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    Structured Review

    New England Biolabs non matrix associated dna
    Growth-dependent and c-Myc-dependent attachment of <t>rDNA</t> to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic <t>DNA</t> from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
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    Images

    1) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.
    Figure Legend Snippet: rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.

    Techniques Used: Migration, Positive Control, Ligation, Amplification, Isolation, Activity Assay, Polymerase Chain Reaction, Activation Assay

    2) Product Images from "Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases"

    Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.2109

    ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and BamH1 restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p
    Figure Legend Snippet: ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and BamH1 restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p

    Techniques Used: Clone Assay, Plasmid Preparation, Fluorescence, Enzyme-linked Immunosorbent Assay, In Vivo, SDS Page, Western Blot, Quantitative RT-PCR, Preserving

    3) Product Images from "Expression of RCK2 MAPKAP (MAPK-activated protein kinase) rescues yeast cells sensitivity to osmotic stress"

    Article Title: Expression of RCK2 MAPKAP (MAPK-activated protein kinase) rescues yeast cells sensitivity to osmotic stress

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-015-0276-7

    Phenotypic microarray analysis for S. cerevisiae Δ rck2 , S. cerevisiae Δ rck2 pCM161 and S. cerevisiae Δ rck2 pCM161: RCK2 under osmotic stress a 10% sorbitol stress, b 4% glucose stress, c 1.0 M glycerol stress. Mean ± SD (n = 3).
    Figure Legend Snippet: Phenotypic microarray analysis for S. cerevisiae Δ rck2 , S. cerevisiae Δ rck2 pCM161 and S. cerevisiae Δ rck2 pCM161: RCK2 under osmotic stress a 10% sorbitol stress, b 4% glucose stress, c 1.0 M glycerol stress. Mean ± SD (n = 3).

    Techniques Used: Microarray

    4) Product Images from "The Role of Genetically Modified Mesenchymal Stem Cells in Urinary Bladder Regeneration"

    Article Title: The Role of Genetically Modified Mesenchymal Stem Cells in Urinary Bladder Regeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138643

    Effects of Cyr61 and Wnt5a on urodynamics. (A) Data shown as means ± SEM (range) for pre- and post-augment capacity (volume infused prior to first urine leak), voiding pressure (highest pressure at time of void) and compliance (percentage of bladder volume infused at pressures ≤ 20 cmH 2 O). By 4 weeks, Cyr61KD, Cyr61OX and Wnt5aOX groups all demonstrated > 90% mean percent recovery of pre-augment bladder capacity [(post-augment capacity–pre-augment capacity)*100]. (B) Representative tracings from urodynamics evaluation. Voiding pressures averaged 40 cm H 2 O.
    Figure Legend Snippet: Effects of Cyr61 and Wnt5a on urodynamics. (A) Data shown as means ± SEM (range) for pre- and post-augment capacity (volume infused prior to first urine leak), voiding pressure (highest pressure at time of void) and compliance (percentage of bladder volume infused at pressures ≤ 20 cmH 2 O). By 4 weeks, Cyr61KD, Cyr61OX and Wnt5aOX groups all demonstrated > 90% mean percent recovery of pre-augment bladder capacity [(post-augment capacity–pre-augment capacity)*100]. (B) Representative tracings from urodynamics evaluation. Voiding pressures averaged 40 cm H 2 O.

    Techniques Used:

    Effects of Cyr61 and Wnt5a on regenerating vasculature and musculature. (A) Cyr61OX and Wnt5aOX showed early and persistent increased vasculature, comparably greater than unmanipulated MSCs, with numbers of vessels/mm 2 similar to MSC/CD34 + HSPC grafts at 4 weeks. (§ Unseeded, MSC and MSC/CD34 + HSPC data, shown as solid and dotted lines, previously reported [ 7 ]). At both 4 and 10 weeks, Cyr61KD demonstrated significantly decreased vasculature as compared to Cyr61OX. Data shown as means ± SEM; ***P
    Figure Legend Snippet: Effects of Cyr61 and Wnt5a on regenerating vasculature and musculature. (A) Cyr61OX and Wnt5aOX showed early and persistent increased vasculature, comparably greater than unmanipulated MSCs, with numbers of vessels/mm 2 similar to MSC/CD34 + HSPC grafts at 4 weeks. (§ Unseeded, MSC and MSC/CD34 + HSPC data, shown as solid and dotted lines, previously reported [ 7 ]). At both 4 and 10 weeks, Cyr61KD demonstrated significantly decreased vasculature as compared to Cyr61OX. Data shown as means ± SEM; ***P

    Techniques Used:

    Effects of Cyr61 and Wnt5a on peripheral nerve regeneration. (A) Cyr61KD and MSC demonstrate no nerve regeneration at 4 weeks and poor nerve regeneration at 10 weeks. Wnt5aOX and MSC/CD34 + HSPC demonstrate increased early and robust nerve regeneration. Cyr61OX demonstrates nerve regeneration that continues to improve from 4 to 10 weeks. Data shown as means ± SEM (range). MSC and MSC/CD34 + HSPC data represent new quantitative assessment for a subset of samples from a previous study.[ 7 ] (B) Sample photomicrographs demonstrate βIII tubulin (+) (green) neuronal staining (rows 2 and 4, blue: DAPI, green arrows: regenerated nerves, white arrows: transition between native and regenerated tissue, R: regenerated tissue, N: native tissue). Masson’s trichrome-stained images are of a serial section of tissue for each sample (rows 1 and 3; black arrows: transition between native and regenerated tissue). Scale bar, 200 μm. (Unseeded, MSC and MSC/CD34 + HSPC images shown in S2 Fig ).
    Figure Legend Snippet: Effects of Cyr61 and Wnt5a on peripheral nerve regeneration. (A) Cyr61KD and MSC demonstrate no nerve regeneration at 4 weeks and poor nerve regeneration at 10 weeks. Wnt5aOX and MSC/CD34 + HSPC demonstrate increased early and robust nerve regeneration. Cyr61OX demonstrates nerve regeneration that continues to improve from 4 to 10 weeks. Data shown as means ± SEM (range). MSC and MSC/CD34 + HSPC data represent new quantitative assessment for a subset of samples from a previous study.[ 7 ] (B) Sample photomicrographs demonstrate βIII tubulin (+) (green) neuronal staining (rows 2 and 4, blue: DAPI, green arrows: regenerated nerves, white arrows: transition between native and regenerated tissue, R: regenerated tissue, N: native tissue). Masson’s trichrome-stained images are of a serial section of tissue for each sample (rows 1 and 3; black arrows: transition between native and regenerated tissue). Scale bar, 200 μm. (Unseeded, MSC and MSC/CD34 + HSPC images shown in S2 Fig ).

    Techniques Used: Staining

    Effects of Cyr61 and Wnt5a on urothelium growth. (A) Wnt5aOX resulted in mean urothelium width similar to MSC/CD34 + HSPCs. Cyr61OX resulted in urothelium width significantly greater than Cyr61KD at both time points. Cyr61KD and unseeded grafts had the thinnest urothelium. Data shown as means ± SEM; *P
    Figure Legend Snippet: Effects of Cyr61 and Wnt5a on urothelium growth. (A) Wnt5aOX resulted in mean urothelium width similar to MSC/CD34 + HSPCs. Cyr61OX resulted in urothelium width significantly greater than Cyr61KD at both time points. Cyr61KD and unseeded grafts had the thinnest urothelium. Data shown as means ± SEM; *P

    Techniques Used:

    MSC construct validation. (A) Western blot analysis of the Cyr61OX MSC construct demonstrates significant over-expression of Cyr61 as compared to unmanipulated MSCs (expected molecular weight of ~40kDa). The Cyr61KD MSC construct demonstrates significantly reduced expression of Cyr61. β-tubulin loading control confirmed equivalent protein loading amongst samples. Unmanipulated MSCs were cultured in MSC Growth Media (Lonza). (B) Western blot analysis of the Wnt5aOX MSC construct demonstrates significant over-expression as compared to unmanipulated MSCs (expected molecular weight of 45kDa). The minimal expression of Wnt5a in unmanipulated MSCs is readily apparent upon longer exposure times. Protein lysate from the fibroblast cell line fHs 173We was used as a negative control compared to Wnt5a constructs. β-tubulin loading control confirmed equivalent protein loading amongst samples.
    Figure Legend Snippet: MSC construct validation. (A) Western blot analysis of the Cyr61OX MSC construct demonstrates significant over-expression of Cyr61 as compared to unmanipulated MSCs (expected molecular weight of ~40kDa). The Cyr61KD MSC construct demonstrates significantly reduced expression of Cyr61. β-tubulin loading control confirmed equivalent protein loading amongst samples. Unmanipulated MSCs were cultured in MSC Growth Media (Lonza). (B) Western blot analysis of the Wnt5aOX MSC construct demonstrates significant over-expression as compared to unmanipulated MSCs (expected molecular weight of 45kDa). The minimal expression of Wnt5a in unmanipulated MSCs is readily apparent upon longer exposure times. Protein lysate from the fibroblast cell line fHs 173We was used as a negative control compared to Wnt5a constructs. β-tubulin loading control confirmed equivalent protein loading amongst samples.

    Techniques Used: Construct, Western Blot, Over Expression, Molecular Weight, Expressing, Cell Culture, Negative Control

    5) Product Images from "Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle"

    Article Title: Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136679

    EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.
    Figure Legend Snippet: EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.

    Techniques Used: In Vivo

    EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.
    Figure Legend Snippet: EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.

