Structured Review

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Phylogenetic tree of tul4 (17 kDa lipoprotein) gene of Francisella spp. obtained from 11 nucleotide sequences (200 bp) identified in feeding D. marginatus and D. reticulatus ticks, respectively collected from horse and wild boar in our study areas (AC: Alpi Cozie regional park; TP: Po Torinese natural park). Reference sequences are identified by GenBank accession number enclosed in parentheses. Bootstrap values (1000 replications) above 70 are shown next to the internal nodes. <t>Amplicons</t> from this study are indicated with a black circle (●).
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Images

1) Product Images from "Dermacentor marginatus and Dermacentor reticulatus, and Their Infection by SFG Rickettsiae and Francisella-Like Endosymbionts, in Mountain and Periurban Habitats of Northwestern Italy"

Article Title: Dermacentor marginatus and Dermacentor reticulatus, and Their Infection by SFG Rickettsiae and Francisella-Like Endosymbionts, in Mountain and Periurban Habitats of Northwestern Italy

Journal: Veterinary Sciences

doi: 10.3390/vetsci7040157

Phylogenetic tree of tul4 (17 kDa lipoprotein) gene of Francisella spp. obtained from 11 nucleotide sequences (200 bp) identified in feeding D. marginatus and D. reticulatus ticks, respectively collected from horse and wild boar in our study areas (AC: Alpi Cozie regional park; TP: Po Torinese natural park). Reference sequences are identified by GenBank accession number enclosed in parentheses. Bootstrap values (1000 replications) above 70 are shown next to the internal nodes. Amplicons from this study are indicated with a black circle (●).
Figure Legend Snippet: Phylogenetic tree of tul4 (17 kDa lipoprotein) gene of Francisella spp. obtained from 11 nucleotide sequences (200 bp) identified in feeding D. marginatus and D. reticulatus ticks, respectively collected from horse and wild boar in our study areas (AC: Alpi Cozie regional park; TP: Po Torinese natural park). Reference sequences are identified by GenBank accession number enclosed in parentheses. Bootstrap values (1000 replications) above 70 are shown next to the internal nodes. Amplicons from this study are indicated with a black circle (●).

Techniques Used:

Phylogenetic tree of OmpA gene of Rickettsia spp. obtained from 32 nucleotide sequences (426 bp) from D. marginatus and D. reticulatus ticks collected in the study areas (AC: Alpi Cozie regional park; TP: Po Torinese natural park). Reference sequences are identified by the GenBank accession number enclosed in parentheses. Bootstrap values (1000 replications) above 70 are shown next to the internal nodes. Amplicons obtained in this study are indicated with a black symbol: ● Feeding ticks from wild boar, red deer, Northern chamois, and humans; ▲ Dermacentor ticks collected from the vegetation; and ■ Ear biopsy collected from wild boar.
Figure Legend Snippet: Phylogenetic tree of OmpA gene of Rickettsia spp. obtained from 32 nucleotide sequences (426 bp) from D. marginatus and D. reticulatus ticks collected in the study areas (AC: Alpi Cozie regional park; TP: Po Torinese natural park). Reference sequences are identified by the GenBank accession number enclosed in parentheses. Bootstrap values (1000 replications) above 70 are shown next to the internal nodes. Amplicons obtained in this study are indicated with a black symbol: ● Feeding ticks from wild boar, red deer, Northern chamois, and humans; ▲ Dermacentor ticks collected from the vegetation; and ■ Ear biopsy collected from wild boar.

Techniques Used: Northern Blot

2) Product Images from "Step-by-step evolution of neo-sex chromosomes in geographical populations of wild silkmoths, Samia cynthia ssp."

Article Title: Step-by-step evolution of neo-sex chromosomes in geographical populations of wild silkmoths, Samia cynthia ssp.

Journal: Heredity

doi: 10.1038/hdy.2010.94

FISH mapping of S. cynthia orthologues of B. mori autosomal genes on female pachytene chromosome complements of S. cynthia subspecies. Red signals (arrowheads) are Cy3-labelled orthologous probes of the B. mori genes RpL4 ( a ), eEF-2 ( b ) and hemolin ( c ). GISH with Green-labelled female genomic probe (green signals) identified the original W-heterochromatin compartment of the neo-W chromosome and also highlighted a heterochromatin block on the NOR bivalent (asterisk) in S. cynthia subsp. indet. ( a , b ) and S. c. walkeri ( c ). Chromosomes were counterstained with DAPI (light blue). In S. cynthia subsp. indet., the RpL4 orthologue mapped to the NOR bivalent ( a ), whereas the eEF-2 orthologue mapped to an autosome bivalent ( b ). The hemolin orthologue mapped to the NOR bivalent in S. c. walkeri ( c ); note a conspicuous nucleolus (N) associated with the NOR bivalent. Bar represents 10 μm.
Figure Legend Snippet: FISH mapping of S. cynthia orthologues of B. mori autosomal genes on female pachytene chromosome complements of S. cynthia subspecies. Red signals (arrowheads) are Cy3-labelled orthologous probes of the B. mori genes RpL4 ( a ), eEF-2 ( b ) and hemolin ( c ). GISH with Green-labelled female genomic probe (green signals) identified the original W-heterochromatin compartment of the neo-W chromosome and also highlighted a heterochromatin block on the NOR bivalent (asterisk) in S. cynthia subsp. indet. ( a , b ) and S. c. walkeri ( c ). Chromosomes were counterstained with DAPI (light blue). In S. cynthia subsp. indet., the RpL4 orthologue mapped to the NOR bivalent ( a ), whereas the eEF-2 orthologue mapped to an autosome bivalent ( b ). The hemolin orthologue mapped to the NOR bivalent in S. c. walkeri ( c ); note a conspicuous nucleolus (N) associated with the NOR bivalent. Bar represents 10 μm.

Techniques Used: Fluorescence In Situ Hybridization, Blocking Assay

FISH identification of autosomal parts of the S. c. walkeri and S. cynthia subsp. indet. neo-sex chromosomes on female pachytene chromosomes of three subspecies of S. cynthia . The Cy3-labelled orthologous probe of the B. mori XDH 1 gene (red signals, arrowheads) hybridized to an autosome bivalent, but not to the ‘U-shaped' univalent of the Z chromosome in the female pachytene complement of S. c. ricini ( a ), whereas the XDH 1 orthologous probe mapped to autosomal parts of both the neo-W and neo-Z chromosomes in S. c. walkeri ( b ) and to autosomal parts of both the neo-W and Z 1 chromosomes in S. cynthia subsp. indet. ( c ). The Cy3-labelled orthologous probe of the B. mori lysozyme gene (red signals, arrowhead) hybridized to the same autosomal segment of both the neo-W and neo-Z chromosomes in S. c. walkeri ( d ). GISH with Green-labelled female genomic probe (green signals) identified the original W-heterochromatin parts of the neo-W chromosome in S. c. walkeri ( b , d ) and S. cynthia subsp. indet. ( c ). Chromosomes were counterstained with DAPI (light blue). Bar represents 10 μm.
Figure Legend Snippet: FISH identification of autosomal parts of the S. c. walkeri and S. cynthia subsp. indet. neo-sex chromosomes on female pachytene chromosomes of three subspecies of S. cynthia . The Cy3-labelled orthologous probe of the B. mori XDH 1 gene (red signals, arrowheads) hybridized to an autosome bivalent, but not to the ‘U-shaped' univalent of the Z chromosome in the female pachytene complement of S. c. ricini ( a ), whereas the XDH 1 orthologous probe mapped to autosomal parts of both the neo-W and neo-Z chromosomes in S. c. walkeri ( b ) and to autosomal parts of both the neo-W and Z 1 chromosomes in S. cynthia subsp. indet. ( c ). The Cy3-labelled orthologous probe of the B. mori lysozyme gene (red signals, arrowhead) hybridized to the same autosomal segment of both the neo-W and neo-Z chromosomes in S. c. walkeri ( d ). GISH with Green-labelled female genomic probe (green signals) identified the original W-heterochromatin parts of the neo-W chromosome in S. c. walkeri ( b , d ) and S. cynthia subsp. indet. ( c ). Chromosomes were counterstained with DAPI (light blue). Bar represents 10 μm.

Techniques Used: Fluorescence In Situ Hybridization

FISH identification of the autosomal segment of the neo-W chromosome homologous to the Z 2 chromosome in S. cynthia subsp. indet. on female pachytene chromosomes of S. c. walkeri ( a ) and S. cynthia subsp. indet. ( b , c ). The Cy3-labelled orthologous probe of the B. mori Topo II gene (red signals, arrowheads) hybridized to an autosomal bivalent but not to the neo-Wneo-Z bivalent in a female pachytene complement of S. c. walkeri ( a ), whereas in S. cynthia subsp. indet., the probe mapped to the Z 2 chromosome and the neo-W chromosome of the neo-WZ 1 Z 2 trivalent ( b ). The Cy3-labelled orthologous probe of the B. mori RpL18 gene (red signal, arrowhead) also hybridized to the Z 2 chromosome and the neo-W chromosome of the neo-WZ 1 Z 2 trivalent in S. cynthia subsp. indet. ( c ). GISH with Green-labelled female genomic probe (green signals) identified the original W-heterochromatin parts of the neo-W chromosome in S. c. walkeri ( a ) and S. cynthia subsp. indet. ( b , c ). Chromosomes were counterstained with DAPI (light blue). Bar represents 10 μm.
Figure Legend Snippet: FISH identification of the autosomal segment of the neo-W chromosome homologous to the Z 2 chromosome in S. cynthia subsp. indet. on female pachytene chromosomes of S. c. walkeri ( a ) and S. cynthia subsp. indet. ( b , c ). The Cy3-labelled orthologous probe of the B. mori Topo II gene (red signals, arrowheads) hybridized to an autosomal bivalent but not to the neo-Wneo-Z bivalent in a female pachytene complement of S. c. walkeri ( a ), whereas in S. cynthia subsp. indet., the probe mapped to the Z 2 chromosome and the neo-W chromosome of the neo-WZ 1 Z 2 trivalent ( b ). The Cy3-labelled orthologous probe of the B. mori RpL18 gene (red signal, arrowhead) also hybridized to the Z 2 chromosome and the neo-W chromosome of the neo-WZ 1 Z 2 trivalent in S. cynthia subsp. indet. ( c ). GISH with Green-labelled female genomic probe (green signals) identified the original W-heterochromatin parts of the neo-W chromosome in S. c. walkeri ( a ) and S. cynthia subsp. indet. ( b , c ). Chromosomes were counterstained with DAPI (light blue). Bar represents 10 μm.

