/2J mice ( n = 5 [A to D] or n = 15 [E to G]) were intranasally inoculated with 100 μl of inoculum containing 5 × 10 6 293T cells transfected 6 h earlier with bcmd-P8PR8 (H1N1 PR8 ) or the corresponding 8-plasmid RG DNAs. Negative control mice received 5 × 10 6 mock-transfected 293T cells or cells transfected with either bcmd-P6PR8 without HA and NA cassettes or bcmd-DH10 empty DNA. Positive control mice were intranasally inoculated with wt PR8 virus (10 4 TCID 50 /100 μl). Mice were monitored for clinical signs, including body weight changes (A and E) and survival (B and F). At the time of death (C) or at 5 dpi (G), mice were necropsied or euthanized and lungs and brain were collected and analyzed for levels of virus in tissues by a TCID 50 assay in MDCK cells. *, P

    Techniques Used: Transfection, Mouse Assay, Plasmid Preparation, Negative Control, Positive Control

    Transfection-based inoculation of all-in-one bacmids leads to lethal PR8 virus replication in BALB/c mice. Five-week-old BALB/c mice ( n = 15) were intranasally inoculated with 100 μl of inoculum containing 5 × 10 6 293T cells, which had been previously transfected 6 h earlier with either 25 μg of bcmd-P8PR8 (H1N1 PR8 ) or 40 μg of the corresponding 8-plasmid RG set. Negative control mice received 5 × 10 6 mock-transfected 293T cells, or transfected bcmd-P6P without HA and NA cassettes, or bcmd-DH10. Positive control mice were intranasally inoculated with wt PR8 virus (10 4 TCID 50 /100 μl). Mice were monitored for clinical signs, including body weight changes (A) and survival (B). (C) Five mice were necropsied at 5 dpi, and lungs were collected and analyzed for virus levels by titration on MDCK cells. *, P
    Figure Legend Snippet: Transfection-based inoculation of all-in-one bacmids leads to lethal PR8 virus replication in BALB/c mice. Five-week-old BALB/c mice ( n = 15) were intranasally inoculated with 100 μl of inoculum containing 5 × 10 6 293T cells, which had been previously transfected 6 h earlier with either 25 μg of bcmd-P8PR8 (H1N1 PR8 ) or 40 μg of the corresponding 8-plasmid RG set. Negative control mice received 5 × 10 6 mock-transfected 293T cells, or transfected bcmd-P6P without HA and NA cassettes, or bcmd-DH10. Positive control mice were intranasally inoculated with wt PR8 virus (10 4 TCID 50 /100 μl). Mice were monitored for clinical signs, including body weight changes (A) and survival (B). (C) Five mice were necropsied at 5 dpi, and lungs were collected and analyzed for virus levels by titration on MDCK cells. *, P

    Techniques Used: Transfection, Mouse Assay, Plasmid Preparation, Negative Control, Positive Control, Titration

    10) Product Images from "Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1"