    Techniques Used: In Vivo, Imaging, Electroporation, Expressing, Staining

    6) Product Images from "Expression of Myocilin Mutants Sensitizes Cells to Oxidative Stress-Induced Apoptosis "

    Article Title: Expression of Myocilin Mutants Sensitizes Cells to Oxidative Stress-Induced Apoptosis

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.090853

    CHOP is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. Eye sections of aged wild-type ( upper row ) and transgenic ( lower row ) mice were stained with anti-CHOP antibody and DAPI ( right panel ). Left panels show the bright field images and middle panels show high magnification of TM surrounding region from the white boxes in the left panels . CB, ciliary body. Scale bar = 50 μm.
    Figure Legend Snippet: CHOP is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. Eye sections of aged wild-type ( upper row ) and transgenic ( lower row ) mice were stained with anti-CHOP antibody and DAPI ( right panel ). Left panels show the bright field images and middle panels show high magnification of TM surrounding region from the white boxes in the left panels . CB, ciliary body. Scale bar = 50 μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Mutagenesis, Staining

    Characterization of stably transfected HEK293 cell lines expressing wild-type and mutated myocilins. A: Established Tet-on HEK293 cells harboring a plasmid encoding wild-type, Y437H mutant, or I477N mutant myocilin were cultured in the presence of indicated concentration of DOX for 48 hours. Total RNA was isolated and the MYOC gene expression levels were quantified by quantitative PCR with a MYOC -specific primer set. Asterisks indicate the selected concentrations of DOX that were used in most subsequent experiments. Error bars represent ± SD of triplicate cultures. B: Tet-on HEK293 cells were cultured in media containing high concentration (5 μg/ml) of DOX for 48 hours. For the thapsigargin (TG) treatment, vector control cells were incubated with 3 μmol/L TG for 24 hours. Apoptotic cells were examined by the TUNEL assay. Dark spots represent apoptotic cells. Scale bar = 100 μm. C: Cell lysates from the HEK293 cells that were cultured as in B were immunoblotted with anti-myocilin, anit-GRP78, anti-cleaved PARP, and anti-GAPDH antibodies. D: The Tet-on HEK293 cells were cultured for 10 days in the medium containing 1 μg/ml DOX for vector, wild-type, and Y437H mutant myocilin cell lines or 0.2 μg/ml DOX for the I477N mutant myocilin cell line. The media were replaced every two days. The ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: The Tet-on HEK293 cells were cultured in the presence of indicated concentration of DOX for 48 hours. Cell lysates or conditioned media were immunoblotted with anti-myocilin, anti-GRP78, anti-PDI, and anti-GAPDH antibodies. Lower mobility of wild-type and the I477N myocilin mutant is explained by the presence of the FLAG tag at their C-terminus. F: Band densities for GRP78 and PDI in E were quantified by using Image J software. Comparisons of densitometry values were made relative to their value in Y437H mutant myocilin cell line. Error bars represent ± SD.
    Figure Legend Snippet: Characterization of stably transfected HEK293 cell lines expressing wild-type and mutated myocilins. A: Established Tet-on HEK293 cells harboring a plasmid encoding wild-type, Y437H mutant, or I477N mutant myocilin were cultured in the presence of indicated concentration of DOX for 48 hours. Total RNA was isolated and the MYOC gene expression levels were quantified by quantitative PCR with a MYOC -specific primer set. Asterisks indicate the selected concentrations of DOX that were used in most subsequent experiments. Error bars represent ± SD of triplicate cultures. B: Tet-on HEK293 cells were cultured in media containing high concentration (5 μg/ml) of DOX for 48 hours. For the thapsigargin (TG) treatment, vector control cells were incubated with 3 μmol/L TG for 24 hours. Apoptotic cells were examined by the TUNEL assay. Dark spots represent apoptotic cells. Scale bar = 100 μm. C: Cell lysates from the HEK293 cells that were cultured as in B were immunoblotted with anti-myocilin, anit-GRP78, anti-cleaved PARP, and anti-GAPDH antibodies. D: The Tet-on HEK293 cells were cultured for 10 days in the medium containing 1 μg/ml DOX for vector, wild-type, and Y437H mutant myocilin cell lines or 0.2 μg/ml DOX for the I477N mutant myocilin cell line. The media were replaced every two days. The ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: The Tet-on HEK293 cells were cultured in the presence of indicated concentration of DOX for 48 hours. Cell lysates or conditioned media were immunoblotted with anti-myocilin, anti-GRP78, anti-PDI, and anti-GAPDH antibodies. Lower mobility of wild-type and the I477N myocilin mutant is explained by the presence of the FLAG tag at their C-terminus. F: Band densities for GRP78 and PDI in E were quantified by using Image J software. Comparisons of densitometry values were made relative to their value in Y437H mutant myocilin cell line. Error bars represent ± SD.

    Techniques Used: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Cell Culture, Concentration Assay, Isolation, Real-time Polymerase Chain Reaction, Incubation, TUNEL Assay, Staining, FLAG-tag, Software

    GRP78 is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young (four months old) and aged (16 months old) wild-type or transgenic mice were immunoblotted with anti-myocilin, anti-GRP78, and anti-HSC70 antibodies. HSC70 was used for internal control. B: Eye sections of aged wild-type and transgenic mice were stained with anti-GRP78 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.
    Figure Legend Snippet: GRP78 is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young (four months old) and aged (16 months old) wild-type or transgenic mice were immunoblotted with anti-myocilin, anti-GRP78, and anti-HSC70 antibodies. HSC70 was used for internal control. B: Eye sections of aged wild-type and transgenic mice were stained with anti-GRP78 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Mutagenesis, Staining

    Decrease in the levels of antioxidant proteins in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young and aged wild-type or transgenic mice were immunoblotted with anti-PON2, anti-GPx-3, and anti-HSC70 antibodies. Two pairs of mice for PON2 and three pairs of mice for GPx-3 were analyzed. HSC70 was used for internal control. B: Quantification of the results shown in A . Error bars represent ± SD. C: Eye sections of wild-type and transgenic mice were stained with anti-GPx-3 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.
    Figure Legend Snippet: Decrease in the levels of antioxidant proteins in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young and aged wild-type or transgenic mice were immunoblotted with anti-PON2, anti-GPx-3, and anti-HSC70 antibodies. Two pairs of mice for PON2 and three pairs of mice for GPx-3 were analyzed. HSC70 was used for internal control. B: Quantification of the results shown in A . Error bars represent ± SD. C: Eye sections of wild-type and transgenic mice were stained with anti-GPx-3 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Mutagenesis, Staining

    Accumulation of mutant myocilins sensitizes cells to oxidative stress. A: Vector control cells were cultured in the media containing indicated concentration of H 2 O 2 for 24 hours. Apoptotic cells (dark cells) were detected by the TUNEL assay. Only few apoptotic cells were detected when concentration of H 2 O 2 was not more than 200 μmol/L. B: Tet-on HEK293 cells were pre-incubated for 24 hours with 1 μg/ml DOX for vector, wild-type, and the Y437H mutant myocilin cell line or 0.2 μg/ml DOX for the I477N mutant myocilin cell line, and then they were cultured in the media containing 100 μmol/L H 2 O 2 and DOX for additional 24 hours. Apoptotic cells were identified by the TUNEL assay. Scale bar = 100 μm. C: TUNEL positive cells in B were counted. Error bars represent ± SD of triplicate cultures. D: Dead cells were counted by using a hemocytometer after Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: Cells were incubated as in B . Cell lysates were immunoblotted with anti-myocilin, anti-cleaved PARP, and anti-GAPDH antibodies. F: Band densities for cleaved PARP in E were quantified by using Image J software. Comparisons were made relative to the Y437H mutant myocilin cell line. Error bars represent ± SD. G: HEK293 cells were transiently transfected with constructs expressing wild-type or one of four different mutant myocilins. Cell lysates were prepared 48 hours after transfection and were immunoblotted with anti-myocilin and GAPDH antibodies. H: Transiently transfected HEK293 cells were cultured with 100 μmol/L H 2 O 2 for 24 hours, and then the ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures.
    Figure Legend Snippet: Accumulation of mutant myocilins sensitizes cells to oxidative stress. A: Vector control cells were cultured in the media containing indicated concentration of H 2 O 2 for 24 hours. Apoptotic cells (dark cells) were detected by the TUNEL assay. Only few apoptotic cells were detected when concentration of H 2 O 2 was not more than 200 μmol/L. B: Tet-on HEK293 cells were pre-incubated for 24 hours with 1 μg/ml DOX for vector, wild-type, and the Y437H mutant myocilin cell line or 0.2 μg/ml DOX for the I477N mutant myocilin cell line, and then they were cultured in the media containing 100 μmol/L H 2 O 2 and DOX for additional 24 hours. Apoptotic cells were identified by the TUNEL assay. Scale bar = 100 μm. C: TUNEL positive cells in B were counted. Error bars represent ± SD of triplicate cultures. D: Dead cells were counted by using a hemocytometer after Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: Cells were incubated as in B . Cell lysates were immunoblotted with anti-myocilin, anti-cleaved PARP, and anti-GAPDH antibodies. F: Band densities for cleaved PARP in E were quantified by using Image J software. Comparisons were made relative to the Y437H mutant myocilin cell line. Error bars represent ± SD. G: HEK293 cells were transiently transfected with constructs expressing wild-type or one of four different mutant myocilins. Cell lysates were prepared 48 hours after transfection and were immunoblotted with anti-myocilin and GAPDH antibodies. H: Transiently transfected HEK293 cells were cultured with 100 μmol/L H 2 O 2 for 24 hours, and then the ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures.

    Techniques Used: Mutagenesis, Plasmid Preparation, Cell Culture, Concentration Assay, TUNEL Assay, Incubation, Staining, Software, Transfection, Construct, Expressing

    7) Product Images from "Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle"

    Article Title: Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136679

    EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.
    Figure Legend Snippet: EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.

    Techniques Used: In Vivo

    EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.
    Figure Legend Snippet: EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.

    Techniques Used: In Vivo, Imaging, Electroporation, Expressing, Staining

    8) Product Images from "Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome"

    Article Title: Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25606-2

    Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .
    Figure Legend Snippet: Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .

    Techniques Used: Expressing, In Situ, Hybridization, Staining, Incubation

    Localisation of ArLQP expression in the tube feet and stomach of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the central disk region and an adjoining arm in starfish. ( B ) Longitudinal section of a tube foot showing stained cells (arrowhead) associated with the basal nerve ring in the disk region. ( C ) High magnification image showing stained cells (arrowhead) associated with the basal nerve ring in the disk region of a tube foot. ( D ) Transverse section of the central disk region showing stained cells in the cardiac stomach and pyloric stomach. A higher magnification of the boxed region of the cardiac stomach is shown in ( E ), where stained cells can be seen in the mucosal layer of the cardiac stomach, with some cells (arrowheads) in close proximity to the basi-epithelial nerve plexus. A higher magnification image of a stained cell in the pyloric stomach is shown in ( F ). a, anus ; amp, ampullae ; bnr, basal nerve ring ; conr, circumoral nerve ring ; cs, cardiac stomach ; g, gonad ; gcc, general coelomic cavity ; l, lumen ; m, mouth ; md, madreporite ; o, ossicle ; oa, organ axial; p, papillae ; pc, pyloric caecum ; pd, pyloric duct ; pm, peristomial membrane ; ps, pyloric stomach ; rc, rectal caecum ; rca, ring canal ; rn, radial nerve ; rw, radial water vascular canal ; sa, sinus of axial organ; sc, stone canal ; tb, Tiedemann’s bodies ; tf, tube foot ; tfd, tube foot disc . Scale bars: 50 μm in B , D ; 20 μm in C ; 10 μm in E and F .
    Figure Legend Snippet: Localisation of ArLQP expression in the tube feet and stomach of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the central disk region and an adjoining arm in starfish. ( B ) Longitudinal section of a tube foot showing stained cells (arrowhead) associated with the basal nerve ring in the disk region. ( C ) High magnification image showing stained cells (arrowhead) associated with the basal nerve ring in the disk region of a tube foot. ( D ) Transverse section of the central disk region showing stained cells in the cardiac stomach and pyloric stomach. A higher magnification of the boxed region of the cardiac stomach is shown in ( E ), where stained cells can be seen in the mucosal layer of the cardiac stomach, with some cells (arrowheads) in close proximity to the basi-epithelial nerve plexus. A higher magnification image of a stained cell in the pyloric stomach is shown in ( F ). a, anus ; amp, ampullae ; bnr, basal nerve ring ; conr, circumoral nerve ring ; cs, cardiac stomach ; g, gonad ; gcc, general coelomic cavity ; l, lumen ; m, mouth ; md, madreporite ; o, ossicle ; oa, organ axial; p, papillae ; pc, pyloric caecum ; pd, pyloric duct ; pm, peristomial membrane ; ps, pyloric stomach ; rc, rectal caecum ; rca, ring canal ; rn, radial nerve ; rw, radial water vascular canal ; sa, sinus of axial organ; sc, stone canal ; tb, Tiedemann’s bodies ; tf, tube foot ; tfd, tube foot disc . Scale bars: 50 μm in B , D ; 20 μm in C ; 10 μm in E and F .