Techniques Used: Fluorescence In Situ Hybridization

FISH mapping of S. cynthia orthologues of B. mori Z-linked genes on female pachytene chromosomes of three subspecies of S. cynthia. Red signals (arrowheads) are Cy3-labelled probes of BYB ( a – e ) and kettin ( f – h ) orthologous genes. GISH with Green-labelled female genomic probe (green signals) identified the original W compartment composed of heterochromatin ( b – e , g , h ) and also highlighted a block of heterochromatin in the NOR autosome (asterisk in b , d ). Chromosomes were counterstained with DAPI (light blue). S. c. ricini ( a , f ): the U-shaped, apparently self-paired univalent of the Z chromosome with BYB orthologue signals on both chromatids is seen in the pachytene complement besides 13 autosome bivalents; also note a conspicuous nucleolus (N) associated with one end of the NOR autosome ( a ); and a Z-chromosome univalent with kettin orthologue signals on both chromatids ( f ). S. c. walkeri ( b , c , g ): a pachytene complement composed of a neo-Wneo-Z sex chromosome bivalent and 12 autosome bivalents ( b ); signals of the BYB ( b , c ) and kettin ( g ) orthologous probes are located on the neo-Z chromosome. S. cynthia subsp. indet. ( d , e , h ): a pachytene complement consists of a neo-sex chromosome trivalent (neo-WZ 1 Z 2 ) and 11 autosome bivalents ( d ); signals of the BYB ( d , e ) and kettin ( h ) orthologous probes are located on the Z 1 chromosome. Bar represents 10 μm ( a – e , h ), 7.5 μm ( g ) and 5 μm ( f ).
Figure Legend Snippet: FISH mapping of S. cynthia orthologues of B. mori Z-linked genes on female pachytene chromosomes of three subspecies of S. cynthia. Red signals (arrowheads) are Cy3-labelled probes of BYB ( a – e ) and kettin ( f – h ) orthologous genes. GISH with Green-labelled female genomic probe (green signals) identified the original W compartment composed of heterochromatin ( b – e , g , h ) and also highlighted a block of heterochromatin in the NOR autosome (asterisk in b , d ). Chromosomes were counterstained with DAPI (light blue). S. c. ricini ( a , f ): the U-shaped, apparently self-paired univalent of the Z chromosome with BYB orthologue signals on both chromatids is seen in the pachytene complement besides 13 autosome bivalents; also note a conspicuous nucleolus (N) associated with one end of the NOR autosome ( a ); and a Z-chromosome univalent with kettin orthologue signals on both chromatids ( f ). S. c. walkeri ( b , c , g ): a pachytene complement composed of a neo-Wneo-Z sex chromosome bivalent and 12 autosome bivalents ( b ); signals of the BYB ( b , c ) and kettin ( g ) orthologous probes are located on the neo-Z chromosome. S. cynthia subsp. indet. ( d , e , h ): a pachytene complement consists of a neo-sex chromosome trivalent (neo-WZ 1 Z 2 ) and 11 autosome bivalents ( d ); signals of the BYB ( d , e ) and kettin ( h ) orthologous probes are located on the Z 1 chromosome. Bar represents 10 μm ( a – e , h ), 7.5 μm ( g ) and 5 μm ( f ).

Techniques Used: Fluorescence In Situ Hybridization, Blocking Assay

3) Product Images from "Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides New Insights into Sex Chromosome Evolution in Paradoxical Frogs"

Article Title: Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides New Insights into Sex Chromosome Evolution in Paradoxical Frogs

Journal: Genes

doi: 10.3390/genes10020160

Phylogenetic relationships of Pseudis and Lysapsus inferred by Bayesian analysis of the mitochondrial H1 and cytb fragments. Numbers at the nodes indicate posterior probability values. Pseudis fusca from Carlos Chagas – MG is in bold.
Figure Legend Snippet: Phylogenetic relationships of Pseudis and Lysapsus inferred by Bayesian analysis of the mitochondrial H1 and cytb fragments. Numbers at the nodes indicate posterior probability values. Pseudis fusca from Carlos Chagas – MG is in bold.

Techniques Used:

4) Product Images from "Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing"

Article Title: Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing

Journal: Viruses

doi: 10.3390/v6031219

Percentage of mappable DNA sequence reads (Y axis) at different levels of sequence depth contributing to the agro-begomovirus-betatsatellite clones used to inoculate the positive control plants, and the resultant contigs containing the begomovirus genomic or betasatellite sequences (X axis) produced by the de novo and reference-guided assemblies. 10 M = 10 million reads, 100 K = 100 thousand reads, TYLCV-OM = Tomato yellow leaf curl virus from Oman, TYLCB = Tomato yellow leaf curl betasatellite, CLCuGB = Cotton leaf curl Gezira betasatellite.
Figure Legend Snippet: Percentage of mappable DNA sequence reads (Y axis) at different levels of sequence depth contributing to the agro-begomovirus-betatsatellite clones used to inoculate the positive control plants, and the resultant contigs containing the begomovirus genomic or betasatellite sequences (X axis) produced by the de novo and reference-guided assemblies. 10 M = 10 million reads, 100 K = 100 thousand reads, TYLCV-OM = Tomato yellow leaf curl virus from Oman, TYLCB = Tomato yellow leaf curl betasatellite, CLCuGB = Cotton leaf curl Gezira betasatellite.

Techniques Used: Sequencing, Clone Assay, Positive Control, Produced

Phylogenetic relationships of helper begomovirus DNA sequences obtained from field samples, and from agro-inoculated positive control test plants, obtained by deep sequencing (in red), in relation to selected begomovirus reference sequences. The virus acronyms and accession numbers are as described by Idris et al ., (2011) [ 11 ].
Figure Legend Snippet: Phylogenetic relationships of helper begomovirus DNA sequences obtained from field samples, and from agro-inoculated positive control test plants, obtained by deep sequencing (in red), in relation to selected begomovirus reference sequences. The virus acronyms and accession numbers are as described by Idris et al ., (2011) [ 11 ].

Techniques Used: Positive Control, Sequencing

Percentage of mappable DNA sequence reads (Y axis) at different levels of sequence depth that contributed to sample G11 contigs containing the begomovirus genomic sequence (X axis) resulting from the de novo and reference-guided assemblies. 10 M = 10 million reads, 100 K = 100 thousand reads, ToLCSDV = Tomato leaf curl Sudan virus .
Figure Legend Snippet: Percentage of mappable DNA sequence reads (Y axis) at different levels of sequence depth that contributed to sample G11 contigs containing the begomovirus genomic sequence (X axis) resulting from the de novo and reference-guided assemblies. 10 M = 10 million reads, 100 K = 100 thousand reads, ToLCSDV = Tomato leaf curl Sudan virus .

Techniques Used: Sequencing

Phylogenetic relationships of alphasatellite DNA sequences obtained from field samples, and agro-inoculated positive control test plants, obtained by de novo assembly of the resultant DNA obtained from deep sequencing (in red), in relation to selected, reference alphasatellites. The alphasatellite acronyms and accession numbers are as described by Idris et al ., (2011) [ 11 ].
Figure Legend Snippet: Phylogenetic relationships of alphasatellite DNA sequences obtained from field samples, and agro-inoculated positive control test plants, obtained by de novo assembly of the resultant DNA obtained from deep sequencing (in red), in relation to selected, reference alphasatellites. The alphasatellite acronyms and accession numbers are as described by Idris et al ., (2011) [ 11 ].