    Article Title: Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1

    Journal: Genes & Development

    doi: 10.1101/gad.310995.117

    Nucleosomes are excluded from a > 1-kb region surrounding a mismatch. ( A ) The DNA substrate used in this study. The 3011-base-pair (bp) DNA carries an A:T base pair (pMM1 homo ) or an A:C mispair (pMM1 AC ) at position 1. Positions of restriction enzyme sites used in this study, the site of biotin modification, and amplicons for quantitative PCR (qPCR) (P1: 2950–61, P2: 253–383, P3: 476–602, P4: 728–860, P5: 1498–1628, P6: 2266–2397, and P7: 2413–2537) are indicated. ( B ) Supercoiling assay in NPE. Covalently closed pMM1 homo (lanes 2 – 8 ) or pMM1 AC (lanes 9 – 15 ) was incubated in NPE and sampled at the indicated times. (Lane 1 ) Supercoiled pMM1 homo purified from Escherichia coli was used as a size standard. (oc/r) Open circular or relaxed DNA; (sc) supercoiled DNA. ( C ) pMM1 homo (lanes 1 – 4 ) or pMM1 AC (lanes 5 – 8 ) was incubated in NPE for 60 min and digested by micrococcal nuclease (MNase). DNA samples stained with SYBR Gold ( top ) and Southern blotting with the PvuII–PvuII probe ( middle ) and the DraI–DraI probe ( bottom ) are shown. ( D – F ) The MNase assay described in C was repeated in the presence of a control plasmid (pControl), and undigested DNA was quantified by qPCR. The amount of DNA relative to the input ( D ) and normalized to pControl ( E ) and pMM1 homo ( F ) is presented. Mean ± one standard deviation (SD) is shown. n = 3.
    Figure Legend Snippet: Nucleosomes are excluded from a > 1-kb region surrounding a mismatch. ( A ) The DNA substrate used in this study. The 3011-base-pair (bp) DNA carries an A:T base pair (pMM1 homo ) or an A:C mispair (pMM1 AC ) at position 1. Positions of restriction enzyme sites used in this study, the site of biotin modification, and amplicons for quantitative PCR (qPCR) (P1: 2950–61, P2: 253–383, P3: 476–602, P4: 728–860, P5: 1498–1628, P6: 2266–2397, and P7: 2413–2537) are indicated. ( B ) Supercoiling assay in NPE. Covalently closed pMM1 homo (lanes 2 – 8 ) or pMM1 AC (lanes 9 – 15 ) was incubated in NPE and sampled at the indicated times. (Lane 1 ) Supercoiled pMM1 homo purified from Escherichia coli was used as a size standard. (oc/r) Open circular or relaxed DNA; (sc) supercoiled DNA. ( C ) pMM1 homo (lanes 1 – 4 ) or pMM1 AC (lanes 5 – 8 ) was incubated in NPE for 60 min and digested by micrococcal nuclease (MNase). DNA samples stained with SYBR Gold ( top ) and Southern blotting with the PvuII–PvuII probe ( middle ) and the DraI–DraI probe ( bottom ) are shown. ( D – F ) The MNase assay described in C was repeated in the presence of a control plasmid (pControl), and undigested DNA was quantified by qPCR. The amount of DNA relative to the input ( D ) and normalized to pControl ( E ) and pMM1 homo ( F ) is presented. Mean ± one standard deviation (SD) is shown. n = 3.

    Techniques Used: Modification, Real-time Polymerase Chain Reaction, Incubation, Purification, Staining, Southern Blot, Plasmid Preparation, Standard Deviation

    11) Product Images from "Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells"

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085740

    Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the  CD4  gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.
    Figure Legend Snippet: Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the CD4 gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.

    Techniques Used: Polymerase Chain Reaction, Magnetic Beads, Amplification, Real-time Polymerase Chain Reaction, Isolation

    Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.
    Figure Legend Snippet: Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Molecular Weight, Derivative Assay, Amplification

    Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.
    Figure Legend Snippet: Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Isolation, Magnetic Beads, Amplification

    DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.
    Figure Legend Snippet: DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Purification, FACS

    12) Product Images from "Maturation and electrophysiological properties of human pluripotent stem cell‐derived oligodendrocytes"

    Article Title: Maturation and electrophysiological properties of human pluripotent stem cell‐derived oligodendrocytes