    Techniques Used: Expressing, In Situ, Hybridization, Staining

    9) Product Images from "Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome"

    Article Title: Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25606-2

    Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .
    Figure Legend Snippet: Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .

    Techniques Used: Expressing, In Situ, Hybridization, Staining, Incubation

    Localisation of ArLQP expression in the tube feet and stomach of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the central disk region and an adjoining arm in starfish. ( B ) Longitudinal section of a tube foot showing stained cells (arrowhead) associated with the basal nerve ring in the disk region. ( C ) High magnification image showing stained cells (arrowhead) associated with the basal nerve ring in the disk region of a tube foot. ( D ) Transverse section of the central disk region showing stained cells in the cardiac stomach and pyloric stomach. A higher magnification of the boxed region of the cardiac stomach is shown in ( E ), where stained cells can be seen in the mucosal layer of the cardiac stomach, with some cells (arrowheads) in close proximity to the basi-epithelial nerve plexus. A higher magnification image of a stained cell in the pyloric stomach is shown in ( F ). a, anus ; amp, ampullae ; bnr, basal nerve ring ; conr, circumoral nerve ring ; cs, cardiac stomach ; g, gonad ; gcc, general coelomic cavity ; l, lumen ; m, mouth ; md, madreporite ; o, ossicle ; oa, organ axial; p, papillae ; pc, pyloric caecum ; pd, pyloric duct ; pm, peristomial membrane ; ps, pyloric stomach ; rc, rectal caecum ; rca, ring canal ; rn, radial nerve ; rw, radial water vascular canal ; sa, sinus of axial organ; sc, stone canal ; tb, Tiedemann’s bodies ; tf, tube foot ; tfd, tube foot disc . Scale bars: 50 μm in B , D ; 20 μm in C ; 10 μm in E and F .
    Figure Legend Snippet: Localisation of ArLQP expression in the tube feet and stomach of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the central disk region and an adjoining arm in starfish. ( B ) Longitudinal section of a tube foot showing stained cells (arrowhead) associated with the basal nerve ring in the disk region. ( C ) High magnification image showing stained cells (arrowhead) associated with the basal nerve ring in the disk region of a tube foot. ( D ) Transverse section of the central disk region showing stained cells in the cardiac stomach and pyloric stomach. A higher magnification of the boxed region of the cardiac stomach is shown in ( E ), where stained cells can be seen in the mucosal layer of the cardiac stomach, with some cells (arrowheads) in close proximity to the basi-epithelial nerve plexus. A higher magnification image of a stained cell in the pyloric stomach is shown in ( F ). a, anus ; amp, ampullae ; bnr, basal nerve ring ; conr, circumoral nerve ring ; cs, cardiac stomach ; g, gonad ; gcc, general coelomic cavity ; l, lumen ; m, mouth ; md, madreporite ; o, ossicle ; oa, organ axial; p, papillae ; pc, pyloric caecum ; pd, pyloric duct ; pm, peristomial membrane ; ps, pyloric stomach ; rc, rectal caecum ; rca, ring canal ; rn, radial nerve ; rw, radial water vascular canal ; sa, sinus of axial organ; sc, stone canal ; tb, Tiedemann’s bodies ; tf, tube foot ; tfd, tube foot disc . Scale bars: 50 μm in B , D ; 20 μm in C ; 10 μm in E and F .

    Techniques Used: Expressing, In Situ, Hybridization, Staining

    10) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    11) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    12) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.
    Figure Legend Snippet: rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.

    Techniques Used: Migration, Positive Control, Ligation, Amplification, Isolation, Activity Assay, Polymerase Chain Reaction, Activation Assay

    13) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.
    Figure Legend Snippet: rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.

    Techniques Used: Migration, Positive Control, Ligation, Amplification, Isolation, Activity Assay, Polymerase Chain Reaction, Activation Assay

    14) Product Images from "Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis"

    Article Title: Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis

    Journal: eLife

    doi: 10.7554/eLife.17557

    Cp110 localizes to cilia-forming basal bodies and ciliary tips of monociliated GRP cells. ( A–D ) GFP-Cp110 localizes to cilia-forming basal bodies in GRP cells injected with gfp-cp110 (green) and centrin4-cfp (basal body/daughter centriole, blue) and immunostained for cilia (Acetylated-α-tubulin, red). ( A ) Single GRP cilium of normal length (approximately 4 µm) with two GFP-Cp110 foci (yellow arrowheads; mother centriole/basal body and daughter centriole) overlapping with Centrin4-CFP at the base of the cilium. (n = 3). ( B ), Differential effects of gfp-cp110 expression in GRP cells. Basal bodies (blue) and cilia (red) in GRP cells expressing different amounts of GFP-Cp110 (green). ( B’ ), Magnification of area depicted in ( B ) showing cilia of different length with different amounts of GFP signal at their base. ( C ) In some GRPs, a subset of cilia displayed GFP-Cp110 localization to the ciliary tip (boxes and yellow arrows). Three individual cases are shown in ( C’–C’’’ ), where yellow arrows indicate ciliary tip. (n = 9 for B and C combined). DOI: http://dx.doi.org/10.7554/eLife.17557.012
    Figure Legend Snippet: Cp110 localizes to cilia-forming basal bodies and ciliary tips of monociliated GRP cells. ( A–D ) GFP-Cp110 localizes to cilia-forming basal bodies in GRP cells injected with gfp-cp110 (green) and centrin4-cfp (basal body/daughter centriole, blue) and immunostained for cilia (Acetylated-α-tubulin, red). ( A ) Single GRP cilium of normal length (approximately 4 µm) with two GFP-Cp110 foci (yellow arrowheads; mother centriole/basal body and daughter centriole) overlapping with Centrin4-CFP at the base of the cilium. (n = 3). ( B ), Differential effects of gfp-cp110 expression in GRP cells. Basal bodies (blue) and cilia (red) in GRP cells expressing different amounts of GFP-Cp110 (green). ( B’ ), Magnification of area depicted in ( B ) showing cilia of different length with different amounts of GFP signal at their base. ( C ) In some GRPs, a subset of cilia displayed GFP-Cp110 localization to the ciliary tip (boxes and yellow arrows). Three individual cases are shown in ( C’–C’’’ ), where yellow arrows indicate ciliary tip. (n = 9 for B and C combined). DOI: http://dx.doi.org/10.7554/eLife.17557.012

    Techniques Used: Injection, Expressing

    Alignment of the cp110 region, where the missing Adenine was identified in the FS-clone. BC167469, Xenopus tropicalis genome 9.0 sequence and the current Xenopus tropicalis cp110 reference sequence (XM_0129708) are shown.
    Figure Legend Snippet: Alignment of the cp110 region, where the missing Adenine was identified in the FS-clone. BC167469, Xenopus tropicalis genome 9.0 sequence and the current Xenopus tropicalis cp110 reference sequence (XM_0129708) are shown.

    Techniques Used: Sequencing

    Schematic depiction of Cp110 localization sites at centrioles, basal bodies and cilia. ( A ) Cp110 caps the distal ends of centrioles. ( B ) Cp110 localizes adjacent to the basal body at a posterior domain as well as to the tip of the rootlet. Additionally, Cp110 can localize to ciliary tips. DOI: http://dx.doi.org/10.7554/eLife.17557.014
    Figure Legend Snippet: Schematic depiction of Cp110 localization sites at centrioles, basal bodies and cilia. ( A ) Cp110 caps the distal ends of centrioles. ( B ) Cp110 localizes adjacent to the basal body at a posterior domain as well as to the tip of the rootlet. Additionally, Cp110 can localize to ciliary tips. DOI: http://dx.doi.org/10.7554/eLife.17557.014

    Techniques Used:

    Schematic representation of Cp110 domains and their proposed function. Cp110 domains are depicted as described in Figure 5D . Proposed functions are indicated. DOI: http://dx.doi.org/10.7554/eLife.17557.022
    Figure Legend Snippet: Schematic representation of Cp110 domains and their proposed function. Cp110 domains are depicted as described in Figure 5D . Proposed functions are indicated. DOI: http://dx.doi.org/10.7554/eLife.17557.022

    Techniques Used:

    Cp110 is required for ciliary adhesion complex formation in MCCs. ( A ) Greater area view of gfp-cp110 (green), FAK-mKate (magenta) and centrin4-cfp (blue) overexpression in MCCs. Green circles in magenta channel indicate position of Cp110. Related to Figure 4A . ( B )FAK-GFP (green) levels were greatly reduced in cp110 morphants, while Centrin4-CFP (blue) and Clamp-RFP (red) levels remained largely unchanged. n embryos/MCCs: control (6/18), cp110 MO (9/27). ( B’ ) Apical (0–1.82 μm) and deep ( > 1.82 μm) localized basal bodies are shown from MCC presented in ( B ). DOI: http://dx.doi.org/10.7554/eLife.17557.016
    Figure Legend Snippet: Cp110 is required for ciliary adhesion complex formation in MCCs. ( A ) Greater area view of gfp-cp110 (green), FAK-mKate (magenta) and centrin4-cfp (blue) overexpression in MCCs. Green circles in magenta channel indicate position of Cp110. Related to Figure 4A . ( B )FAK-GFP (green) levels were greatly reduced in cp110 morphants, while Centrin4-CFP (blue) and Clamp-RFP (red) levels remained largely unchanged. n embryos/MCCs: control (6/18), cp110 MO (9/27). ( B’ ) Apical (0–1.82 μm) and deep ( > 1.82 μm) localized basal bodies are shown from MCC presented in ( B ). DOI: http://dx.doi.org/10.7554/eLife.17557.016