Techniques Used: Positive Control, Sequencing

5) Product Images from "A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA"

Article Title: A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000380

Breakpoint identification by PAMP with an INK4A minigenomic tiling array. (A) Five groups of primers (F A , F B , R x , R Y and R Z , the small arrows and arrow heads) near the potential breakpoints were generated for PAMP based on our previous mapping [3] . The mapped CDKN2A breakpoints of the Detroit 562 cell line ( Figure 5 ) are indicated for clarification. The “E1”, “E2” and “E3” designations (blue fonts) are the relative positions of INK4A exons. The first exon of ARF is further to the right of this diagram and is not covered by this array. The tiling probes for the array are indicated with two alternating colors (short black and orange lines) for ease of identification. (B) The first row of the INK4A minigenomic array was spotted with the tiling probes shown in panel A. Cot-1 DNA (repetitive sequence of genomic DNA) spots are indicated on this array. The rest of the spots are herring sperm DNA. Both Cot-1 and herring sperm DNA are used as nonspecific controls. This array was hybridized with labeled samples derived from two cell lines. The same sets of primers (F A , F B , R Y and R Z ) were used for PAMP reactions on Detroit 562 (mutant) and HEK293 (wild type) genomic DNA to map the potential CDKN2A breakpoints. The amplicons were labeled with different dyes, yielding a green signal (Cy-3) for the mutant sample and a red signal (Cy-5) for the wild type sample, to be simultaneously hybridized on the array (two-color array). The two green spots on the first row revealed the breakpoint location as been discussed in Figure 2 .
Figure Legend Snippet: Breakpoint identification by PAMP with an INK4A minigenomic tiling array. (A) Five groups of primers (F A , F B , R x , R Y and R Z , the small arrows and arrow heads) near the potential breakpoints were generated for PAMP based on our previous mapping [3] . The mapped CDKN2A breakpoints of the Detroit 562 cell line ( Figure 5 ) are indicated for clarification. The “E1”, “E2” and “E3” designations (blue fonts) are the relative positions of INK4A exons. The first exon of ARF is further to the right of this diagram and is not covered by this array. The tiling probes for the array are indicated with two alternating colors (short black and orange lines) for ease of identification. (B) The first row of the INK4A minigenomic array was spotted with the tiling probes shown in panel A. Cot-1 DNA (repetitive sequence of genomic DNA) spots are indicated on this array. The rest of the spots are herring sperm DNA. Both Cot-1 and herring sperm DNA are used as nonspecific controls. This array was hybridized with labeled samples derived from two cell lines. The same sets of primers (F A , F B , R Y and R Z ) were used for PAMP reactions on Detroit 562 (mutant) and HEK293 (wild type) genomic DNA to map the potential CDKN2A breakpoints. The amplicons were labeled with different dyes, yielding a green signal (Cy-3) for the mutant sample and a red signal (Cy-5) for the wild type sample, to be simultaneously hybridized on the array (two-color array). The two green spots on the first row revealed the breakpoint location as been discussed in Figure 2 .

Techniques Used: Generated, Clarification Assay, Sequencing, Labeling, Derivative Assay, Mutagenesis

6) Product Images from "Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models"

Article Title: Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models

Journal: Nature Communications

doi: 10.1038/s41467-020-19396-3

Sig-1R, but not the Sig-1R- E 102 Q mutant, rescues the phenotype of flies expressing expanded (G4C2) repeats. a Western blot of human Sig-1R (Sigma-1 receptor) in Drosophila ; n = 3 independent experiments with similar results from biologically independent preparations. b Representative external eye morphology of flies (20 days after eclosion) expressing no transgene (control), 30 G4C2 repeats ((G4C2) 30 ) alone or together with human Sig-1R. c Quantification of flies presenting necrotic spots in the eyes; n = 4 independent sets of studies from a total of 91 flies in control, 48 in (G4C2) 30 , and 68 in (G4C2) 30 + Sig-1R group; statistics at the end. d Climbing performances (observation starts at 1 min) of 4-day-old flies expressing no transgene (control), 3 ((G4C2) 3 ) or 30 G4C2 repeats ((G4C2) 30 ). n = 8 flies/group; number of trials: control, 4; (G4C2) 3 , 5; (G4C2) 30 , 5. *** p
Figure Legend Snippet: Sig-1R, but not the Sig-1R- E 102 Q mutant, rescues the phenotype of flies expressing expanded (G4C2) repeats. a Western blot of human Sig-1R (Sigma-1 receptor) in Drosophila ; n = 3 independent experiments with similar results from biologically independent preparations. b Representative external eye morphology of flies (20 days after eclosion) expressing no transgene (control), 30 G4C2 repeats ((G4C2) 30 ) alone or together with human Sig-1R. c Quantification of flies presenting necrotic spots in the eyes; n = 4 independent sets of studies from a total of 91 flies in control, 48 in (G4C2) 30 , and 68 in (G4C2) 30 + Sig-1R group; statistics at the end. d Climbing performances (observation starts at 1 min) of 4-day-old flies expressing no transgene (control), 3 ((G4C2) 3 ) or 30 G4C2 repeats ((G4C2) 30 ). n = 8 flies/group; number of trials: control, 4; (G4C2) 3 , 5; (G4C2) 30 , 5. *** p

Techniques Used: Mutagenesis, Expressing, Western Blot

Sig-1R knockdown exacerbates the increase of cytoplasmic Ran caused by (G4C2)31-RNA: Imaging analyses. a The shRNA control (sh-C) or Sig-1R-shRNA (shSig-1R) vector was transiently transfected into HeLa cells for 24 h. The (G 4 C 2 ) 31 -RNA vector was then transfected into HeLa cells to produce the (G 4 C 2 ) 31 -RNA in the cell. Distribution of endogenous Ran (in green) was detected by immunostaining and confocal microscopy. b The semi-quantification of cytosolic or nuclear Ran was performed by using NIH Image J. (version 1.51b). Data are presented as means ± SEM; n = 18 for biologically independent control cells receiving scrambled shRNA plus (G 4 C 2 ) 31 -RNA; n = 21 for biologically independent cells receiving shSig-1R plus (G 4 C 2 ) 31 -RNA. Two-tailed unpaired Student’s t test, p = 0.0056. ** p
Figure Legend Snippet: Sig-1R knockdown exacerbates the increase of cytoplasmic Ran caused by (G4C2)31-RNA: Imaging analyses. a The shRNA control (sh-C) or Sig-1R-shRNA (shSig-1R) vector was transiently transfected into HeLa cells for 24 h. The (G 4 C 2 ) 31 -RNA vector was then transfected into HeLa cells to produce the (G 4 C 2 ) 31 -RNA in the cell. Distribution of endogenous Ran (in green) was detected by immunostaining and confocal microscopy. b The semi-quantification of cytosolic or nuclear Ran was performed by using NIH Image J. (version 1.51b). Data are presented as means ± SEM; n = 18 for biologically independent control cells receiving scrambled shRNA plus (G 4 C 2 ) 31 -RNA; n = 21 for biologically independent cells receiving shSig-1R plus (G 4 C 2 ) 31 -RNA. Two-tailed unpaired Student’s t test, p = 0.0056. ** p

Techniques Used: Imaging, shRNA, Plasmid Preparation, Transfection, Immunostaining, Confocal Microscopy, Two Tailed Test

Sig-1R knockout exacerbates whereas Sig-1R overexpression attenuates the cytoplasmic Ran accumulation caused by (G4C2) 31 -RNA: western blot analyses. a Apparent decrease of nuclear Ran in Sig-1R knockout cells when compared to that seen in wild-type cells. Subcellular fractionation followed by western blot was used to examine the level of Ran in the cytoplasm or nucleus in HeLa cells in which Sig-1Rs were depleted by the CRISPR/Cas9 technique. GFP-(G 4 C 2 ) 31 vectors were transiently transfected for 24 h into either wild-type or CRISPR/Cas9 Sig1R-KO cells before subcellular fractionation. b Sig-1R successfully overexpressed in (G4C2)31-RNA-treated cells. c Overexpression of Sig-1R decreased Ran in the cytoplasm while concomitantly increased Ran in the nucleus. d Sig-1R knockout apparently exacerbates the N/C ratio of Ran when compared to that seen in wild type. See Results for explanations. Quantitative summary of results from three sets of independent experiments illustrated in a . Data are presented as means ± SEM; n = 3 for each group; two-way ANOVA followed by Sidak’s multiple comparisons test, p = 0.0027 for wild-type group, p = 0.0003 for Sig-1R-KO group, ** p
Figure Legend Snippet: Sig-1R knockout exacerbates whereas Sig-1R overexpression attenuates the cytoplasmic Ran accumulation caused by (G4C2) 31 -RNA: western blot analyses. a Apparent decrease of nuclear Ran in Sig-1R knockout cells when compared to that seen in wild-type cells. Subcellular fractionation followed by western blot was used to examine the level of Ran in the cytoplasm or nucleus in HeLa cells in which Sig-1Rs were depleted by the CRISPR/Cas9 technique. GFP-(G 4 C 2 ) 31 vectors were transiently transfected for 24 h into either wild-type or CRISPR/Cas9 Sig1R-KO cells before subcellular fractionation. b Sig-1R successfully overexpressed in (G4C2)31-RNA-treated cells. c Overexpression of Sig-1R decreased Ran in the cytoplasm while concomitantly increased Ran in the nucleus. d Sig-1R knockout apparently exacerbates the N/C ratio of Ran when compared to that seen in wild type. See Results for explanations. Quantitative summary of results from three sets of independent experiments illustrated in a . Data are presented as means ± SEM; n = 3 for each group; two-way ANOVA followed by Sidak’s multiple comparisons test, p = 0.0027 for wild-type group, p = 0.0003 for Sig-1R-KO group, ** p

Techniques Used: Knock-Out, Over Expression, Western Blot, Fractionation, CRISPR, Transfection