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2273

    Derivation and specification of oligodendrocytes from human pluripotent stem cell (hPSC)‐derived oligodendrocyte precursor cells (OPCs). (A) : Summary of the protocol used to generate hPSC‐derived oligodendrocytes: (1) hPSCs were neuralized via dual‐SMAD inhibition. (2) NPCs were patterned to ventral spinal cord by exposure to retinoic acid and sonic hedgehog agonists, purmorphamine, and SAG. (3) spinal cord‐patterned NPCs were converted to OPCs by exposure to PDGFα and other mitogens. (4) OPCs could be further expanded by mechanical dissociation. (5) Oligodendrocyte differentiation was induced by mitogen withdrawal. (B) : Representative staining of cells 1 week post‐mitogen removal identifies OLIG2 + ‐progenitors, and cells expressing PDGFRα, O4, and MBP in all lines examined. Scale bar = 50 μm. (C) : Percentage of PDGFRα + ‐cells (ES/iPS1/iPS2: N = 4/4/3) and (D) O4 + ‐cells (ES/iPS1/iPS2: N = 4/13/13) in week 1 cultures. (E) : Percentage O4 + ‐cells that express PDGFRα(ES/iPS1/iPS2: N = 4/4/3) and MBP (ES/iPS1/iPS2: N = 4/5/6). Equivalent cellular specification was observed across all lines. (F) : Example images illustrating no overlap of PDGFRα + ‐ and O4 + ‐cells (upper panels) but substantial overlap of MBP + ‐ and O4 + ‐cells (lower panels). Scale bar = 12 μm. (G) : Sholl analysis performed upon week 1 and 3 O4 + ‐oligodendrocytes. Abbreviations: DAPI, 4',6‐diamidino‐2‐phenylindole; ES, embryonic stem cell; FGF, fibroblast growth factor; hPSC, human pluripotent stem cell; IGF, Insulin‐like growth factor; iPS, induced pluripotent stem cell; MBP, myelin basic protein; NPC, neural precursor cell; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; PDGF, platelet‐derived growth factor; RA, retinoic acid; SAG, smoothened agonist; scNPC, spinal cord‐patterned neural precursor.
    Figure Legend Snippet: Derivation and specification of oligodendrocytes from human pluripotent stem cell (hPSC)‐derived oligodendrocyte precursor cells (OPCs). (A) : Summary of the protocol used to generate hPSC‐derived oligodendrocytes: (1) hPSCs were neuralized via dual‐SMAD inhibition. (2) NPCs were patterned to ventral spinal cord by exposure to retinoic acid and sonic hedgehog agonists, purmorphamine, and SAG. (3) spinal cord‐patterned NPCs were converted to OPCs by exposure to PDGFα and other mitogens. (4) OPCs could be further expanded by mechanical dissociation. (5) Oligodendrocyte differentiation was induced by mitogen withdrawal. (B) : Representative staining of cells 1 week post‐mitogen removal identifies OLIG2 + ‐progenitors, and cells expressing PDGFRα, O4, and MBP in all lines examined. Scale bar = 50 μm. (C) : Percentage of PDGFRα + ‐cells (ES/iPS1/iPS2: N = 4/4/3) and (D) O4 + ‐cells (ES/iPS1/iPS2: N = 4/13/13) in week 1 cultures. (E) : Percentage O4 + ‐cells that express PDGFRα(ES/iPS1/iPS2: N = 4/4/3) and MBP (ES/iPS1/iPS2: N = 4/5/6). Equivalent cellular specification was observed across all lines. (F) : Example images illustrating no overlap of PDGFRα + ‐ and O4 + ‐cells (upper panels) but substantial overlap of MBP + ‐ and O4 + ‐cells (lower panels). Scale bar = 12 μm. (G) : Sholl analysis performed upon week 1 and 3 O4 + ‐oligodendrocytes. Abbreviations: DAPI, 4',6‐diamidino‐2‐phenylindole; ES, embryonic stem cell; FGF, fibroblast growth factor; hPSC, human pluripotent stem cell; IGF, Insulin‐like growth factor; iPS, induced pluripotent stem cell; MBP, myelin basic protein; NPC, neural precursor cell; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; PDGF, platelet‐derived growth factor; RA, retinoic acid; SAG, smoothened agonist; scNPC, spinal cord‐patterned neural precursor.

    Techniques Used: Derivative Assay, Inhibition, Staining, Expressing

    AMPA receptors in human pluripotent stem cell (hPSC)‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes . (A) : AMPA‐mediated whole‐cell current responses in a PDGFRα + ‐OPC are strongly potentiated by cyclothiazide and blocked by CNQX. (B) : Depicts sample recordings in which responses to applications of GABA (100 μM), NMDA (100 μM, in the presence of glycine [50 μM]) were obtained from week 3 O4 + ‐oligodendrocytes. (C) : Mean current densities for AMPA ( n = 10/7/11, N = 2/1/2), NMDA ( n = 6/8, N = 2/2), and GABA responses ( n = 8/19, N = 3/3). (D) : Mean normalized mRNA fold expression data for AMPAR subunits GluA1‐GluA4 in OPC cultures ( N = 3) and week 3 O4 + ‐oligodendrocytes ( N = 13) as assessed by quantitative real‐time polymerase chain reaction. Data were normalized to GluA1 for each maturation stage after normalizing to β‐actin and GAPDH. (E) : Sample nonstationary fluctuation analysis recordings of AMPAR‐mediated currents from a PDGFRα + ‐OPC and, (F) week 3 O4 + ‐oligodendrocyte. (G) : Plot describing the linear relationship of the variance of the AC‐coupled current to the DC‐current amplitude for the former recordings in c and d . The fitted slopes for each plot gave respective unitary single‐channel current amplitude estimates of −0.45 pA and −0.1 pA, respectively, from which the unitary conductance was calculated. (H) : Mean AMPAR conductance for PDGFRα + ‐OPCs and O4 + ‐oligodendrocytes derived from control hPSC lines. (I) : Example recording of 1‐naphthyl acetyl spermine (NASPM) block of steady‐state currents evoked by AMPA in a PDGFRα + ‐OPC and, (J) week 3 O4 + ‐oligodendrocyte. (K) : Mean percentage block of AMPA currents by NASPM. *, p
    Figure Legend Snippet: AMPA receptors in human pluripotent stem cell (hPSC)‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes . (A) : AMPA‐mediated whole‐cell current responses in a PDGFRα + ‐OPC are strongly potentiated by cyclothiazide and blocked by CNQX. (B) : Depicts sample recordings in which responses to applications of GABA (100 μM), NMDA (100 μM, in the presence of glycine [50 μM]) were obtained from week 3 O4 + ‐oligodendrocytes. (C) : Mean current densities for AMPA ( n = 10/7/11, N = 2/1/2), NMDA ( n = 6/8, N = 2/2), and GABA responses ( n = 8/19, N = 3/3). (D) : Mean normalized mRNA fold expression data for AMPAR subunits GluA1‐GluA4 in OPC cultures ( N = 3) and week 3 O4 + ‐oligodendrocytes ( N = 13) as assessed by quantitative real‐time polymerase chain reaction. Data were normalized to GluA1 for each maturation stage after normalizing to β‐actin and GAPDH. (E) : Sample nonstationary fluctuation analysis recordings of AMPAR‐mediated currents from a PDGFRα + ‐OPC and, (F) week 3 O4 + ‐oligodendrocyte. (G) : Plot describing the linear relationship of the variance of the AC‐coupled current to the DC‐current amplitude for the former recordings in c and d . The fitted slopes for each plot gave respective unitary single‐channel current amplitude estimates of −0.45 pA and −0.1 pA, respectively, from which the unitary conductance was calculated. (H) : Mean AMPAR conductance for PDGFRα + ‐OPCs and O4 + ‐oligodendrocytes derived from control hPSC lines. (I) : Example recording of 1‐naphthyl acetyl spermine (NASPM) block of steady‐state currents evoked by AMPA in a PDGFRα + ‐OPC and, (J) week 3 O4 + ‐oligodendrocyte. (K) : Mean percentage block of AMPA currents by NASPM. *, p

    Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Blocking Assay

    Voltage‐gated Na + ‐channel expression in human pluripotent stem cell‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes. (A) : Current‐clamp recording demonstrating that PDGFRα + ‐OPCs exhibited tetrodotoxin (TTX)‐sensitive spikes in response to depolarization by current injection (each current step below trace represents 10 pA). (B) : To isolate and measure Na V ‐channel activity, the membrane potential was initially stepped in 20 mV increments from –84 mV to + 16 mV ( activation ). Scale bars = 500 pA, 5 milliseconds. The protocol was then repeated in the presence of TTX and the current data subtracted from that of the former to yield the TTX‐sensitive Na V ‐specific current. Not all TTX‐sensitive currents are shown for figure clarity. (C) : Normalized current–voltage plot of Na V ‐channel activity expressed by PDGFRα + ‐OPCs. Data were normalized to +16 mV current data. (D) : Decrease in Na V ‐channel expression from PDGFRα + ‐OPCs to O4 + ‐oligodendrocytes ( n = 5–18, N = 3; Mann Whitney U tests). ***, p
    Figure Legend Snippet: Voltage‐gated Na + ‐channel expression in human pluripotent stem cell‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes. (A) : Current‐clamp recording demonstrating that PDGFRα + ‐OPCs exhibited tetrodotoxin (TTX)‐sensitive spikes in response to depolarization by current injection (each current step below trace represents 10 pA). (B) : To isolate and measure Na V ‐channel activity, the membrane potential was initially stepped in 20 mV increments from –84 mV to + 16 mV ( activation ). Scale bars = 500 pA, 5 milliseconds. The protocol was then repeated in the presence of TTX and the current data subtracted from that of the former to yield the TTX‐sensitive Na V ‐specific current. Not all TTX‐sensitive currents are shown for figure clarity. (C) : Normalized current–voltage plot of Na V ‐channel activity expressed by PDGFRα + ‐OPCs. Data were normalized to +16 mV current data. (D) : Decrease in Na V ‐channel expression from PDGFRα + ‐OPCs to O4 + ‐oligodendrocytes ( n = 5–18, N = 3; Mann Whitney U tests). ***, p

    Techniques Used: Expressing, Derivative Assay, Injection, Activity Assay, Activation Assay, MANN-WHITNEY