    Techniques Used: Over Expression

    Cp110 is required for basal body function in MCC ciliogenesis. ( A ) Cp110-deficient MCC cilia fail to beat directionally. gfp-cfap20 injected embryos were used to visualize ciliary beating (10 s projections are shown). (Related to Video 2 – 3 ). ( B ) Quantification of cilia motility data. ***p
    Figure Legend Snippet: Cp110 is required for basal body function in MCC ciliogenesis. ( A ) Cp110-deficient MCC cilia fail to beat directionally. gfp-cfap20 injected embryos were used to visualize ciliary beating (10 s projections are shown). (Related to Video 2 – 3 ). ( B ) Quantification of cilia motility data. ***p

    Techniques Used: Injection

    Model of the transcriptional/post-transcriptional regulatory module required to achieve optimal Cp110 levels in MCC ciliogenesis. A schematic model of ciliary transcription factors and miRNAs is shown. Activation is shown as arrow. Inhibition is shown as T-shaped arrow. DOI: http://dx.doi.org/10.7554/eLife.17557.025
    Figure Legend Snippet: Model of the transcriptional/post-transcriptional regulatory module required to achieve optimal Cp110 levels in MCC ciliogenesis. A schematic model of ciliary transcription factors and miRNAs is shown. Activation is shown as arrow. Inhibition is shown as T-shaped arrow. DOI: http://dx.doi.org/10.7554/eLife.17557.025

    Techniques Used: Activation Assay, Inhibition

    Cp110 central domain deletion enhances centriolar and basal body phenotypes. ( A–E ) Related to Figure 5 and Figure 5—figure supplement 1 . ( A–B ) gfp-cp110ΔCentral overexpression induces supernumerary centrioles, polynucleated cells and severe cytokinesis defects. ( A ) Controls and embryos injected with gfp-cp110ΔCentral (green) were analyzed for ciliation by immunofluorescent staining against Acetylated-α-tubulin (cilia, Ac.-α-tub., red) and nuclei (DAPI, blue). In uninjected control embryos, cell borders were visualized by Actin staining (green, left panel only). n embryos: control, 6; cp110 MO, 5. ( B ) Embryos were injected with centrin4-cfp (centrioles, blue) and gfp-cp110ΔCentral (green). A central region of a non-MCC epidermal cell is shown. All GFP-Cp110ΔCentral foci overlap with Centrin4-CFP foci. ( C–D ) gfp-cp110ΔCentral overexpression induces increased numbers of basal bodies in MCCs, which frequently fail to separate. ( C ) Embryos were injected with centrin4-cfp (basal bodies, blue) and gfp-cp110ΔCentral (green), which caused strongly enlarged MCCs and aggregated basal bodies. ( D ) Magnification of basal body cluster from MCC shown in C . ( E ) Cp110 constructs generated in this study and their effect on epidermal cells. +, phenotype present; ++, strong phenotype; +++, very strong phenotype; -, phenotype not present. DOI: http://dx.doi.org/10.7554/eLife.17557.021
    Figure Legend Snippet: Cp110 central domain deletion enhances centriolar and basal body phenotypes. ( A–E ) Related to Figure 5 and Figure 5—figure supplement 1 . ( A–B ) gfp-cp110ΔCentral overexpression induces supernumerary centrioles, polynucleated cells and severe cytokinesis defects. ( A ) Controls and embryos injected with gfp-cp110ΔCentral (green) were analyzed for ciliation by immunofluorescent staining against Acetylated-α-tubulin (cilia, Ac.-α-tub., red) and nuclei (DAPI, blue). In uninjected control embryos, cell borders were visualized by Actin staining (green, left panel only). n embryos: control, 6; cp110 MO, 5. ( B ) Embryos were injected with centrin4-cfp (centrioles, blue) and gfp-cp110ΔCentral (green). A central region of a non-MCC epidermal cell is shown. All GFP-Cp110ΔCentral foci overlap with Centrin4-CFP foci. ( C–D ) gfp-cp110ΔCentral overexpression induces increased numbers of basal bodies in MCCs, which frequently fail to separate. ( C ) Embryos were injected with centrin4-cfp (basal bodies, blue) and gfp-cp110ΔCentral (green), which caused strongly enlarged MCCs and aggregated basal bodies. ( D ) Magnification of basal body cluster from MCC shown in C . ( E ) Cp110 constructs generated in this study and their effect on epidermal cells. +, phenotype present; ++, strong phenotype; +++, very strong phenotype; -, phenotype not present. DOI: http://dx.doi.org/10.7554/eLife.17557.021

    Techniques Used: Over Expression, Injection, Staining, Construct, Generated

    Cp110 levels in MCCs are controlled by ciliary transcription factors and miR-34/449 microRNAs. ( A ) Related to Figure 6A . cp110 expression in MCCs is regulated through the conserved MCC signaling/transcriptional cascade. Embryos were injected with Notch-icd to inhibit MCC induction (red) or with Notch-icd together with multicilin (mci ) to stimulate MCC induction (green). RNA-sequencing (RNA-seq) was performed on extracts from mucociliary organoids at MCC specification stage (st. 16). Normalized counts are shown as bar graphs. The foxj1 expression analysis confirmed successful manipulation. ( B ) Related to Figure 6B,E–F . Expression of miRNA miR-34a is not activated by ciliary transcription factors. ChIP-seq and RNA-seq was performed as described in Figure 6B . miRNA location is indicated by red box. DOI: http://dx.doi.org/10.7554/eLife.17557.024
    Figure Legend Snippet: Cp110 levels in MCCs are controlled by ciliary transcription factors and miR-34/449 microRNAs. ( A ) Related to Figure 6A . cp110 expression in MCCs is regulated through the conserved MCC signaling/transcriptional cascade. Embryos were injected with Notch-icd to inhibit MCC induction (red) or with Notch-icd together with multicilin (mci ) to stimulate MCC induction (green). RNA-sequencing (RNA-seq) was performed on extracts from mucociliary organoids at MCC specification stage (st. 16). Normalized counts are shown as bar graphs. The foxj1 expression analysis confirmed successful manipulation. ( B ) Related to Figure 6B,E–F . Expression of miRNA miR-34a is not activated by ciliary transcription factors. ChIP-seq and RNA-seq was performed as described in Figure 6B . miRNA location is indicated by red box. DOI: http://dx.doi.org/10.7554/eLife.17557.024

    Techniques Used: Expressing, Injection, RNA Sequencing Assay, Chromatin Immunoprecipitation

    Cp110 coiled-coil domains are required for cilia inhibition and centriolar functions. ( A ) Related to Figure 5A . Embryos injected with gfp-cp110ΔN or gfp-cp110ΔC (all green) were analyzed for ciliation by immunofluorescent staining against Acetylated-α-tubulin (cilia, Ac.-α-tub., red). Upper panels: red fluorescence channel only. Lower panels: green/red merge channels. ( B ) Related to ( A ) and Figure 5A . Quantification of MCC cilia phenotype in controls and after overexpression of full-length gfp-cp110 and deletion constructs. ***p
    Figure Legend Snippet: Cp110 coiled-coil domains are required for cilia inhibition and centriolar functions. ( A ) Related to Figure 5A . Embryos injected with gfp-cp110ΔN or gfp-cp110ΔC (all green) were analyzed for ciliation by immunofluorescent staining against Acetylated-α-tubulin (cilia, Ac.-α-tub., red). Upper panels: red fluorescence channel only. Lower panels: green/red merge channels. ( B ) Related to ( A ) and Figure 5A . Quantification of MCC cilia phenotype in controls and after overexpression of full-length gfp-cp110 and deletion constructs. ***p

    Techniques Used: Inhibition, Injection, Staining, Fluorescence, Over Expression, Construct

    Cp110 is required for primary and motile monocilia. ( A–C ) Related to Figure 2A . Control and cp110 morphant embryos were stained by WMISH and cleared embryos were analyzed for nkx2.2 and pax6 expression in the neural tube. ( A ) Quantification of nkx2.2 expression. ( B ) WMISH for pax6 expression. Normal expression indicated by green arrowhead, reduced expression indicated by red arrowhead. White and black boxes indicate normal and reduced expression, respectively in graph in C . ( C ) Quantification of pax6 expression. In ( A ) and ( C ), ***p
    Figure Legend Snippet: Cp110 is required for primary and motile monocilia. ( A–C ) Related to Figure 2A . Control and cp110 morphant embryos were stained by WMISH and cleared embryos were analyzed for nkx2.2 and pax6 expression in the neural tube. ( A ) Quantification of nkx2.2 expression. ( B ) WMISH for pax6 expression. Normal expression indicated by green arrowhead, reduced expression indicated by red arrowhead. White and black boxes indicate normal and reduced expression, respectively in graph in C . ( C ) Quantification of pax6 expression. In ( A ) and ( C ), ***p

    Techniques Used: Staining, Expressing

    Cp110 localizes to cilia-forming basal bodies in MCCs. ( A–B ) GFP-Cp110 localization to basal bodies in Xenopus MCCs. ( A ) GFP-Cp110 (green) localizes to basal bodies (Centrin4-CFP, blue) prior to apical docking, during the stages of apical basal body transport. Actin staining shown in red. n = 2 embryos, 18 MCCs. ( B ) GFP-Cp110 (green) shows asymmetric localization to basal bodies/rootlets (Clamp-RFP, red) along the anterior-posterior axis. n = 3 embryos, 30 MCCs. ( C ) Immunofluorescent staining for Cp110 (green) and cilia (Acetylated-α-tubulin, red) shows Cp110 localization at the level of basal bodies and at the lateral membrane (white arrows) in human HAEC MCCs. Three levels along apical-basal axis are shown (top, apical ciliary tuft level; middle, apical MCC membrane level; bottom, cytoplasmic level). n = 1 donor, 12 MCCs. ( D–E ) Mouse trachea staining for Cp110 (green), cilia (Acetylated-α-tubulin, red) and nuclei (DAPI, blue). (n = 4). ( D ) Magnified view of MCCs. ( E ) Greater area view of mouse trachea with multiple MCCs. ( E’ ) Negative control immunofluorescent staining as described in ( E ), but without the use of primary anti-Cp110 antibody. (n = 1). DOI: http://dx.doi.org/10.7554/eLife.17557.011
    Figure Legend Snippet: Cp110 localizes to cilia-forming basal bodies in MCCs. ( A–B ) GFP-Cp110 localization to basal bodies in Xenopus MCCs. ( A ) GFP-Cp110 (green) localizes to basal bodies (Centrin4-CFP, blue) prior to apical docking, during the stages of apical basal body transport. Actin staining shown in red. n = 2 embryos, 18 MCCs. ( B ) GFP-Cp110 (green) shows asymmetric localization to basal bodies/rootlets (Clamp-RFP, red) along the anterior-posterior axis. n = 3 embryos, 30 MCCs. ( C ) Immunofluorescent staining for Cp110 (green) and cilia (Acetylated-α-tubulin, red) shows Cp110 localization at the level of basal bodies and at the lateral membrane (white arrows) in human HAEC MCCs. Three levels along apical-basal axis are shown (top, apical ciliary tuft level; middle, apical MCC membrane level; bottom, cytoplasmic level). n = 1 donor, 12 MCCs. ( D–E ) Mouse trachea staining for Cp110 (green), cilia (Acetylated-α-tubulin, red) and nuclei (DAPI, blue). (n = 4). ( D ) Magnified view of MCCs. ( E ) Greater area view of mouse trachea with multiple MCCs. ( E’ ) Negative control immunofluorescent staining as described in ( E ), but without the use of primary anti-Cp110 antibody. (n = 1). DOI: http://dx.doi.org/10.7554/eLife.17557.011