Localization and association of HA-tagged Sig-1R, RanGAP1, and a nuclear pore complex proteins Nup62 in HeLa cells. a Immunohistochemistry followed by confocal microscopic examination indicates perinuclear colocalizations of immunoreactive HA-tagged Sig-1R (green) with RanGAP (red), Nup62 (red) and Nup358 (red). HeLa cells transiently transfected with human HA-Sig-1R vectors were used. b Coimmunoprecipitation (Co-IP) of GFP-tagged Sig-1R with RanGAP and Ran. HeLa cells were transfected with GFP or GFP-Sig-1R vectors for 24 h before the co-IP experiment. Proteins interacting with GFP (control) or GFP-Sig-1R were detected by western blot. c NuPs’ interaction with Sig-1R in a co-IP experiment. The mAb414 pulled down FG repeats-containing Nups together with Sig-1R-V5-His which was transfected into HeLa cells. d Nup50 antibody co-IPed with endogenous Sig-1R which in this experiment was detected by the Santa Cruz B5 anti-Sig-1R antibody (sc137075). Note: all other endogenous Sig-1Rs in western blot in the cell line portion of this study was detected by custom-made anti-Sig-1R antiserum #5460 (see Methods section). The two Sig-1R antibodies have been used interchangeably in the lab to reserve #5460 which is custom-made polyclonal and is limited in quantity (see Methods section). Note: Santa Cruz B5 anti-Sig-1R is monoclonal, thus almost unlimited. Note: colocalization of endogenous Sig-1R with RanGAP1 and Nup62 in HeLa cells is shown in Supplementary Fig. S1 . Sig-1R, Sigma-1 receptor, RanGAP RanGTP-activating protein, Nup nucleoporin. n = 4 ( a ), n = 4 ( b ), n = 3 ( c ), and n = 3 ( d ) independent experiments with similar results each from biologically independent cells or cellular preparations.
Figure Legend Snippet: Localization and association of HA-tagged Sig-1R, RanGAP1, and a nuclear pore complex proteins Nup62 in HeLa cells. a Immunohistochemistry followed by confocal microscopic examination indicates perinuclear colocalizations of immunoreactive HA-tagged Sig-1R (green) with RanGAP (red), Nup62 (red) and Nup358 (red). HeLa cells transiently transfected with human HA-Sig-1R vectors were used. b Coimmunoprecipitation (Co-IP) of GFP-tagged Sig-1R with RanGAP and Ran. HeLa cells were transfected with GFP or GFP-Sig-1R vectors for 24 h before the co-IP experiment. Proteins interacting with GFP (control) or GFP-Sig-1R were detected by western blot. c NuPs’ interaction with Sig-1R in a co-IP experiment. The mAb414 pulled down FG repeats-containing Nups together with Sig-1R-V5-His which was transfected into HeLa cells. d Nup50 antibody co-IPed with endogenous Sig-1R which in this experiment was detected by the Santa Cruz B5 anti-Sig-1R antibody (sc137075). Note: all other endogenous Sig-1Rs in western blot in the cell line portion of this study was detected by custom-made anti-Sig-1R antiserum #5460 (see Methods section). The two Sig-1R antibodies have been used interchangeably in the lab to reserve #5460 which is custom-made polyclonal and is limited in quantity (see Methods section). Note: Santa Cruz B5 anti-Sig-1R is monoclonal, thus almost unlimited. Note: colocalization of endogenous Sig-1R with RanGAP1 and Nup62 in HeLa cells is shown in Supplementary Fig. S1 . Sig-1R, Sigma-1 receptor, RanGAP RanGTP-activating protein, Nup nucleoporin. n = 4 ( a ), n = 4 ( b ), n = 3 ( c ), and n = 3 ( d ) independent experiments with similar results each from biologically independent cells or cellular preparations.

Techniques Used: Immunohistochemistry, Transfection, Co-Immunoprecipitation Assay, Western Blot

Colocalization of HA-tagged Sig-1R, RanGAP, and Nup62 in differentiated NSC-34 motoneuron-like cells. a Colocalization of HA-Sig-1R with RanGAP. Cells were transiently transfected with pcDNA-HA-Sig-1R, using Lipofectamine 2000, which provided ~50% of transfection efficiency in NSC-34 cells ( https://www.thermofisher.com ). Two days after transfection, cells were double-labeled with anti-HA and anti-RanGAP antibodies and examined by confocal microscopy. HA-Sig-1R, green; endogenous RanGAP, red; DNA, blue. b , c Multiple focal planes (Z sections) of the whole nuclear volume were acquired by the DeltaVision microscopy imaging systems. Results of the whole-nucleus image analysis of Sig-1R and RanGAP ( b ) or Nup62 ( c ) are shown. On left panels of b and c , square images are the top-down view ( z -axis), and rectangle panels are a side view ( x -axis) of the 3D reconstruction of images. On right panels of b and c , 20 sections were obtained from a cell. Number 1 is the Z-start at the top surface of the cellular nucleus; number 20 is Z-end at the bottom layer of the nucleus which was near the attachment of the cell to the coverslip. On central panels, white dotted arrows in images of the two sections 11 indicate the track of fluorescence intensity profiles (ImageJ: Plot Profile command) along the arrows. A shift of each focal plane in the Z -axis is 0.25 μm. Three-dimensional reconstructions were made from the Z-series images. Again: HA-Sig-1R, green; endogenous RanGAP/Nup62, red; DNA, blue. Sig-1R, Sigma-1 receptor, RanGAP RanGTP-activating protein, Nup62 nucleoporin 62. n = 3 independent experiments with similar results from biologically independent cells.
Figure Legend Snippet: Colocalization of HA-tagged Sig-1R, RanGAP, and Nup62 in differentiated NSC-34 motoneuron-like cells. a Colocalization of HA-Sig-1R with RanGAP. Cells were transiently transfected with pcDNA-HA-Sig-1R, using Lipofectamine 2000, which provided ~50% of transfection efficiency in NSC-34 cells ( https://www.thermofisher.com ). Two days after transfection, cells were double-labeled with anti-HA and anti-RanGAP antibodies and examined by confocal microscopy. HA-Sig-1R, green; endogenous RanGAP, red; DNA, blue. b , c Multiple focal planes (Z sections) of the whole nuclear volume were acquired by the DeltaVision microscopy imaging systems. Results of the whole-nucleus image analysis of Sig-1R and RanGAP ( b ) or Nup62 ( c ) are shown. On left panels of b and c , square images are the top-down view ( z -axis), and rectangle panels are a side view ( x -axis) of the 3D reconstruction of images. On right panels of b and c , 20 sections were obtained from a cell. Number 1 is the Z-start at the top surface of the cellular nucleus; number 20 is Z-end at the bottom layer of the nucleus which was near the attachment of the cell to the coverslip. On central panels, white dotted arrows in images of the two sections 11 indicate the track of fluorescence intensity profiles (ImageJ: Plot Profile command) along the arrows. A shift of each focal plane in the Z -axis is 0.25 μm. Three-dimensional reconstructions were made from the Z-series images. Again: HA-Sig-1R, green; endogenous RanGAP/Nup62, red; DNA, blue. Sig-1R, Sigma-1 receptor, RanGAP RanGTP-activating protein, Nup62 nucleoporin 62. n = 3 independent experiments with similar results from biologically independent cells.

Techniques Used: Transfection, Labeling, Confocal Microscopy, Microscopy, Imaging, Fluorescence

Human Sig-1R interaction with (G4C2) 10 -RNA: direct binding assay with purified molecules in the in vitro cellular assay. a Purified human sigma-1 receptor (Sig-1R) directly binds (G4C2) 10 -RNA in a chemical reaction in test tube. The biotin pull-down assay plus western-blot analysis were performed to detect the association of biotin-labeled (G 4 C 2 ) 10 -RNA and GST-tagged human Sig-1R. b (G4C2) 10 -RNA binds the endogenous Sig-1R in rat liver microsomal preparation. Biotin-labeled (G4C2) 10 -RNA was incubated with lysates from rat liver microsomes followed by the biotin pull-down assay plus western-blot to detect the association of biotin-labeled (G 4 C 2 ) 10 -RNA and endogenous Sig-1R. c Scrambled RNA repeats failed to bind Sig-1R. Biotin-labeled (A 2 U 2 GC) 10 -RNA was chosen as the scrambled RNA control. HeLa cells were transfected with mouse Sig-1R-YFP (mSig-1R-YFP) vectors for 24 h before the biotin pull-down assay plus western blot. Note: compare lane 4 vs lane 8. d Sig-1R mutation from glutamic acid to glutamine at amino acid 102 (i.e., Sig-1R-E102Q) has a lower affinity for (G4C2) 10 -RNA. NG-108 cells were transiently transfected with wild-type human Sig-1R-YFP or Sig-1R-E102Q-YFP vector for 24 h before the biotin pull-down assay plus western blot. Note: compare lane 5 vs lane 6. Sig-1R, Sigma-1 receptor. n = 4 ( a ), n = 3 ( b ), n = 3 ( c ), and n = 3 ( d ) independent experiments with similar results from biologically independent cellular preparations.
Figure Legend Snippet: Human Sig-1R interaction with (G4C2) 10 -RNA: direct binding assay with purified molecules in the in vitro cellular assay. a Purified human sigma-1 receptor (Sig-1R) directly binds (G4C2) 10 -RNA in a chemical reaction in test tube. The biotin pull-down assay plus western-blot analysis were performed to detect the association of biotin-labeled (G 4 C 2 ) 10 -RNA and GST-tagged human Sig-1R. b (G4C2) 10 -RNA binds the endogenous Sig-1R in rat liver microsomal preparation. Biotin-labeled (G4C2) 10 -RNA was incubated with lysates from rat liver microsomes followed by the biotin pull-down assay plus western-blot to detect the association of biotin-labeled (G 4 C 2 ) 10 -RNA and endogenous Sig-1R. c Scrambled RNA repeats failed to bind Sig-1R. Biotin-labeled (A 2 U 2 GC) 10 -RNA was chosen as the scrambled RNA control. HeLa cells were transfected with mouse Sig-1R-YFP (mSig-1R-YFP) vectors for 24 h before the biotin pull-down assay plus western blot. Note: compare lane 4 vs lane 8. d Sig-1R mutation from glutamic acid to glutamine at amino acid 102 (i.e., Sig-1R-E102Q) has a lower affinity for (G4C2) 10 -RNA. NG-108 cells were transiently transfected with wild-type human Sig-1R-YFP or Sig-1R-E102Q-YFP vector for 24 h before the biotin pull-down assay plus western blot. Note: compare lane 5 vs lane 6. Sig-1R, Sigma-1 receptor. n = 4 ( a ), n = 3 ( b ), n = 3 ( c ), and n = 3 ( d ) independent experiments with similar results from biologically independent cellular preparations.