    Membrane current properties of human pluripotent stem cell‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes. (A) : Whole‐cell current recordings from a PDGFRα + ‐OPC (blue) and week 3 O4 + ‐oligodendrocyte (orange) in response to a voltage‐step protocol that involved incremental application of 20 mV voltage steps from a holding potential of –84 mV. Live‐stained cells are shown inset. (B) : Mean normalized current–voltage plots for PDGFRα + ‐OPCs, week 1 (circles) and week 3 (triangles) O4 + ‐oligodendrocytes ( n = 9–17, N = 3–6) derived from the ES line. Current amplitudes were measured 175 milliseconds after voltage‐step initiation and normalized to –64 mV current data. (C) : Mean PDGFRα + ‐OPC (iPS1/iPS2: n = 10/5, N = 3/1) and week 3 O4 + ‐oligodendrocyte (iPS1/iPS2: n = 8/6, N = 4/2) rectification index data calculated for each line examined. (D) : Mean input resistance measurements for PDGFRα + ‐OPCs (ES/iPS1/iPS2: n = 20/21/8, N = 3/3/3) and O4 + ‐oligodendrocytes (ES/iPS1/iPS2: n = 26‐32/19/7, N = 3/3/3). (E) : Mean whole‐cell capacitance measurements for PDGFRα + ‐OPCs (ES/iPS1/iPS2: n = 22/21/12, N = 3/3/3) and O4 + ‐oligodendrocytes (ES/iPS1/iPS2: n = 32‐41/26/7, N = 3/3/3). *, p
    Figure Legend Snippet: Membrane current properties of human pluripotent stem cell‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes. (A) : Whole‐cell current recordings from a PDGFRα + ‐OPC (blue) and week 3 O4 + ‐oligodendrocyte (orange) in response to a voltage‐step protocol that involved incremental application of 20 mV voltage steps from a holding potential of –84 mV. Live‐stained cells are shown inset. (B) : Mean normalized current–voltage plots for PDGFRα + ‐OPCs, week 1 (circles) and week 3 (triangles) O4 + ‐oligodendrocytes ( n = 9–17, N = 3–6) derived from the ES line. Current amplitudes were measured 175 milliseconds after voltage‐step initiation and normalized to –64 mV current data. (C) : Mean PDGFRα + ‐OPC (iPS1/iPS2: n = 10/5, N = 3/1) and week 3 O4 + ‐oligodendrocyte (iPS1/iPS2: n = 8/6, N = 4/2) rectification index data calculated for each line examined. (D) : Mean input resistance measurements for PDGFRα + ‐OPCs (ES/iPS1/iPS2: n = 20/21/8, N = 3/3/3) and O4 + ‐oligodendrocytes (ES/iPS1/iPS2: n = 26‐32/19/7, N = 3/3/3). (E) : Mean whole‐cell capacitance measurements for PDGFRα + ‐OPCs (ES/iPS1/iPS2: n = 22/21/12, N = 3/3/3) and O4 + ‐oligodendrocytes (ES/iPS1/iPS2: n = 32‐41/26/7, N = 3/3/3). *, p

    Techniques Used: Derivative Assay, Staining

    Voltage‐gated K + ‐channel expression in human pluripotent stem cell‐derived oligodendroglia. (A) : To isolate I k ‐channel activity, 10 mV incremental voltage‐pulses were initially applied to activate I k ‐channels in the presence of tetrodotoxin (TTX) ( activation ). This was then repeated in the presence of TEA and the current data subtracted from that of the former to determine the I K ‐specific current ( subtracted ). I K ‐current amplitudes were measured 200 milliseconds after activation. The examples shown are from PDGFRα + ‐oligodendrocyte precursor cells (OPCs). (B) : Normalized current–voltage plot of I K ‐channel activity measured from PDGFRα + ‐OPCs ( n = 4) and O4 + ‐oligodendrocytes ( n = 4). Data were normalized to +46 mV current data. (C) : Decrease in I K ‐channel expression from PDGFRα + ‐OPCs to O4 + ‐oligodendrocytes ( n = 8–15, N = 3‐4). Current amplitude data were measured from the 100‐mV step. (D) : I A ‐channel activity was measured in the presence of TTX and Cd 2+ (100 μM). The holding potential was pre‐stepped to –124 mV (500 milliseconds) and, there from, the holding potential depolarized in 10 mV increments before returning to –84 mV ( activation ). Since I A ‐channels inactivate rapidly upon depolarization, an inactivation protocol pre‐stepped cells to –34 mV from –84 mV to isolate non‐ I A current ( inactivation ), which was subtracted from the former to generate the I A ‐mediated current ( subtracted ). I A ‐current amplitudes were measured from the transient peak responses. The examples shown are from PDGFRα + ‐OPCs. (E) : Normalized current–voltage plot of I A ‐channel activity measured from PDGFRα + ‐OPCs ( n = 6) and O4 + ‐oligodendrocytes ( n = 4). Data were normalized to +36 mV current data. (F) : Decrease in I A ‐channel expression from PDGFRα + ‐OPCs to O4 + ‐oligodendrocytes ( n = 6‐12, N = 3‐4). Current amplitude data were measured from the depolarization step to +16 mV. *, p
    Figure Legend Snippet: Voltage‐gated K + ‐channel expression in human pluripotent stem cell‐derived oligodendroglia. (A) : To isolate I k ‐channel activity, 10 mV incremental voltage‐pulses were initially applied to activate I k ‐channels in the presence of tetrodotoxin (TTX) ( activation ). This was then repeated in the presence of TEA and the current data subtracted from that of the former to determine the I K ‐specific current ( subtracted ). I K ‐current amplitudes were measured 200 milliseconds after activation. The examples shown are from PDGFRα + ‐oligodendrocyte precursor cells (OPCs). (B) : Normalized current–voltage plot of I K ‐channel activity measured from PDGFRα + ‐OPCs ( n = 4) and O4 + ‐oligodendrocytes ( n = 4). Data were normalized to +46 mV current data. (C) : Decrease in I K ‐channel expression from PDGFRα + ‐OPCs to O4 + ‐oligodendrocytes ( n = 8–15, N = 3‐4). Current amplitude data were measured from the 100‐mV step. (D) : I A ‐channel activity was measured in the presence of TTX and Cd 2+ (100 μM). The holding potential was pre‐stepped to –124 mV (500 milliseconds) and, there from, the holding potential depolarized in 10 mV increments before returning to –84 mV ( activation ). Since I A ‐channels inactivate rapidly upon depolarization, an inactivation protocol pre‐stepped cells to –34 mV from –84 mV to isolate non‐ I A current ( inactivation ), which was subtracted from the former to generate the I A ‐mediated current ( subtracted ). I A ‐current amplitudes were measured from the transient peak responses. The examples shown are from PDGFRα + ‐OPCs. (E) : Normalized current–voltage plot of I A ‐channel activity measured from PDGFRα + ‐OPCs ( n = 6) and O4 + ‐oligodendrocytes ( n = 4). Data were normalized to +36 mV current data. (F) : Decrease in I A ‐channel expression from PDGFRα + ‐OPCs to O4 + ‐oligodendrocytes ( n = 6‐12, N = 3‐4). Current amplitude data were measured from the depolarization step to +16 mV. *, p