    Techniques Used: Staining, Negative Control

    Left panel shows Centrin-CFP (blue) and GFP-Cp110 (green) in images where green channel brightness is low. In this image Cp110 localizes adjacent to the basal body. Right panel shows Centrin-CFP (blue) and GFP-Cp110 (green) in images where green channel brightness is high. In this image we can visualize the low-level localization of GFP-Cp110 to the rootlet tip, which was not visible in the left panel. Bottom panel shows Clamp-RFP staining of the ciliary rootlet (red). Top row, middle panel shows schematic localization of Cp110 relative to the basal body and the rootlet. Green = Cp110, blue = Centrin4, red = Clamp.
    Figure Legend Snippet: Left panel shows Centrin-CFP (blue) and GFP-Cp110 (green) in images where green channel brightness is low. In this image Cp110 localizes adjacent to the basal body. Right panel shows Centrin-CFP (blue) and GFP-Cp110 (green) in images where green channel brightness is high. In this image we can visualize the low-level localization of GFP-Cp110 to the rootlet tip, which was not visible in the left panel. Bottom panel shows Clamp-RFP staining of the ciliary rootlet (red). Top row, middle panel shows schematic localization of Cp110 relative to the basal body and the rootlet. Green = Cp110, blue = Centrin4, red = Clamp.

    Techniques Used: Staining

    Schematic representation of summary model of the roles of Cp110 in MCC ciliation. Cp110 levels in MCCs need to be precisely regulated for successful ciliogenesis and normal cilia function. Please see text for detailed description. DOI: http://dx.doi.org/10.7554/eLife.17557.018
    Figure Legend Snippet: Schematic representation of summary model of the roles of Cp110 in MCC ciliation. Cp110 levels in MCCs need to be precisely regulated for successful ciliogenesis and normal cilia function. Please see text for detailed description. DOI: http://dx.doi.org/10.7554/eLife.17557.018

    Techniques Used:

    Cp110 is required for ciliary adhesion complex formation in MCCs. ( A-D ) Cp110 is required for Vinculin (n embryos/MCCs: control (10/30), cp110 MO (10/30)) and Paxillin (n embryos/MCCs: control (5/15), cp110 MO (5/15)) localization to MCC basal bodies. Control and cp110 morphant embryos were injected with vinculin-gfp ( A ) and ( C ), green) or paxillin-gfp ( B ) and ( D ), green) together with centrin4-cfp (blue). Related to Figure 4C–D . ( E ) Full membranes of co-IP experiment shown in Figure 4B . In addition to FLAG-Cp110 (full length Cp110), a FLAG-Cp110-FSΔCCD1 was overexpressed, which misses the first coiled-coil domain, as well as the truncation of the cp110-fs clone (please compare to Figure 5D ). DOI: http://dx.doi.org/10.7554/eLife.17557.017
    Figure Legend Snippet: Cp110 is required for ciliary adhesion complex formation in MCCs. ( A-D ) Cp110 is required for Vinculin (n embryos/MCCs: control (10/30), cp110 MO (10/30)) and Paxillin (n embryos/MCCs: control (5/15), cp110 MO (5/15)) localization to MCC basal bodies. Control and cp110 morphant embryos were injected with vinculin-gfp ( A ) and ( C ), green) or paxillin-gfp ( B ) and ( D ), green) together with centrin4-cfp (blue). Related to Figure 4C–D . ( E ) Full membranes of co-IP experiment shown in Figure 4B . In addition to FLAG-Cp110 (full length Cp110), a FLAG-Cp110-FSΔCCD1 was overexpressed, which misses the first coiled-coil domain, as well as the truncation of the cp110-fs clone (please compare to Figure 5D ). DOI: http://dx.doi.org/10.7554/eLife.17557.017

    Techniques Used: Injection, Co-Immunoprecipitation Assay

    15) Product Images from "Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome"

    Article Title: Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25606-2

    Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .
    Figure Legend Snippet: Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .

    Techniques Used: Expressing, In Situ, Hybridization, Staining, Incubation

    Localisation of ArLQP expression in the tube feet and stomach of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the central disk region and an adjoining arm in starfish. ( B ) Longitudinal section of a tube foot showing stained cells (arrowhead) associated with the basal nerve ring in the disk region. ( C ) High magnification image showing stained cells (arrowhead) associated with the basal nerve ring in the disk region of a tube foot. ( D ) Transverse section of the central disk region showing stained cells in the cardiac stomach and pyloric stomach. A higher magnification of the boxed region of the cardiac stomach is shown in ( E ), where stained cells can be seen in the mucosal layer of the cardiac stomach, with some cells (arrowheads) in close proximity to the basi-epithelial nerve plexus. A higher magnification image of a stained cell in the pyloric stomach is shown in ( F ). a, anus ; amp, ampullae ; bnr, basal nerve ring ; conr, circumoral nerve ring ; cs, cardiac stomach ; g, gonad ; gcc, general coelomic cavity ; l, lumen ; m, mouth ; md, madreporite ; o, ossicle ; oa, organ axial; p, papillae ; pc, pyloric caecum ; pd, pyloric duct ; pm, peristomial membrane ; ps, pyloric stomach ; rc, rectal caecum ; rca, ring canal ; rn, radial nerve ; rw, radial water vascular canal ; sa, sinus of axial organ; sc, stone canal ; tb, Tiedemann’s bodies ; tf, tube foot ; tfd, tube foot disc . Scale bars: 50 μm in B , D ; 20 μm in C ; 10 μm in E and F .
    Figure Legend Snippet: Localisation of ArLQP expression in the tube feet and stomach of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the central disk region and an adjoining arm in starfish. ( B ) Longitudinal section of a tube foot showing stained cells (arrowhead) associated with the basal nerve ring in the disk region. ( C ) High magnification image showing stained cells (arrowhead) associated with the basal nerve ring in the disk region of a tube foot. ( D ) Transverse section of the central disk region showing stained cells in the cardiac stomach and pyloric stomach. A higher magnification of the boxed region of the cardiac stomach is shown in ( E ), where stained cells can be seen in the mucosal layer of the cardiac stomach, with some cells (arrowheads) in close proximity to the basi-epithelial nerve plexus. A higher magnification image of a stained cell in the pyloric stomach is shown in ( F ). a, anus ; amp, ampullae ; bnr, basal nerve ring ; conr, circumoral nerve ring ; cs, cardiac stomach ; g, gonad ; gcc, general coelomic cavity ; l, lumen ; m, mouth ; md, madreporite ; o, ossicle ; oa, organ axial; p, papillae ; pc, pyloric caecum ; pd, pyloric duct ; pm, peristomial membrane ; ps, pyloric stomach ; rc, rectal caecum ; rca, ring canal ; rn, radial nerve ; rw, radial water vascular canal ; sa, sinus of axial organ; sc, stone canal ; tb, Tiedemann’s bodies ; tf, tube foot ; tfd, tube foot disc . Scale bars: 50 μm in B , D ; 20 μm in C ; 10 μm in E and F .

    Techniques Used: Expressing, In Situ, Hybridization, Staining

    16) Product Images from "Expression of Myocilin Mutants Sensitizes Cells to Oxidative Stress-Induced Apoptosis "

    Article Title: Expression of Myocilin Mutants Sensitizes Cells to Oxidative Stress-Induced Apoptosis

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.090853

    CHOP is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. Eye sections of aged wild-type ( upper row ) and transgenic ( lower row ) mice were stained with anti-CHOP antibody and DAPI ( right panel ). Left panels show the bright field images and middle panels show high magnification of TM surrounding region from the white boxes in the left panels . CB, ciliary body. Scale bar = 50 μm.
    Figure Legend Snippet: CHOP is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. Eye sections of aged wild-type ( upper row ) and transgenic ( lower row ) mice were stained with anti-CHOP antibody and DAPI ( right panel ). Left panels show the bright field images and middle panels show high magnification of TM surrounding region from the white boxes in the left panels . CB, ciliary body. Scale bar = 50 μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Mutagenesis, Staining