Techniques Used: Binding Assay, Purification, In Vitro, Pull Down Assay, Western Blot, Labeling, Incubation, Transfection, Mutagenesis, Plasmid Preparation

Sig-1R regulates nuclear pore protein stability. a Knockdown of sigma-1 receptor (Sig-1R) by shRNA (shSig-1R) dose-dependently caused a reduction of nucleoporins (Nups). HeLa cells were transiently transfected with either shRNA control (sh-C) or different doses of shSig1R vector. Forty-eight hours after transfection, western blot was performed to detect protein expression levels of Sig-1R, Actin, and Nups. In the western blot, Nup62 was probed by mAb414 or a specific Nup62 antibody. b Sig-1R knockdown dose-dependently reduced the level of Nup50. c Faster turnover of Nup358 or Nup214 in Sig-1R knockdown cells. Cycloheximide (100 µg/ml) was added to stop de novo synthesis of proteins. Time-lapsed levels of Nups were examined by western blot probed by mAb414. d – f Summarized results from three sets of independent turnover studies on Nup358, Nup214, and Nup50. Data were subjected to two-way ANOVA followed by Sidak’s multiple comparisons test (Graphpad Prism version 8.2). For d Nup358, p values are 0.022, 0.001, 0.0045, and 0.0801 for 2 h, 4 h, 6 h, and 8 h, respectively; for e Nup214, p values are 0.7858, 0.1538, 0.0004, and
Figure Legend Snippet: Sig-1R regulates nuclear pore protein stability. a Knockdown of sigma-1 receptor (Sig-1R) by shRNA (shSig-1R) dose-dependently caused a reduction of nucleoporins (Nups). HeLa cells were transiently transfected with either shRNA control (sh-C) or different doses of shSig1R vector. Forty-eight hours after transfection, western blot was performed to detect protein expression levels of Sig-1R, Actin, and Nups. In the western blot, Nup62 was probed by mAb414 or a specific Nup62 antibody. b Sig-1R knockdown dose-dependently reduced the level of Nup50. c Faster turnover of Nup358 or Nup214 in Sig-1R knockdown cells. Cycloheximide (100 µg/ml) was added to stop de novo synthesis of proteins. Time-lapsed levels of Nups were examined by western blot probed by mAb414. d – f Summarized results from three sets of independent turnover studies on Nup358, Nup214, and Nup50. Data were subjected to two-way ANOVA followed by Sidak’s multiple comparisons test (Graphpad Prism version 8.2). For d Nup358, p values are 0.022, 0.001, 0.0045, and 0.0801 for 2 h, 4 h, 6 h, and 8 h, respectively; for e Nup214, p values are 0.7858, 0.1538, 0.0004, and

Techniques Used: shRNA, Transfection, Plasmid Preparation, Western Blot, Expressing

7) Product Images from "Cell Type-Specific Replication of Simian Virus 40 Conferred by Hormone Response Elements in the Late Promoter"

Article Title: Cell Type-Specific Replication of Simian Virus 40 Conferred by Hormone Response Elements in the Late Promoter

Journal: Journal of Virology

doi: 10.1128/JVI.76.13.6762-6770.2002

). (B) Sequences of the wild-type and mutant double-stranded HRE-containing oligonucleotides used in this study. Only the strand corresponding to the viral late RNA is shown. The bases altered in the mutants are underlined and in small capitals. The numbers in parentheses indicate the SV40 nucleotides to which the oligonucleotides correspond in the SV numbering system.
Figure Legend Snippet: ). (B) Sequences of the wild-type and mutant double-stranded HRE-containing oligonucleotides used in this study. Only the strand corresponding to the viral late RNA is shown. The bases altered in the mutants are underlined and in small capitals. The numbers in parentheses indicate the SV40 nucleotides to which the oligonucleotides correspond in the SV numbering system.

Techniques Used: Mutagenesis

8) Product Images from "CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals"

Article Title: CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals

Journal: bioRxiv

doi: 10.1101/2020.06.25.171538

Schematic representation of the screening pipeline. Starting from hundreds of samples the amplicon generation takes place by preparing the individuals for sequencing. By the end of the sequencing run the demultiplexing and analysis can take place that can lead to the identification of the desired edited individuals.
Figure Legend Snippet: Schematic representation of the screening pipeline. Starting from hundreds of samples the amplicon generation takes place by preparing the individuals for sequencing. By the end of the sequencing run the demultiplexing and analysis can take place that can lead to the identification of the desired edited individuals.

Techniques Used: Amplification, Sequencing

Amplicon preparation. (a) Diagram of the targeted gene, ISOCHORISMATE SYNTHASE 1 ( ICS1 ). Black boxes indicate exons, and grey boxes untranslated regions. The arrow shows the direction of transcription. (b) - (d) Amplicon preparation. (b) The first PCR step to amplify a specific region of the genome. The oligonucleotide primers in this step fuse the first part of the TruSeq adapters (grey) and the frame shifting nucleotides (red). (c) The second PCR amplification adds the last part of the TruSeq adapters (purple) and one of the 96 barcodes (orange). (d) The final amplicon with frameshifting base pairs(s) (red), TruSeq adapters (grey and purple) and barcode (orange).
Figure Legend Snippet: Amplicon preparation. (a) Diagram of the targeted gene, ISOCHORISMATE SYNTHASE 1 ( ICS1 ). Black boxes indicate exons, and grey boxes untranslated regions. The arrow shows the direction of transcription. (b) - (d) Amplicon preparation. (b) The first PCR step to amplify a specific region of the genome. The oligonucleotide primers in this step fuse the first part of the TruSeq adapters (grey) and the frame shifting nucleotides (red). (c) The second PCR amplification adds the last part of the TruSeq adapters (purple) and one of the 96 barcodes (orange). (d) The final amplicon with frameshifting base pairs(s) (red), TruSeq adapters (grey and purple) and barcode (orange).

Techniques Used: Amplification, Polymerase Chain Reaction

9) Product Images from "Novel DNA Microarray System for Analysis of Nascent mRNAs"

Article Title: Novel DNA Microarray System for Analysis of Nascent mRNAs

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn015

( A ) Eluted RNAs were converted to cDNA, and cDNA was amplified by TS-PCR. ( B ) Validation with quantitative RT–PCR of the DNA microarray data using each amplification method. Genes expressed at various levels in the non-amplification method, TS-PCR amplification method, and T7 in vitro transcription (IVT) amplification method were confirmed using quantitative RT–PCR. Quantitative RT–PCR was performed using the total RNA of these transcription factors. Gapd was used as the control. The same total RNA was used for quantitative RT–PCR and DNA microarray experiments. The ratio of the total target RNA levels was normalized by using those of the Tbp ( Mus musculus TATA box binding protein) gene, and the fold change for each gene in the differentiated ES cells compared to the control ES cells was calculated. Error bars represent standard deviations of log (base 2) of ratio. ( C ) Amplified cDNA labeled with Cy3/Cy5 underwent self-self hybridization.
Figure Legend Snippet: ( A ) Eluted RNAs were converted to cDNA, and cDNA was amplified by TS-PCR. ( B ) Validation with quantitative RT–PCR of the DNA microarray data using each amplification method. Genes expressed at various levels in the non-amplification method, TS-PCR amplification method, and T7 in vitro transcription (IVT) amplification method were confirmed using quantitative RT–PCR. Quantitative RT–PCR was performed using the total RNA of these transcription factors. Gapd was used as the control. The same total RNA was used for quantitative RT–PCR and DNA microarray experiments. The ratio of the total target RNA levels was normalized by using those of the Tbp ( Mus musculus TATA box binding protein) gene, and the fold change for each gene in the differentiated ES cells compared to the control ES cells was calculated. Error bars represent standard deviations of log (base 2) of ratio. ( C ) Amplified cDNA labeled with Cy3/Cy5 underwent self-self hybridization.

Techniques Used: Amplification, Polymerase Chain Reaction, Quantitative RT-PCR, Microarray, In Vitro, Binding Assay, Labeling, Hybridization

10) Product Images from "The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer"

Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1515

LARP1 regulates stability at the level of the 3′ UTR and binds the BCL2 3′ UTR via its DM15 domain. ( A ) Schematics outlining construction of 3′-untranslated region (3′ UTR) reporter constructs for BIK and BCL2. Both were compared relative to a control vector with no additional 3′ UTR sequence. ( B ) OVCAR8 and SKOV3 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector. Renilla luciferase activity following LARP1 knockdown was determined for each 3′ UTR construct cells. Data was normalized to Firefly luciferase activity. ( C ) OVCAR8 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector and Renilla luciferase mRNA abundance following LARP1 knockdown was determined using RT-qPCR. ( D ) Electrophoretic mobility shift assay (EMSA) of LARP1 with a fragment of the BCL2 3′ UTR (construct 3). The affinity of this interaction is indicated. ( E ) Competition experiment analysed by EMSA of LARP1 pre-bound to radiolabelled RNA representing the 5′ TOP of RPS6 and competed with cold competitors, as indicated. ( F ) Confocal immunofluorescence microscopy of SKOV3 cells treated with sodium arsenite to trigger aggregation of mRNP bodies. Cells were stained for LARP1 protein (green) and either the P-body marker DCP1a or stress granule marker PABP (both red). Scale bar 10 μm (top) and 25 μm (bottom).
Figure Legend Snippet: LARP1 regulates stability at the level of the 3′ UTR and binds the BCL2 3′ UTR via its DM15 domain. ( A ) Schematics outlining construction of 3′-untranslated region (3′ UTR) reporter constructs for BIK and BCL2. Both were compared relative to a control vector with no additional 3′ UTR sequence. ( B ) OVCAR8 and SKOV3 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector. Renilla luciferase activity following LARP1 knockdown was determined for each 3′ UTR construct cells. Data was normalized to Firefly luciferase activity. ( C ) OVCAR8 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector and Renilla luciferase mRNA abundance following LARP1 knockdown was determined using RT-qPCR. ( D ) Electrophoretic mobility shift assay (EMSA) of LARP1 with a fragment of the BCL2 3′ UTR (construct 3). The affinity of this interaction is indicated. ( E ) Competition experiment analysed by EMSA of LARP1 pre-bound to radiolabelled RNA representing the 5′ TOP of RPS6 and competed with cold competitors, as indicated. ( F ) Confocal immunofluorescence microscopy of SKOV3 cells treated with sodium arsenite to trigger aggregation of mRNP bodies. Cells were stained for LARP1 protein (green) and either the P-body marker DCP1a or stress granule marker PABP (both red). Scale bar 10 μm (top) and 25 μm (bottom).