    Techniques Used: Expressing, Derivative Assay, Activity Assay, Activation Assay

    Oligodendrocytes derived from mutant C9ORF72 patients. (A) : Representative staining of iPS C9 1 and iPS C9 2 cells 1 week post‐mitogen removal identifies OLIG2 + ‐progenitors, and cells expressing PDGFRα, O4, and MBP. Scale bar = 50 μm. (B) : MACS‐sorted week 3 C9ORF72 mutant (iPS C9 1 and iPS C9 2) oligodendrocytes display comparable C9ORF72 ‐v2 and C9ORF72 ‐total (v1, v2, and v3 isoforms) expression compared with controls (iPS1 and iPS2) as showed by quantitative real‐time polymerase chain reaction (qRT‐PCR) ( N = 5 for each line, unpaired t tests). (C) : Fluorescence in situ hybridization showing the presence of nuclear GGGGCC RNA‐containing foci in both iPS C9 1 ( N = 3) and iPS C9 2 ( N = 3) O4 + ‐oligodendrocytes. Scale bar = 10 μm. (D) : Percentage O4 + ‐oligodendrocytes derived from the iPS C9 1 and iPS C9 2 lines that express nuclear GGGGCC RNA‐containing foci at week 1 (iPS C9 1/iPS C9 2: N = 3/3) and 3 (iPS C9 1/iPS C9 2: N = 3/3). (E) : Flow cytometry quantification of O4 + and caspase3‐7 + cells showing no differences in cell death across the mutant (iPS C9 1 and iPS C9 2) and control (iPS1 and iPS2) lines in basal conditions ( N = 3 for each line, unpaired t tests). (F) : MACS‐sorted week 3 C9ORF72 mutant (iPS C9 1 and iPS C9 2) oligodendrocytes display no differences in MCT1 expression compared with controls (iPS1 and iPS2) as showed by qRT‐PCR ( N = 5 for each line, unpaired t tests). (G) : Mean percentage reduction in rectification indices of currents recorded from week 3 O4 + ‐oligodendrocytes with respect to oligodendrocyte precursor cells (OPCs) (unpaired t tests). (H) : Mean AMPAR conductance for PDGFRα + ‐OPCs (iPS C9 1/iPS C9 2: n = 12/11, N = 3/3) and O4 + ‐oligodendrocytes (iPS C9 1/iPS C9 2: n = 4/5, N = 2/1) derived from the iPS C9 lines. *, p
    Figure Legend Snippet: Oligodendrocytes derived from mutant C9ORF72 patients. (A) : Representative staining of iPS C9 1 and iPS C9 2 cells 1 week post‐mitogen removal identifies OLIG2 + ‐progenitors, and cells expressing PDGFRα, O4, and MBP. Scale bar = 50 μm. (B) : MACS‐sorted week 3 C9ORF72 mutant (iPS C9 1 and iPS C9 2) oligodendrocytes display comparable C9ORF72 ‐v2 and C9ORF72 ‐total (v1, v2, and v3 isoforms) expression compared with controls (iPS1 and iPS2) as showed by quantitative real‐time polymerase chain reaction (qRT‐PCR) ( N = 5 for each line, unpaired t tests). (C) : Fluorescence in situ hybridization showing the presence of nuclear GGGGCC RNA‐containing foci in both iPS C9 1 ( N = 3) and iPS C9 2 ( N = 3) O4 + ‐oligodendrocytes. Scale bar = 10 μm. (D) : Percentage O4 + ‐oligodendrocytes derived from the iPS C9 1 and iPS C9 2 lines that express nuclear GGGGCC RNA‐containing foci at week 1 (iPS C9 1/iPS C9 2: N = 3/3) and 3 (iPS C9 1/iPS C9 2: N = 3/3). (E) : Flow cytometry quantification of O4 + and caspase3‐7 + cells showing no differences in cell death across the mutant (iPS C9 1 and iPS C9 2) and control (iPS1 and iPS2) lines in basal conditions ( N = 3 for each line, unpaired t tests). (F) : MACS‐sorted week 3 C9ORF72 mutant (iPS C9 1 and iPS C9 2) oligodendrocytes display no differences in MCT1 expression compared with controls (iPS1 and iPS2) as showed by qRT‐PCR ( N = 5 for each line, unpaired t tests). (G) : Mean percentage reduction in rectification indices of currents recorded from week 3 O4 + ‐oligodendrocytes with respect to oligodendrocyte precursor cells (OPCs) (unpaired t tests). (H) : Mean AMPAR conductance for PDGFRα + ‐OPCs (iPS C9 1/iPS C9 2: n = 12/11, N = 3/3) and O4 + ‐oligodendrocytes (iPS C9 1/iPS C9 2: n = 4/5, N = 2/1) derived from the iPS C9 lines. *, p