    Characterization of stably transfected HEK293 cell lines expressing wild-type and mutated myocilins. A: Established Tet-on HEK293 cells harboring a plasmid encoding wild-type, Y437H mutant, or I477N mutant myocilin were cultured in the presence of indicated concentration of DOX for 48 hours. Total RNA was isolated and the MYOC gene expression levels were quantified by quantitative PCR with a MYOC -specific primer set. Asterisks indicate the selected concentrations of DOX that were used in most subsequent experiments. Error bars represent ± SD of triplicate cultures. B: Tet-on HEK293 cells were cultured in media containing high concentration (5 μg/ml) of DOX for 48 hours. For the thapsigargin (TG) treatment, vector control cells were incubated with 3 μmol/L TG for 24 hours. Apoptotic cells were examined by the TUNEL assay. Dark spots represent apoptotic cells. Scale bar = 100 μm. C: Cell lysates from the HEK293 cells that were cultured as in B were immunoblotted with anti-myocilin, anit-GRP78, anti-cleaved PARP, and anti-GAPDH antibodies. D: The Tet-on HEK293 cells were cultured for 10 days in the medium containing 1 μg/ml DOX for vector, wild-type, and Y437H mutant myocilin cell lines or 0.2 μg/ml DOX for the I477N mutant myocilin cell line. The media were replaced every two days. The ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: The Tet-on HEK293 cells were cultured in the presence of indicated concentration of DOX for 48 hours. Cell lysates or conditioned media were immunoblotted with anti-myocilin, anti-GRP78, anti-PDI, and anti-GAPDH antibodies. Lower mobility of wild-type and the I477N myocilin mutant is explained by the presence of the FLAG tag at their C-terminus. F: Band densities for GRP78 and PDI in E were quantified by using Image J software. Comparisons of densitometry values were made relative to their value in Y437H mutant myocilin cell line. Error bars represent ± SD.
    Figure Legend Snippet: Characterization of stably transfected HEK293 cell lines expressing wild-type and mutated myocilins. A: Established Tet-on HEK293 cells harboring a plasmid encoding wild-type, Y437H mutant, or I477N mutant myocilin were cultured in the presence of indicated concentration of DOX for 48 hours. Total RNA was isolated and the MYOC gene expression levels were quantified by quantitative PCR with a MYOC -specific primer set. Asterisks indicate the selected concentrations of DOX that were used in most subsequent experiments. Error bars represent ± SD of triplicate cultures. B: Tet-on HEK293 cells were cultured in media containing high concentration (5 μg/ml) of DOX for 48 hours. For the thapsigargin (TG) treatment, vector control cells were incubated with 3 μmol/L TG for 24 hours. Apoptotic cells were examined by the TUNEL assay. Dark spots represent apoptotic cells. Scale bar = 100 μm. C: Cell lysates from the HEK293 cells that were cultured as in B were immunoblotted with anti-myocilin, anit-GRP78, anti-cleaved PARP, and anti-GAPDH antibodies. D: The Tet-on HEK293 cells were cultured for 10 days in the medium containing 1 μg/ml DOX for vector, wild-type, and Y437H mutant myocilin cell lines or 0.2 μg/ml DOX for the I477N mutant myocilin cell line. The media were replaced every two days. The ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: The Tet-on HEK293 cells were cultured in the presence of indicated concentration of DOX for 48 hours. Cell lysates or conditioned media were immunoblotted with anti-myocilin, anti-GRP78, anti-PDI, and anti-GAPDH antibodies. Lower mobility of wild-type and the I477N myocilin mutant is explained by the presence of the FLAG tag at their C-terminus. F: Band densities for GRP78 and PDI in E were quantified by using Image J software. Comparisons of densitometry values were made relative to their value in Y437H mutant myocilin cell line. Error bars represent ± SD.

    Techniques Used: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Cell Culture, Concentration Assay, Isolation, Real-time Polymerase Chain Reaction, Incubation, TUNEL Assay, Staining, FLAG-tag, Software

    GRP78 is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young (four months old) and aged (16 months old) wild-type or transgenic mice were immunoblotted with anti-myocilin, anti-GRP78, and anti-HSC70 antibodies. HSC70 was used for internal control. B: Eye sections of aged wild-type and transgenic mice were stained with anti-GRP78 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.
    Figure Legend Snippet: GRP78 is up-regulated in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young (four months old) and aged (16 months old) wild-type or transgenic mice were immunoblotted with anti-myocilin, anti-GRP78, and anti-HSC70 antibodies. HSC70 was used for internal control. B: Eye sections of aged wild-type and transgenic mice were stained with anti-GRP78 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Mutagenesis, Staining

    Decrease in the levels of antioxidant proteins in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young and aged wild-type or transgenic mice were immunoblotted with anti-PON2, anti-GPx-3, and anti-HSC70 antibodies. Two pairs of mice for PON2 and three pairs of mice for GPx-3 were analyzed. HSC70 was used for internal control. B: Quantification of the results shown in A . Error bars represent ± SD. C: Eye sections of wild-type and transgenic mice were stained with anti-GPx-3 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.
    Figure Legend Snippet: Decrease in the levels of antioxidant proteins in the eye angle tissue of aged transgenic mice expressing Y437H mutant myocilin. A: Lysates from the dissected angle tissue of young and aged wild-type or transgenic mice were immunoblotted with anti-PON2, anti-GPx-3, and anti-HSC70 antibodies. Two pairs of mice for PON2 and three pairs of mice for GPx-3 were analyzed. HSC70 was used for internal control. B: Quantification of the results shown in A . Error bars represent ± SD. C: Eye sections of wild-type and transgenic mice were stained with anti-GPx-3 antibody and DAPI ( lower row ). Upper row shows the bright field images. CB, ciliary body. Scale bar = 50 μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Mutagenesis, Staining

    Accumulation of mutant myocilins sensitizes cells to oxidative stress. A: Vector control cells were cultured in the media containing indicated concentration of H 2 O 2 for 24 hours. Apoptotic cells (dark cells) were detected by the TUNEL assay. Only few apoptotic cells were detected when concentration of H 2 O 2 was not more than 200 μmol/L. B: Tet-on HEK293 cells were pre-incubated for 24 hours with 1 μg/ml DOX for vector, wild-type, and the Y437H mutant myocilin cell line or 0.2 μg/ml DOX for the I477N mutant myocilin cell line, and then they were cultured in the media containing 100 μmol/L H 2 O 2 and DOX for additional 24 hours. Apoptotic cells were identified by the TUNEL assay. Scale bar = 100 μm. C: TUNEL positive cells in B were counted. Error bars represent ± SD of triplicate cultures. D: Dead cells were counted by using a hemocytometer after Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: Cells were incubated as in B . Cell lysates were immunoblotted with anti-myocilin, anti-cleaved PARP, and anti-GAPDH antibodies. F: Band densities for cleaved PARP in E were quantified by using Image J software. Comparisons were made relative to the Y437H mutant myocilin cell line. Error bars represent ± SD. G: HEK293 cells were transiently transfected with constructs expressing wild-type or one of four different mutant myocilins. Cell lysates were prepared 48 hours after transfection and were immunoblotted with anti-myocilin and GAPDH antibodies. H: Transiently transfected HEK293 cells were cultured with 100 μmol/L H 2 O 2 for 24 hours, and then the ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures.
    Figure Legend Snippet: Accumulation of mutant myocilins sensitizes cells to oxidative stress. A: Vector control cells were cultured in the media containing indicated concentration of H 2 O 2 for 24 hours. Apoptotic cells (dark cells) were detected by the TUNEL assay. Only few apoptotic cells were detected when concentration of H 2 O 2 was not more than 200 μmol/L. B: Tet-on HEK293 cells were pre-incubated for 24 hours with 1 μg/ml DOX for vector, wild-type, and the Y437H mutant myocilin cell line or 0.2 μg/ml DOX for the I477N mutant myocilin cell line, and then they were cultured in the media containing 100 μmol/L H 2 O 2 and DOX for additional 24 hours. Apoptotic cells were identified by the TUNEL assay. Scale bar = 100 μm. C: TUNEL positive cells in B were counted. Error bars represent ± SD of triplicate cultures. D: Dead cells were counted by using a hemocytometer after Trypan Blue staining. Error bars represent ± SD of triplicate cultures. E: Cells were incubated as in B . Cell lysates were immunoblotted with anti-myocilin, anti-cleaved PARP, and anti-GAPDH antibodies. F: Band densities for cleaved PARP in E were quantified by using Image J software. Comparisons were made relative to the Y437H mutant myocilin cell line. Error bars represent ± SD. G: HEK293 cells were transiently transfected with constructs expressing wild-type or one of four different mutant myocilins. Cell lysates were prepared 48 hours after transfection and were immunoblotted with anti-myocilin and GAPDH antibodies. H: Transiently transfected HEK293 cells were cultured with 100 μmol/L H 2 O 2 for 24 hours, and then the ratio of dead cells to total cells was determined by Trypan Blue staining. Error bars represent ± SD of triplicate cultures.

    Techniques Used: Mutagenesis, Plasmid Preparation, Cell Culture, Concentration Assay, TUNEL Assay, Incubation, Staining, Software, Transfection, Construct, Expressing

    17) Product Images from "Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome"

    Article Title: Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25606-2

    Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .
    Figure Legend Snippet: Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .

    Techniques Used: Expressing, In Situ, Hybridization, Staining, Incubation

    18) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.
    Figure Legend Snippet: rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.

    Techniques Used: Migration, Positive Control, Ligation, Amplification, Isolation, Activity Assay, Polymerase Chain Reaction, Activation Assay

    19) Product Images from "Significance of 1B and 2B domains in modulating elastic properties of lamin A"

    Article Title: Significance of 1B and 2B domains in modulating elastic properties of lamin A

    Journal: Scientific Reports

    doi: 10.1038/srep27879

    Coiled-coil unzipping of 2B domain. Panels ( A,B ) show representative FX traces for the two-state and three-state unfolding of 2B. Similar to 1B domain lamin 2B dimers were also unfolding via several intermediates. The molecular design for SMFS experiment was (I27) 3 -2B-1C. Average unfolding forces for both the intermediate and main peaks were similar, at 75 pN. Cartoon picture represents the dimer of the 2B domain. In ( A ) first unfolding peak with 52 nm contour length increase corresponds to lamin 2B domain two-state unfolding and I27 domains are folding with 28 nm spacing at ~180 pN force. Panel ( B ) is representing the three state unzipping events. In ( C , D ) histograms for the contour length and unfolding force analysis has been shown respectively. The black bar is representing the value for the main peak whereas red is depicting the intermediate peaks. Average contour length increments for the main and intermediate peaks were ~52 nm and 25 nm, respectively. Almost 40% molecules were unzipping via an intermediate pathway. All these unfolding experiments for the 2B domains were done at 5–10 μM concentration using 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer at 25 °C.
    Figure Legend Snippet: Coiled-coil unzipping of 2B domain. Panels ( A,B ) show representative FX traces for the two-state and three-state unfolding of 2B. Similar to 1B domain lamin 2B dimers were also unfolding via several intermediates. The molecular design for SMFS experiment was (I27) 3 -2B-1C. Average unfolding forces for both the intermediate and main peaks were similar, at 75 pN. Cartoon picture represents the dimer of the 2B domain. In ( A ) first unfolding peak with 52 nm contour length increase corresponds to lamin 2B domain two-state unfolding and I27 domains are folding with 28 nm spacing at ~180 pN force. Panel ( B ) is representing the three state unzipping events. In ( C , D ) histograms for the contour length and unfolding force analysis has been shown respectively. The black bar is representing the value for the main peak whereas red is depicting the intermediate peaks. Average contour length increments for the main and intermediate peaks were ~52 nm and 25 nm, respectively. Almost 40% molecules were unzipping via an intermediate pathway. All these unfolding experiments for the 2B domains were done at 5–10 μM concentration using 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer at 25 °C.