Techniques Used: Construct, Plasmid Preparation, Sequencing, Transfection, Luciferase, Activity Assay, Quantitative RT-PCR, Electrophoretic Mobility Shift Assay, Immunofluorescence, Microscopy, Staining, Marker

LARP1 is a component of BCL2-containing mRNP complexes and promotes transcript stability. ( A ) Schematic of LARP1 RNA-immunoprecipitation (RIP) with representative western blot of LARP1 protein following LARP1-immunoprecipiation in OVCAR8 and SKOV3 cells. ( B ) RT-qPCR analysis of BCL2 and BIK mRNA obtained from RIP with anti-LARP1 and IgG control antibodies in OVCAR8 cells. OZA1 and 28S were included as negative controls. ( C ) Representative cell images following LARP1 knockdown and 8 h exposure to actinomycin D (Scale bar 200 μm). ( D ) Following transient knockdown of LARP1, OVCAR8 cells were treated with actinomycin D to halt transcription and apoptosis (as determined by cleaved Caspase 3/7) was monitored using the CaspaseGlo assay (data normalized to T = 0, at the time of actinomycin-D administration). ** P
Figure Legend Snippet: LARP1 is a component of BCL2-containing mRNP complexes and promotes transcript stability. ( A ) Schematic of LARP1 RNA-immunoprecipitation (RIP) with representative western blot of LARP1 protein following LARP1-immunoprecipiation in OVCAR8 and SKOV3 cells. ( B ) RT-qPCR analysis of BCL2 and BIK mRNA obtained from RIP with anti-LARP1 and IgG control antibodies in OVCAR8 cells. OZA1 and 28S were included as negative controls. ( C ) Representative cell images following LARP1 knockdown and 8 h exposure to actinomycin D (Scale bar 200 μm). ( D ) Following transient knockdown of LARP1, OVCAR8 cells were treated with actinomycin D to halt transcription and apoptosis (as determined by cleaved Caspase 3/7) was monitored using the CaspaseGlo assay (data normalized to T = 0, at the time of actinomycin-D administration). ** P

Techniques Used: Immunoprecipitation, Western Blot, Quantitative RT-PCR, Caspase-Glo Assay

11) Product Images from "RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells"

Article Title: RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells

Journal: Nature Communications

doi: 10.1038/ncomms6220

RNaseH1 functions at ALT telomeres. ( a ) Quantification of ChIP dot blot experiments performed in the indicated cell lines using antibodies against endogenous RNaseH1. DNA was first hybridized with telomeric probes and then with Alu repeat probes for specificity. Immunoprecipitated DNA is expressed as fraction of input DNA after subtraction of the background signal associated to control ChIPs using normal IgGs. Bars and error bars are averages and s.d. from three to five experiments. ( b ) Quantification of telomeric hybrids from the indicated chromosome ends and actin locus measured in S9.6 DIP experiments using cells transfected for 72 h with control siRNAs (siCtrl, set to 1) or siRNAs against RNaseH1 (siRH1a and c). Hybrids are expressed as fractions of the input material after subtraction of values from control immunoprecipitations with only beads. Bars and error bars are averages and s.d. from at least three experiments. P -values were computed using the Student’s t -test. * P
Figure Legend Snippet: RNaseH1 functions at ALT telomeres. ( a ) Quantification of ChIP dot blot experiments performed in the indicated cell lines using antibodies against endogenous RNaseH1. DNA was first hybridized with telomeric probes and then with Alu repeat probes for specificity. Immunoprecipitated DNA is expressed as fraction of input DNA after subtraction of the background signal associated to control ChIPs using normal IgGs. Bars and error bars are averages and s.d. from three to five experiments. ( b ) Quantification of telomeric hybrids from the indicated chromosome ends and actin locus measured in S9.6 DIP experiments using cells transfected for 72 h with control siRNAs (siCtrl, set to 1) or siRNAs against RNaseH1 (siRH1a and c). Hybrids are expressed as fractions of the input material after subtraction of values from control immunoprecipitations with only beads. Bars and error bars are averages and s.d. from at least three experiments. P -values were computed using the Student’s t -test. * P

Techniques Used: Chromatin Immunoprecipitation, Dot Blot, Immunoprecipitation, Transfection

12) Product Images from "Leptospira interrogans serovar Copenhageni Harbors Two lexA Genes Involved in SOS Response"

Article Title: Leptospira interrogans serovar Copenhageni Harbors Two lexA Genes Involved in SOS Response

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076419

Genomic and transcriptional organization of the lexA2 region. (A) Schematic representation of the lexA2 genomic region from L. interrogans serovar Copenhageni (upper) compared to the equivalent region of serovar Lai (lower). Arrows represent predicted genes and transcription orientation. Light grey arrows represent genes orthologous between genomes, dark grey genes that are specific to Copenhageni and black arrows indicate genes encoding transposases. The white arrows represent genes with truncated versions in Lai genome (traced arrows) by insertion of IS elements. Remnants of a phage integrase are indicated by a traced line. The numbered bars below the genes indicate the amplified fragments corresponding to the primer pairs used in the RT-PCR analyses. (B) RT-PCR reactions, using either genomic DNA (gDNA), RNA (RT-) or cDNA (RT+) as templates, and primers flanking intergenic regions. The numbers refer to the respective fragments shown in (A).
Figure Legend Snippet: Genomic and transcriptional organization of the lexA2 region. (A) Schematic representation of the lexA2 genomic region from L. interrogans serovar Copenhageni (upper) compared to the equivalent region of serovar Lai (lower). Arrows represent predicted genes and transcription orientation. Light grey arrows represent genes orthologous between genomes, dark grey genes that are specific to Copenhageni and black arrows indicate genes encoding transposases. The white arrows represent genes with truncated versions in Lai genome (traced arrows) by insertion of IS elements. Remnants of a phage integrase are indicated by a traced line. The numbered bars below the genes indicate the amplified fragments corresponding to the primer pairs used in the RT-PCR analyses. (B) RT-PCR reactions, using either genomic DNA (gDNA), RNA (RT-) or cDNA (RT+) as templates, and primers flanking intergenic regions. The numbers refer to the respective fragments shown in (A).

Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

Genomic and transcriptional organization of the lexA1 region. (A) Schematic representation of the lexA1 genomic region. The arrows indicate the direction of transcription. The fragments amplified by the primer pairs used for the RT-PCR analysis are indicated by numbered lines below the genes. (B) Composite image of agarose gels from resulting RT-PCR reactions, using either genomic DNA (DNA), RNA (RT-) or cDNA (RT+) as templates. The numbers refer to the respective fragments shown in (A).
Figure Legend Snippet: Genomic and transcriptional organization of the lexA1 region. (A) Schematic representation of the lexA1 genomic region. The arrows indicate the direction of transcription. The fragments amplified by the primer pairs used for the RT-PCR analysis are indicated by numbered lines below the genes. (B) Composite image of agarose gels from resulting RT-PCR reactions, using either genomic DNA (DNA), RNA (RT-) or cDNA (RT+) as templates. The numbers refer to the respective fragments shown in (A).

Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

13) Product Images from "High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions"

Article Title: High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky410

Production of a low-density functional MicA sa RNA array. ( A ) Generation of a Cy3-labelled MicA sa RNA array using a DNA template array of Alexa647-labelled MicA and MicA sa . ( B ) A non-labelled MicA sa RNA array (generated as in (A) but without labelling) probed with Cy5-labelled ompA .
Figure Legend Snippet: Production of a low-density functional MicA sa RNA array. ( A ) Generation of a Cy3-labelled MicA sa RNA array using a DNA template array of Alexa647-labelled MicA and MicA sa . ( B ) A non-labelled MicA sa RNA array (generated as in (A) but without labelling) probed with Cy5-labelled ompA .