    Techniques Used: Derivative Assay, Mutagenesis, Staining, Expressing, Magnetic Cell Separation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Flow Cytometry, Cytometry

    Inwardly rectifying K + ‐channel expression in human pluripotent stem cell‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes. (A) : Representative images of K ir 4.1 subunit immunostaining in PDGFRα + ‐OPCs ( arrowhead ), and O4 + ‐ ( arrow ) and MBP + ‐oligodendrocytes. Scale bar = 30 μm. (B) : K ir ‐channel measurements were performed using an extracellular solution in which KCl (50 mM) replaced an equimolar amount of NaCl. To isolate of K ir ‐channel activity initially 10 mV incremental voltage‐steps were applied in the range –134 mV to + 6 mV from a holding potential of –74 mV and then repeated in the presence of Ba 2+ (1 mM). Note that only selected voltage‐step current recordings are shown in the figure for clarity. Leak‐subtraction of K ir ‐channel current data were performed using the pre‐pulse current amplitude in the presence of Ba 2+ as zero current. It was not possible to extract a current–voltage (I‐V) plot from PDGFRα + ‐cells given the very low K ir channel current amplitudes. Scale bars = 100 pA, 50 milliseconds. (C) : Normalized I‐V of K ir ‐channel activity obtained from O4 + ‐oligodendrocytes ( n = 7). Data were normalized to –114 mV current data. (D) : An increase in mean K ir ‐channel expression in week 3 O4 + ‐oligodendrocytes ( n = 8, N = 3) from that of week 1 O4 + ‐oligodendrocytes ( n = 10, N = 2) and PDGFRα + ‐OPCs ( n = 9, N = 3). Current amplitude data were measured from the depolarization step to –134 mV. Note that the increase in current density also factors the whole‐cell capacitance. (E) : The mean resting membrane potential PDGFRα + ‐OPCs ( n = 19, N = 3), week 1 O4 + ‐oligodendrocytes ( n = 29, N = 7) and week 3 O4 + ‐oligodendrocytes ( n = 20, N = 4). Error bars are obscured by the mean data point. *, p
    Figure Legend Snippet: Inwardly rectifying K + ‐channel expression in human pluripotent stem cell‐derived oligodendrocyte precursor cells (OPCs) and oligodendrocytes. (A) : Representative images of K ir 4.1 subunit immunostaining in PDGFRα + ‐OPCs ( arrowhead ), and O4 + ‐ ( arrow ) and MBP + ‐oligodendrocytes. Scale bar = 30 μm. (B) : K ir ‐channel measurements were performed using an extracellular solution in which KCl (50 mM) replaced an equimolar amount of NaCl. To isolate of K ir ‐channel activity initially 10 mV incremental voltage‐steps were applied in the range –134 mV to + 6 mV from a holding potential of –74 mV and then repeated in the presence of Ba 2+ (1 mM). Note that only selected voltage‐step current recordings are shown in the figure for clarity. Leak‐subtraction of K ir ‐channel current data were performed using the pre‐pulse current amplitude in the presence of Ba 2+ as zero current. It was not possible to extract a current–voltage (I‐V) plot from PDGFRα + ‐cells given the very low K ir channel current amplitudes. Scale bars = 100 pA, 50 milliseconds. (C) : Normalized I‐V of K ir ‐channel activity obtained from O4 + ‐oligodendrocytes ( n = 7). Data were normalized to –114 mV current data. (D) : An increase in mean K ir ‐channel expression in week 3 O4 + ‐oligodendrocytes ( n = 8, N = 3) from that of week 1 O4 + ‐oligodendrocytes ( n = 10, N = 2) and PDGFRα + ‐OPCs ( n = 9, N = 3). Current amplitude data were measured from the depolarization step to –134 mV. Note that the increase in current density also factors the whole‐cell capacitance. (E) : The mean resting membrane potential PDGFRα + ‐OPCs ( n = 19, N = 3), week 1 O4 + ‐oligodendrocytes ( n = 29, N = 7) and week 3 O4 + ‐oligodendrocytes ( n = 20, N = 4). Error bars are obscured by the mean data point. *, p