    Techniques Used: Concentration Assay

    Viscoelastic measurements of mini-lamins. The elasticity of the half-lamins formed by 1B and 2B domains were shown using rheology. The elastic storage moduli of 1B and 2B domain at 1, 5, 10 and 20 μΜ concentrations are shown in panel A and C whereas corresponding elastic loss moduli are depicted in panel B and D respectively. Ratios of the corresponding storage moduli to loss moduli are shown in panel E. Network yield-up experiments varying strain from 0–500% are shown in panel F.
    Figure Legend Snippet: Viscoelastic measurements of mini-lamins. The elasticity of the half-lamins formed by 1B and 2B domains were shown using rheology. The elastic storage moduli of 1B and 2B domain at 1, 5, 10 and 20 μΜ concentrations are shown in panel A and C whereas corresponding elastic loss moduli are depicted in panel B and D respectively. Ratios of the corresponding storage moduli to loss moduli are shown in panel E. Network yield-up experiments varying strain from 0–500% are shown in panel F.

    Techniques Used:

    Thermal denaturation of lamin 1B and 2B domain. The thermal denaturation studies of the 1B and 2B domains were performed using differential scanning calorimetry in panels ( A B ). 1B domain (panel A ) was unfolding via an intermediate pathway whereas 2B (panel B ) was denatured via the two-state mechanism. The first transition temperature for the 1B was at 63 °C and the final transition was at 72 °C, but 2B domain unfolded at 61 °C. Black curves represent the thermogram whereas red and blue curves were depicting the two-state and three-state fitting. All the fittings were performed using origin 6.0. Thermal unfolding experiments were performed at 20 μM and 100 μM concentration for 1B and 2B respectively at a scan rate of 30 °C/hr in 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer.
    Figure Legend Snippet: Thermal denaturation of lamin 1B and 2B domain. The thermal denaturation studies of the 1B and 2B domains were performed using differential scanning calorimetry in panels ( A B ). 1B domain (panel A ) was unfolding via an intermediate pathway whereas 2B (panel B ) was denatured via the two-state mechanism. The first transition temperature for the 1B was at 63 °C and the final transition was at 72 °C, but 2B domain unfolded at 61 °C. Black curves represent the thermogram whereas red and blue curves were depicting the two-state and three-state fitting. All the fittings were performed using origin 6.0. Thermal unfolding experiments were performed at 20 μM and 100 μM concentration for 1B and 2B respectively at a scan rate of 30 °C/hr in 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer.

    Techniques Used: Concentration Assay

    20) Product Images from "Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases"

    Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.2109

    ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and BamH1 restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p
    Figure Legend Snippet: ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and BamH1 restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p

    Techniques Used: Clone Assay, Plasmid Preparation, Fluorescence, Enzyme-linked Immunosorbent Assay, In Vivo, SDS Page, Western Blot, Quantitative RT-PCR, Preserving

    21) Product Images from "Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle"

    Article Title: Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136679

    EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.
    Figure Legend Snippet: EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.

    Techniques Used: In Vivo

    EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.
    Figure Legend Snippet: EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.

    Techniques Used: In Vivo, Imaging, Electroporation, Expressing, Staining

    22) Product Images from "Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle"

    Article Title: Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136679

    EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.
    Figure Legend Snippet: EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo . Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.

    Techniques Used: In Vivo

    EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.
    Figure Legend Snippet: EHD1 modulates BIN1 mediated tubule formation in vivo . Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.

    Techniques Used: In Vivo, Imaging, Electroporation, Expressing, Staining

    23) Product Images from "TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells"

    Article Title: TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

    Journal: bioRxiv

    doi: 10.1101/2020.05.25.115329

    Identification of clonal dynamics of HBB-targeted HSPCs. a Top: Experimental schematic. Middle: Flow cytometry plots representing robust bi-lineage engraftment in primary transplant (left, week 18 post-transplant) and secondary transplant (right, week 12 post-transplant). Bottom: Bubble plots representing barcode alleles as unique colors from each indicated sorted population. Shown are the three most abundant clones from all six populations. All other barcodes represented as grey bubbles. b Normalized output of barcode alleles with respect to lineage contribution. Total cell output (bar graphs) from indicated barcodes adjusted for both differential lineage output and genome editing efficiency within each subset. Examples of various lineage skewing depicted, with cell counts proportional to the absolute contribution to the xenograft. Skewed output defined as 5-fold or greater bias in absolute cell counts towards lymphoid or myeloid lineages.
    Figure Legend Snippet: Identification of clonal dynamics of HBB-targeted HSPCs. a Top: Experimental schematic. Middle: Flow cytometry plots representing robust bi-lineage engraftment in primary transplant (left, week 18 post-transplant) and secondary transplant (right, week 12 post-transplant). Bottom: Bubble plots representing barcode alleles as unique colors from each indicated sorted population. Shown are the three most abundant clones from all six populations. All other barcodes represented as grey bubbles. b Normalized output of barcode alleles with respect to lineage contribution. Total cell output (bar graphs) from indicated barcodes adjusted for both differential lineage output and genome editing efficiency within each subset. Examples of various lineage skewing depicted, with cell counts proportional to the absolute contribution to the xenograft. Skewed output defined as 5-fold or greater bias in absolute cell counts towards lymphoid or myeloid lineages.

    Techniques Used: Flow Cytometry, Clone Assay

    Bubble plots depicting shared and unique barcode representation in all HBB barcoded mice at sacrifice. a Simpson diversity, representing barcode richness and evenness, for each lineage. b Left: representative quantification of all barcodes in descending order by read proportion. Right: Top four barcodes depicted as stacked bar chart. c-k Visualization of top 3 barcodes from all sorted populations (similar to Figure 4a ) from indicated mice. All other barcodes represented as grey bubbles. Error bars depict mean ± SEM. Note: Despite some colors appearing similar, no top barcodes are shared between distinct mice. See Supplemental Table 3 for individual HBB sequences and barcode counts.
    Figure Legend Snippet: Bubble plots depicting shared and unique barcode representation in all HBB barcoded mice at sacrifice. a Simpson diversity, representing barcode richness and evenness, for each lineage. b Left: representative quantification of all barcodes in descending order by read proportion. Right: Top four barcodes depicted as stacked bar chart. c-k Visualization of top 3 barcodes from all sorted populations (similar to Figure 4a ) from indicated mice. All other barcodes represented as grey bubbles. Error bars depict mean ± SEM. Note: Despite some colors appearing similar, no top barcodes are shared between distinct mice. See Supplemental Table 3 for individual HBB sequences and barcode counts.

    Techniques Used: Mouse Assay

    Design and production of barcoded AAV6 donors for long-term genetic tracking of gene targeted cells and their progeny. a Schematic of HBB targeting strategy. Top: Unmodified (WT) and barcoded HBB alleles depicted, with location of the E6V (GAG - > GTG) sickle cell disease mutation and CRISPR/Cas9 target sites labeled. Bottom: β-globin ORF translation with four barcode pools representing all possible silent mutations encoding amino acids 1-9. b Schematic of barcode library generation and experimental design. c/d Percentages of reads from each valid barcode identified through amplicon sequencing of plasmids (c) and AAV (d) pools 1, 2, and 4. e Recovery of barcodes from untreated genomic DNA containing 1, 3, 10, 30, and 95 individual plasmids containing HBB barcodes. Expected number of barcodes are plotted against the number of barcodes called by the TRACE-seq pipeline after filtering.
    Figure Legend Snippet: Design and production of barcoded AAV6 donors for long-term genetic tracking of gene targeted cells and their progeny. a Schematic of HBB targeting strategy. Top: Unmodified (WT) and barcoded HBB alleles depicted, with location of the E6V (GAG - > GTG) sickle cell disease mutation and CRISPR/Cas9 target sites labeled. Bottom: β-globin ORF translation with four barcode pools representing all possible silent mutations encoding amino acids 1-9. b Schematic of barcode library generation and experimental design. c/d Percentages of reads from each valid barcode identified through amplicon sequencing of plasmids (c) and AAV (d) pools 1, 2, and 4. e Recovery of barcodes from untreated genomic DNA containing 1, 3, 10, 30, and 95 individual plasmids containing HBB barcodes. Expected number of barcodes are plotted against the number of barcodes called by the TRACE-seq pipeline after filtering.

    Techniques Used: Mutagenesis, CRISPR, Labeling, Amplification, Sequencing

    24) Product Images from "Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome"

    Article Title: Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25606-2

    Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .
    Figure Legend Snippet: Localisation of ArLQP expression in the nervous system of A . rubens using mRNA in situ hybridisation. ( A ) Schematic showing the anatomy of the starfish arm as seen from a transverse section. ( B ) Schematic showing the anatomy of a radial nerve cord as seen in transverse section. ( C ) Transverse section of a radial nerve cord showing stained cells concentrated in the lateral parts of the ectoneural region. Higher magnification images of the boxed regions are shown in the panels ( D ) and ( E ). The inset shows absence of staining in a transverse section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. ( F ). Longitudinal parasagittal section of a radial nerve cord showing stained cells in the ectoneural region (arrowheads). A higher magnification of the boxed region is shown in the panel ( G ). ( H ) Transverse section of the circumoral nerve ring showing stained cells concentrated the lateral part of the ectoneural region. The boxed region is shown at higher magnification in panel I . am, apical muscle ; conr, circumoral nerve ring ; cut, cuticle ; ec, ectoneural region ; g, gonads ; hy, hyponeural region ; mn, marginal nerve ; pc, pyloric caeca ; pm, peristomial membrane ; rhs, radial hemal sinus ; rnc, radial nerve cord . Scale bars: 50 μm in C , C inset, F , H ; 10 μm in D , E , G , I .

    Techniques Used: Expressing, In Situ, Hybridization, Staining, Incubation

    25) Product Images from "Significance of 1B and 2B domains in modulating elastic properties of lamin A"

    Article Title: Significance of 1B and 2B domains in modulating elastic properties of lamin A

    Journal: Scientific Reports

    doi: 10.1038/srep27879

    Coiled-coil unzipping of 2B domain. Panels ( A,B ) show representative FX traces for the two-state and three-state unfolding of 2B. Similar to 1B domain lamin 2B dimers were also unfolding via several intermediates. The molecular design for SMFS experiment was (I27) 3 -2B-1C. Average unfolding forces for both the intermediate and main peaks were similar, at 75 pN. Cartoon picture represents the dimer of the 2B domain. In ( A ) first unfolding peak with 52 nm contour length increase corresponds to lamin 2B domain two-state unfolding and I27 domains are folding with 28 nm spacing at ~180 pN force. Panel ( B ) is representing the three state unzipping events. In ( C , D ) histograms for the contour length and unfolding force analysis has been shown respectively. The black bar is representing the value for the main peak whereas red is depicting the intermediate peaks. Average contour length increments for the main and intermediate peaks were ~52 nm and 25 nm, respectively. Almost 40% molecules were unzipping via an intermediate pathway. All these unfolding experiments for the 2B domains were done at 5–10 μM concentration using 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer at 25 °C.
    Figure Legend Snippet: Coiled-coil unzipping of 2B domain. Panels ( A,B ) show representative FX traces for the two-state and three-state unfolding of 2B. Similar to 1B domain lamin 2B dimers were also unfolding via several intermediates. The molecular design for SMFS experiment was (I27) 3 -2B-1C. Average unfolding forces for both the intermediate and main peaks were similar, at 75 pN. Cartoon picture represents the dimer of the 2B domain. In ( A ) first unfolding peak with 52 nm contour length increase corresponds to lamin 2B domain two-state unfolding and I27 domains are folding with 28 nm spacing at ~180 pN force. Panel ( B ) is representing the three state unzipping events. In ( C , D ) histograms for the contour length and unfolding force analysis has been shown respectively. The black bar is representing the value for the main peak whereas red is depicting the intermediate peaks. Average contour length increments for the main and intermediate peaks were ~52 nm and 25 nm, respectively. Almost 40% molecules were unzipping via an intermediate pathway. All these unfolding experiments for the 2B domains were done at 5–10 μM concentration using 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer at 25 °C.