Techniques Used: Functional Assay, Generated

14) Product Images from "Nonencapsidated 5′ Copy-Back Defective Interfering Genomes Produced by Recombinant Measles Viruses Are Recognized by RIG-I and LGP2 but Not MDA5"

Article Title: Nonencapsidated 5′ Copy-Back Defective Interfering Genomes Produced by Recombinant Measles Viruses Are Recognized by RIG-I and LGP2 but Not MDA5

Journal: Journal of Virology

doi: 10.1128/JVI.00643-17

Variation in type I IFN response activation by DI-RNAs in the presence of the MV N and P proteins. (A) Western blot analysis of T7 polymerase, MV-N, MV-P, and β-actin in HEK293-T7 and HEK293-T7-NP cells. A total of 1 × 10 6 cells were plated, and lysis was performed 12 h later. (B) Absolute quantification of 1,212-, 450-, and 1,260-nt DI-RNAs produced by HEK293-T7 and HEK293-T7-NP cells transfected with the p2RZ-DI1212, p2RZ-DI450, and p2RZ-DI1260 vectors. Total RNA (400 ng) purified from cells at 24 h posttransfection was treated with RQ1 RNase-free DNase, and 100 ng was analyzed by RT-qPCR. Absolute quantification was performed using serial dilutions of in vitro -transcribed MV DI-RNAs and normalized with actin. Results are expressed in copy numbers of RNA molecules. Experiments were performed two times, and samples were tested in triplicate. (C and D) HEK293-T7 and HEK293-T7-NP cells were transfected with p2RZ vectors encompassing DI-RNAs or an actin sequence (C) or with in vitro -transcribed RNAs (actin, 5′3P, or 3 different IVT DI-RNAs) (D). Relative IFN-β mRNA expression levels were measured at 24 h posttransfection, normalized to GAPDH, and compared to those of mock-transfected cells. Experiments were performed two times, and the upper and lower 95% confidence intervals are shown as error bars for technical triplicates of the most representative experiment.
Figure Legend Snippet: Variation in type I IFN response activation by DI-RNAs in the presence of the MV N and P proteins. (A) Western blot analysis of T7 polymerase, MV-N, MV-P, and β-actin in HEK293-T7 and HEK293-T7-NP cells. A total of 1 × 10 6 cells were plated, and lysis was performed 12 h later. (B) Absolute quantification of 1,212-, 450-, and 1,260-nt DI-RNAs produced by HEK293-T7 and HEK293-T7-NP cells transfected with the p2RZ-DI1212, p2RZ-DI450, and p2RZ-DI1260 vectors. Total RNA (400 ng) purified from cells at 24 h posttransfection was treated with RQ1 RNase-free DNase, and 100 ng was analyzed by RT-qPCR. Absolute quantification was performed using serial dilutions of in vitro -transcribed MV DI-RNAs and normalized with actin. Results are expressed in copy numbers of RNA molecules. Experiments were performed two times, and samples were tested in triplicate. (C and D) HEK293-T7 and HEK293-T7-NP cells were transfected with p2RZ vectors encompassing DI-RNAs or an actin sequence (C) or with in vitro -transcribed RNAs (actin, 5′3P, or 3 different IVT DI-RNAs) (D). Relative IFN-β mRNA expression levels were measured at 24 h posttransfection, normalized to GAPDH, and compared to those of mock-transfected cells. Experiments were performed two times, and the upper and lower 95% confidence intervals are shown as error bars for technical triplicates of the most representative experiment.

Techniques Used: Activation Assay, Western Blot, Lysis, Produced, Transfection, Purification, Quantitative RT-PCR, In Vitro, Sequencing, Expressing

15) Product Images from "SelexGLM differentiates androgen and glucocorticoid receptor DNA-binding preference over an extended binding site"

Article Title: SelexGLM differentiates androgen and glucocorticoid receptor DNA-binding preference over an extended binding site

Journal: Genome Research

doi: 10.1101/gr.222844.117

SelexGLM shows differences in DNA recognition between AR and GR throughout their binding sites. ( A – D ) Energy logos for AR-DBD ( top ) and GR-DBD ( bottom ), obtained by fitting biophysical models for protein–DNA interaction to the SELEX read counts using an iterative generalized linear modeling approach based on Poisson regression, implemented as SelexGLM for logos generated using round 4 to 8 data. ( E ) Cumulative distribution functions for the contribution of half-site (squares), spacer (triangles), and flanking (circles) sequences on AR-DBD (red) and GR-DBD (blue) binding energy. ( F ) Validation of the contribution of flanking A tracts and spacer to AR- and GR-DBD binding performed by quantitative electrophoretic mobility shift assay (EMSA). Loss of flanking A tracts is more detrimental to AR- than GR-DBD (one vs. two), whereas changing spacer can have detrimental effects on the binding of both (one vs. three). Error bars, SEM based on at least three repeats of each experiment. (*) P -value ≤0.05, (**) P -value ≤0.01, two-sided t -test.
Figure Legend Snippet: SelexGLM shows differences in DNA recognition between AR and GR throughout their binding sites. ( A – D ) Energy logos for AR-DBD ( top ) and GR-DBD ( bottom ), obtained by fitting biophysical models for protein–DNA interaction to the SELEX read counts using an iterative generalized linear modeling approach based on Poisson regression, implemented as SelexGLM for logos generated using round 4 to 8 data. ( E ) Cumulative distribution functions for the contribution of half-site (squares), spacer (triangles), and flanking (circles) sequences on AR-DBD (red) and GR-DBD (blue) binding energy. ( F ) Validation of the contribution of flanking A tracts and spacer to AR- and GR-DBD binding performed by quantitative electrophoretic mobility shift assay (EMSA). Loss of flanking A tracts is more detrimental to AR- than GR-DBD (one vs. two), whereas changing spacer can have detrimental effects on the binding of both (one vs. three). Error bars, SEM based on at least three repeats of each experiment. (*) P -value ≤0.05, (**) P -value ≤0.01, two-sided t -test.

Techniques Used: Binding Assay, Generated, Electrophoretic Mobility Shift Assay

ITC analysis reveals distinct DNA-binding thermodynamics between AR- and GR-DBD. ( A ) The raw heat titration signals ( top ) and normalized heat of injection profiles ( bottom ). ( B ) The K D of AR-DBD and GR-DBD for four sets of sequences fit from the ITC data. AR-DBD affinity is increased with flanking As and an optimal spacer, whereas GR is insensitive. ( C ) Enthalpy, ΔH, is calculated from the heat of binding for each DNA sequence. Flanking sequences decrease ΔH for AR-DBD, enhancing affinity. Smaller indicates a greater contribution to affinity. ( D ) Entropy, ΔS, is calculated from the K D (thus ΔG) and ΔH. GR-DBD affinity is more entropically driven. Larger indicates a greater contribution to affinity. (*) P -value ≤0.05, (**) P -value ≤0.01, (***) P -value ≤0.001, (****) P -value ≤0.0001, two-sided t -test. Error bars, SD represent the standard deviation from at least three experiments.
Figure Legend Snippet: ITC analysis reveals distinct DNA-binding thermodynamics between AR- and GR-DBD. ( A ) The raw heat titration signals ( top ) and normalized heat of injection profiles ( bottom ). ( B ) The K D of AR-DBD and GR-DBD for four sets of sequences fit from the ITC data. AR-DBD affinity is increased with flanking As and an optimal spacer, whereas GR is insensitive. ( C ) Enthalpy, ΔH, is calculated from the heat of binding for each DNA sequence. Flanking sequences decrease ΔH for AR-DBD, enhancing affinity. Smaller indicates a greater contribution to affinity. ( D ) Entropy, ΔS, is calculated from the K D (thus ΔG) and ΔH. GR-DBD affinity is more entropically driven. Larger indicates a greater contribution to affinity. (*) P -value ≤0.05, (**) P -value ≤0.01, (***) P -value ≤0.001, (****) P -value ≤0.0001, two-sided t -test. Error bars, SD represent the standard deviation from at least three experiments.

Techniques Used: Binding Assay, Titration, Injection, Sequencing, Standard Deviation

SELEX-seq reveals differences in AR- and GR-DBD (DNA-binding domain) DNA-binding specificity. ( A ) SELEX-seq. A 70-bp dsDNA library with 23-bp randomized region was incubated with the DBD of AR or GR and separated into monomer and dimer species by EMSA. Dimer-bound DNA was recovered, quantified by qPCR, amplified as the library for the next round, and repeated for eight rounds. Each round of library, including the initial dsDNA library, was sequenced. ( B ) EMSA gel showing the enrichment of dimer-bound sequences after each round of selection for GR-DBD. The intensity of the shifted band plateaus after round 7. A high-affinity palindromic sequence served as a control to locate the dimer band. (*) An artifact during the synthesis of control sequence but not observed in the SELEX library. ( C , D ) Information gain, or Kullback-Leibler divergence, from R0 to R8, as a function of oligonucleotide length. ( E ) Boxplot showing the multiplicity of unique 23-mers in each of the last three rounds of SELEX-seq selection for AR and GR. Even for the most highly selected library (AR R8) fewer than 10% of all reads have 10 copies or more, indicating that the libraries are not overselected. ( F ) Venn diagram showing the overlap of sequences for AR- and GR-DBD with at least 100 sequencing counts. ( G ) Scatterplot of sequences that were commonly bound (yellow from F ) by AR- and GR-DBD.
Figure Legend Snippet: SELEX-seq reveals differences in AR- and GR-DBD (DNA-binding domain) DNA-binding specificity. ( A ) SELEX-seq. A 70-bp dsDNA library with 23-bp randomized region was incubated with the DBD of AR or GR and separated into monomer and dimer species by EMSA. Dimer-bound DNA was recovered, quantified by qPCR, amplified as the library for the next round, and repeated for eight rounds. Each round of library, including the initial dsDNA library, was sequenced. ( B ) EMSA gel showing the enrichment of dimer-bound sequences after each round of selection for GR-DBD. The intensity of the shifted band plateaus after round 7. A high-affinity palindromic sequence served as a control to locate the dimer band. (*) An artifact during the synthesis of control sequence but not observed in the SELEX library. ( C , D ) Information gain, or Kullback-Leibler divergence, from R0 to R8, as a function of oligonucleotide length. ( E ) Boxplot showing the multiplicity of unique 23-mers in each of the last three rounds of SELEX-seq selection for AR and GR. Even for the most highly selected library (AR R8) fewer than 10% of all reads have 10 copies or more, indicating that the libraries are not overselected. ( F ) Venn diagram showing the overlap of sequences for AR- and GR-DBD with at least 100 sequencing counts. ( G ) Scatterplot of sequences that were commonly bound (yellow from F ) by AR- and GR-DBD.