    Techniques Used: Expressing, Derivative Assay, Immunostaining, Activity Assay

    13) Product Images from "Involvement of unconventional myosin VI in myoblast function and myotube formation"

    Article Title: Involvement of unconventional myosin VI in myoblast function and myotube formation

    Journal: Histochemistry and Cell Biology

    doi: 10.1007/s00418-015-1322-6

    MVI localization to different compartments of undifferentiated myoblasts. MVI is visualized in green with anti-MVI antibody ( a – d ) or as GFP-associated fluorescence ( e – h ), and nuclei were stained with DAPI ( a – h ). a MVI is present in the regions next to cortical actin (in red , stained with Alexa Flour 536-conjugated phalloidin). b , c MVI is in the close proximity to calreticulin (in red ), an endoplasmic reticulum marker, and to GM130 (in red ), a Golgi apparatus marker, respectively. d MVI is present next to vinculin-containing (in red ) adhesive structures. Regions indicated in the merged images are shown at higher magnification in the right panel . e Overexpression of H246R MVI mutant (in green ) myoblasts with MVI knockdown; right panel , merged with DAPI staining. f Overexpression of H246R MVI mutant (in green ) in 2-day rat neonatal cardiomyocytes. In red , staining for α-actinin, the marker of Z lines. Arrows on e and f indicate vacuole-like structures. g , h , Day-0 myoblasts overexpressing the GFP-fused wild-type MVI (GFP-MVI) or H246R mutant, respectively. Golgi cisternae (in yellow ) were stained with anti-GM130 monoclonal antibody. Arrows in ( g ), Golgi cisternae in cells overexpressing GFP-MVI, and arrowhead in ( h ), Golgi cisternae in the cell overexpressing the MVI mutant. Bars 20 μm
    Figure Legend Snippet: MVI localization to different compartments of undifferentiated myoblasts. MVI is visualized in green with anti-MVI antibody ( a – d ) or as GFP-associated fluorescence ( e – h ), and nuclei were stained with DAPI ( a – h ). a MVI is present in the regions next to cortical actin (in red , stained with Alexa Flour 536-conjugated phalloidin). b , c MVI is in the close proximity to calreticulin (in red ), an endoplasmic reticulum marker, and to GM130 (in red ), a Golgi apparatus marker, respectively. d MVI is present next to vinculin-containing (in red ) adhesive structures. Regions indicated in the merged images are shown at higher magnification in the right panel . e Overexpression of H246R MVI mutant (in green ) myoblasts with MVI knockdown; right panel , merged with DAPI staining. f Overexpression of H246R MVI mutant (in green ) in 2-day rat neonatal cardiomyocytes. In red , staining for α-actinin, the marker of Z lines. Arrows on e and f indicate vacuole-like structures. g , h , Day-0 myoblasts overexpressing the GFP-fused wild-type MVI (GFP-MVI) or H246R mutant, respectively. Golgi cisternae (in yellow ) were stained with anti-GM130 monoclonal antibody. Arrows in ( g ), Golgi cisternae in cells overexpressing GFP-MVI, and arrowhead in ( h ), Golgi cisternae in the cell overexpressing the MVI mutant. Bars 20 μm

    Techniques Used: Fluorescence, Staining, Marker, Over Expression, Mutagenesis

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