    Techniques Used: Concentration Assay

    SMFS experiments of the coiled-coil 1B domain. ( A ) Cartoon represents the molecular construct used for the coiled-coil unzipping of 1B domain. The molecular construct for SMFS experiments was (I27) 3 -1B-1C. Representative force-extension (F-X) traces are shown in ( B , C ). ( B ) is representing the end-to-end two-state unzipping of the 1B dimer whereas ( C ) is showing the three-state unfolding. In both cases, the lamin force peak followed by six I27 force peaks with ~28 nm spacing in the F-X traces. Histograms for the contour length increment and the unfolding forces are shown in ( D ) and E respectively. Average uncoiling force for the 1B dimer is ~70 pN and contour length increase (ΔL c ) is ~85 nm. Almost 60% dimers were unfolding via several intermediates. Average unfolding force and (ΔL c ) for the intermediate peaks were ~84 pN and 45 nm respectively. WLC fits are also shown in FX curves. All the SMFS experiments for 1B were done at ~5 μM concentration in 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer at 25 °C.
    Figure Legend Snippet: SMFS experiments of the coiled-coil 1B domain. ( A ) Cartoon represents the molecular construct used for the coiled-coil unzipping of 1B domain. The molecular construct for SMFS experiments was (I27) 3 -1B-1C. Representative force-extension (F-X) traces are shown in ( B , C ). ( B ) is representing the end-to-end two-state unzipping of the 1B dimer whereas ( C ) is showing the three-state unfolding. In both cases, the lamin force peak followed by six I27 force peaks with ~28 nm spacing in the F-X traces. Histograms for the contour length increment and the unfolding forces are shown in ( D ) and E respectively. Average uncoiling force for the 1B dimer is ~70 pN and contour length increase (ΔL c ) is ~85 nm. Almost 60% dimers were unfolding via several intermediates. Average unfolding force and (ΔL c ) for the intermediate peaks were ~84 pN and 45 nm respectively. WLC fits are also shown in FX curves. All the SMFS experiments for 1B were done at ~5 μM concentration in 25 mM Tris-Cl (pH 8.5), 250 mM NaCl buffer at 25 °C.

    Techniques Used: Construct, Concentration Assay

    26) Product Images from "TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells"

    Article Title: TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

    Journal: bioRxiv

    doi: 10.1101/2020.05.25.115329

    Correction of the Sickle Cell Disease-causing E6V mutation using barcoded AAV6 donors in SCD-derived CD34 + HSPCs. a Experimental design – SCD patient derived CD34 + HSPCs edited with CRISPR/Cas9 RNP and electroporation only (mock), single donor (non-BC), or barcode donor (BC) AAV6 HDR templates. b SCD correction efficiency (percentage of corrected sickle cell alleles) of non-BC and BC treated groups as a fraction of total NGS reads (e.g. HR reads / [sum of HR reads + unmodified reads].) c Representative example of barcode fractions in descending order from one donor at day 14 time point. Right: Top 20 clones represented as stacked bar graph (representing 11.4% of reads). d Number of unique barcode alleles comprising the top 50% and top 90% of reads from each treatment condition, sampling approximately 1000 cells per condition (see Supplemental Table 1 ). e Representative hemoglobin tetramer HPLC chromatograms of RBC differentiated cell lysates at day 14 post treatment. f Quantification of total hemoglobin protein expression in each group. Each data point represents an individual biological replicate. HgbA: adult hemoglobin HgF: fetal hemoglobin HbS: sickle hemoglobin. AAV6: Recombinant AAV2/6 vector.
    Figure Legend Snippet: Correction of the Sickle Cell Disease-causing E6V mutation using barcoded AAV6 donors in SCD-derived CD34 + HSPCs. a Experimental design – SCD patient derived CD34 + HSPCs edited with CRISPR/Cas9 RNP and electroporation only (mock), single donor (non-BC), or barcode donor (BC) AAV6 HDR templates. b SCD correction efficiency (percentage of corrected sickle cell alleles) of non-BC and BC treated groups as a fraction of total NGS reads (e.g. HR reads / [sum of HR reads + unmodified reads].) c Representative example of barcode fractions in descending order from one donor at day 14 time point. Right: Top 20 clones represented as stacked bar graph (representing 11.4% of reads). d Number of unique barcode alleles comprising the top 50% and top 90% of reads from each treatment condition, sampling approximately 1000 cells per condition (see Supplemental Table 1 ). e Representative hemoglobin tetramer HPLC chromatograms of RBC differentiated cell lysates at day 14 post treatment. f Quantification of total hemoglobin protein expression in each group. Each data point represents an individual biological replicate. HgbA: adult hemoglobin HgF: fetal hemoglobin HbS: sickle hemoglobin. AAV6: Recombinant AAV2/6 vector.

    Techniques Used: Mutagenesis, Derivative Assay, CRISPR, Electroporation, Next-Generation Sequencing, Clone Assay, Sampling, High Performance Liquid Chromatography, Expressing, Recombinant, Plasmid Preparation

    Related Articles

    Ligation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells
    Article Snippet: .. The remaining CCGG sites and CATG sites were then digested with 21 μl of MspI (New England Biolabs) and NlaIII (New England Biolabs) digestion reaction mixture at 37°C for 3 h, and the enzymes were inactivated at 65°C for 20 min. MspI and NlaIII digested sites were then ligated with 7 μl of M-tag/N-tag adapter ligation reaction mixture at 25°C for 2 h and T4 DNA ligase HC (Thermo Scientific) was inactivated at 65°C for 20 min. 1 μl of Thermolabile USER® II enzyme (New England Biolabs) was used to remove excess M-tag adapters and N-tag adapters at 37°C for 20 min, 25°C for 20 min, and inactivated at 65°C for 20 min. .. Single stranded excess adapter oligonucleotides were eliminated by the addition of 1 μl of Exonuclease I (Enzymatics) at 37°C for 30 min.

    Magnetic Beads:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Lambda DNA Preparation:

    Article Title: Global DNA methylation in old subjects is correlated with frailty
    Article Snippet: .. One hundred nanograms of methylated and unmethylated lambda DNA were separately incubated with 5 U of HpaII and MspI restriction endonucleases (New England Biolabs) at 37°C overnight and successively at 65°C for 20 min to inactivate the endonucleases. .. The samples were loaded on a 1.4% agarose gel, electrophoresed in TAE (Tris/Acetate/EDTA) buffer and stained with ethidium bromide.

    SYBR Green Assay:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Incubation:

    Article Title: Global DNA methylation in old subjects is correlated with frailty
    Article Snippet: .. One hundred nanograms of methylated and unmethylated lambda DNA were separately incubated with 5 U of HpaII and MspI restriction endonucleases (New England Biolabs) at 37°C overnight and successively at 65°C for 20 min to inactivate the endonucleases. .. The samples were loaded on a 1.4% agarose gel, electrophoresed in TAE (Tris/Acetate/EDTA) buffer and stained with ethidium bromide.

    Methylation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Article Title: Global DNA methylation in old subjects is correlated with frailty
    Article Snippet: .. One hundred nanograms of methylated and unmethylated lambda DNA were separately incubated with 5 U of HpaII and MspI restriction endonucleases (New England Biolabs) at 37°C overnight and successively at 65°C for 20 min to inactivate the endonucleases. .. The samples were loaded on a 1.4% agarose gel, electrophoresed in TAE (Tris/Acetate/EDTA) buffer and stained with ethidium bromide.

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function
    Article Snippet: .. Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S). .. The reaction buffer included Cut Smart buffer 1 × (New England Biolabs), spermidine 0.1 M, DTT 0.1 M and 0.25 μl of RNaseH in 50 μl final volume.

    Sequencing:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Plasmid Preparation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

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    New England Biolabs bamh1 restriction enzymes
    ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and <t>BamH1</t> restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p
    Bamh1 Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bamh1 restriction enzymes/product/New England Biolabs
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    bamh1 restriction enzymes - by Bioz Stars, 2020-09
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    90
    New England Biolabs restriction enzyme bamh1
    ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and <t>BamH1</t> restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p
    Restriction Enzyme Bamh1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme bamh1/product/New England Biolabs
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    ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and BamH1 restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases

    doi: 10.1002/stem.2109

    Figure Lengend Snippet: ]. The cDNA was cloned in PLVX-IRES-ZsGreen-1 vector using flanking primers. Left panel indicates the vector map and right panel represents release of anti-HER2Ab cDNA followed by digestion of PLVX vector with EcoR1 and BamH1 restriction endonucleases. The lower panel shows PLVX vector control NSCs and NSCs endoding anti-HER2Ab, sorted based on GFP fluorescence. (B) Temporal secretion of anti-HER2Ab by HER2Ab-NSCs in cell supernatants using ELISA. Note a high production of anti-HER2Ab (~1μg) in 48 hrs. The experiments were repeated three times in triplicates and before in vivo injections of NSCs . (C) Determination of stable assembly of anti-HER2Ab secreted by NSC. First two panel shows SDS-PAGE seperation of trastuzumab (T) and anti-HER2Ab released by NSCs (N) under non-reducing and reducing condition respectively. Next two panels demonstrates western blotting of T and N using anti-Human-HRPO. (D) Quantitative RT-PCR of Nestin, Oct4 and βIII tubulin demonstrating preservation of stemness of HER2Ab-NSCs. The experiments were repeated three times in triplicates. * indicates p

    Article Snippet: Following amplification, the PLVX-IRES-ZsGreen1 plasmid (Clonetech, Mountain View, CA) and the PCR product was digested with EcoR1 and BamH1 restriction enzymes (NEB, Ipswich, MA).

    Techniques: Clone Assay, Plasmid Preparation, Fluorescence, Enzyme-linked Immunosorbent Assay, In Vivo, SDS Page, Western Blot, Quantitative RT-PCR, Preserving