Techniques Used: Binding Assay, Incubation, Real-time Polymerase Chain Reaction, Amplification, Selection, Sequencing

16) Product Images from "A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA"

Article Title: A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000380

Breakpoint identification by PAMP with an INK4A minigenomic tiling array. (A) Five groups of primers (F A , F B , R x , R Y and R Z , the small arrows and arrow heads) near the potential breakpoints were generated for PAMP based on our previous mapping [3] . The mapped CDKN2A breakpoints of the Detroit 562 cell line ( Figure 5 ) are indicated for clarification. The “E1”, “E2” and “E3” designations (blue fonts) are the relative positions of INK4A exons. The first exon of ARF is further to the right of this diagram and is not covered by this array. The tiling probes for the array are indicated with two alternating colors (short black and orange lines) for ease of identification. (B) The first row of the INK4A minigenomic array was spotted with the tiling probes shown in panel A. Cot-1 DNA (repetitive sequence of genomic DNA) spots are indicated on this array. The rest of the spots are herring sperm DNA. Both Cot-1 and herring sperm DNA are used as nonspecific controls. This array was hybridized with labeled samples derived from two cell lines. The same sets of primers (F A , F B , R Y and R Z ) were used for PAMP reactions on Detroit 562 (mutant) and HEK293 (wild type) genomic DNA to map the potential CDKN2A breakpoints. The amplicons were labeled with different dyes, yielding a green signal (Cy-3) for the mutant sample and a red signal (Cy-5) for the wild type sample, to be simultaneously hybridized on the array (two-color array). The two green spots on the first row revealed the breakpoint location as been discussed in Figure 2 .
Figure Legend Snippet: Breakpoint identification by PAMP with an INK4A minigenomic tiling array. (A) Five groups of primers (F A , F B , R x , R Y and R Z , the small arrows and arrow heads) near the potential breakpoints were generated for PAMP based on our previous mapping [3] . The mapped CDKN2A breakpoints of the Detroit 562 cell line ( Figure 5 ) are indicated for clarification. The “E1”, “E2” and “E3” designations (blue fonts) are the relative positions of INK4A exons. The first exon of ARF is further to the right of this diagram and is not covered by this array. The tiling probes for the array are indicated with two alternating colors (short black and orange lines) for ease of identification. (B) The first row of the INK4A minigenomic array was spotted with the tiling probes shown in panel A. Cot-1 DNA (repetitive sequence of genomic DNA) spots are indicated on this array. The rest of the spots are herring sperm DNA. Both Cot-1 and herring sperm DNA are used as nonspecific controls. This array was hybridized with labeled samples derived from two cell lines. The same sets of primers (F A , F B , R Y and R Z ) were used for PAMP reactions on Detroit 562 (mutant) and HEK293 (wild type) genomic DNA to map the potential CDKN2A breakpoints. The amplicons were labeled with different dyes, yielding a green signal (Cy-3) for the mutant sample and a red signal (Cy-5) for the wild type sample, to be simultaneously hybridized on the array (two-color array). The two green spots on the first row revealed the breakpoint location as been discussed in Figure 2 .

Techniques Used: Generated, Clarification Assay, Sequencing, Labeling, Derivative Assay, Mutagenesis

17) Product Images from "Novel DNA Microarray System for Analysis of Nascent mRNAs"

Article Title: Novel DNA Microarray System for Analysis of Nascent mRNAs

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn015

( A ) Eluted RNAs were converted to cDNA, and cDNA was amplified by TS-PCR. ( B ) Validation with quantitative RT–PCR of the DNA microarray data using each amplification method. Genes expressed at various levels in the non-amplification method, TS-PCR amplification method, and T7 in vitro transcription (IVT) amplification method were confirmed using quantitative RT–PCR. Quantitative RT–PCR was performed using the total RNA of these transcription factors. Gapd was used as the control. The same total RNA was used for quantitative RT–PCR and DNA microarray experiments. The ratio of the total target RNA levels was normalized by using those of the Tbp ( Mus musculus TATA box binding protein) gene, and the fold change for each gene in the differentiated ES cells compared to the control ES cells was calculated. Error bars represent standard deviations of log (base 2) of ratio. ( C ) Amplified cDNA labeled with Cy3/Cy5 underwent self-self hybridization.
Figure Legend Snippet: ( A ) Eluted RNAs were converted to cDNA, and cDNA was amplified by TS-PCR. ( B ) Validation with quantitative RT–PCR of the DNA microarray data using each amplification method. Genes expressed at various levels in the non-amplification method, TS-PCR amplification method, and T7 in vitro transcription (IVT) amplification method were confirmed using quantitative RT–PCR. Quantitative RT–PCR was performed using the total RNA of these transcription factors. Gapd was used as the control. The same total RNA was used for quantitative RT–PCR and DNA microarray experiments. The ratio of the total target RNA levels was normalized by using those of the Tbp ( Mus musculus TATA box binding protein) gene, and the fold change for each gene in the differentiated ES cells compared to the control ES cells was calculated. Error bars represent standard deviations of log (base 2) of ratio. ( C ) Amplified cDNA labeled with Cy3/Cy5 underwent self-self hybridization.

Techniques Used: Amplification, Polymerase Chain Reaction, Quantitative RT-PCR, Microarray, In Vitro, Binding Assay, Labeling, Hybridization

18) Product Images from "CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals"

Article Title: CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals

Journal: bioRxiv

doi: 10.1101/2020.06.25.171538

Schematic representation of the screening pipeline. Starting from hundreds of samples the amplicon generation takes place by preparing the individuals for sequencing. By the end of the sequencing run the demultiplexing and analysis can take place that can lead to the identification of the desired edited individuals.
Figure Legend Snippet: Schematic representation of the screening pipeline. Starting from hundreds of samples the amplicon generation takes place by preparing the individuals for sequencing. By the end of the sequencing run the demultiplexing and analysis can take place that can lead to the identification of the desired edited individuals.

Techniques Used: Amplification, Sequencing

Amplicon preparation. (a) Diagram of the targeted gene, ISOCHORISMATE SYNTHASE 1 ( ICS1 ). Black boxes indicate exons, and grey boxes untranslated regions. The arrow shows the direction of transcription. (b) - (d) Amplicon preparation. (b) The first PCR step to amplify a specific region of the genome. The oligonucleotide primers in this step fuse the first part of the TruSeq adapters (grey) and the frame shifting nucleotides (red). (c) The second PCR amplification adds the last part of the TruSeq adapters (purple) and one of the 96 barcodes (orange). (d) The final amplicon with frameshifting base pairs(s) (red), TruSeq adapters (grey and purple) and barcode (orange).
Figure Legend Snippet: Amplicon preparation. (a) Diagram of the targeted gene, ISOCHORISMATE SYNTHASE 1 ( ICS1 ). Black boxes indicate exons, and grey boxes untranslated regions. The arrow shows the direction of transcription. (b) - (d) Amplicon preparation. (b) The first PCR step to amplify a specific region of the genome. The oligonucleotide primers in this step fuse the first part of the TruSeq adapters (grey) and the frame shifting nucleotides (red). (c) The second PCR amplification adds the last part of the TruSeq adapters (purple) and one of the 96 barcodes (orange). (d) The final amplicon with frameshifting base pairs(s) (red), TruSeq adapters (grey and purple) and barcode (orange).

Techniques Used: Amplification, Polymerase Chain Reaction

19) Product Images from "DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific gene expression"

Article Title: DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific gene expression

Journal: Genome Research

doi: 10.1101/gr.074070.107

DNA methylation profiles were analyzed by D-REAM. ( A ) Illustration of the D-REAM method. Genomic DNA was digested with methylation-sensitive restriction enzyme HpyCH4IV and amplified by modified LM-PCR (Supplemental Fig. S1). Amplified fragments (gray bars) were hybridized with mouse promoter tiling array ( upper panel). Array signal intensities (vertical bars) were analyzed to identify regions corresponding to fragments in unmethylated HpyCH4IV loci. Comparison of signals from different samples enabled identification of differentially methylated regions ( lower panel). HpyCH4IV loci overlapping with regions yielding differential signals were defined as T-DMRtags. ( B ) Agarose gel electrophoresis of undigested (lane 2 ), HpyCH4IV-digested (lane 3 ), and HpyCH4IV–TaqI-digested (lane 4 ) mouse liver DNA. Positions corresponding to 0.1, 0.5, 1.0, and 2.0 kbp (lanes 1 , 5 ) are indicated on one side of the gel image. ( C ) Venn diagram of DNA methylation status at HpyCH4IV sites in mouse liver and cerebrum. Numbers without parentheses represent numbers of HpyCH4IV sites, while Ensembl transcripts IDs are in parentheses. Outer and inner rectangles represent whole mouse genome and regions covered by the promoter tiling array, respectively. Ovals indicate unmethylated HpyCH4IV sites of liver and cerebrum identified by D-REAM. ( D ) Correlation of microarray probe intensities in duplicate mouse liver experiments, plotted on logarithmic axes (base 2). ( E ) MATscore distribution of array regions corresponding to the TaqI–TaqI fragments (gray) and HpyCH4IV-digested fragments (black). The dotted line represents the MATscore cutoff value. ( F ) Reliability of comparative MAT analysis. Bar-plots of MATscores of the hypomethylated regions identified by MAT ( P
Figure Legend Snippet: DNA methylation profiles were analyzed by D-REAM. ( A ) Illustration of the D-REAM method. Genomic DNA was digested with methylation-sensitive restriction enzyme HpyCH4IV and amplified by modified LM-PCR (Supplemental Fig. S1). Amplified fragments (gray bars) were hybridized with mouse promoter tiling array ( upper panel). Array signal intensities (vertical bars) were analyzed to identify regions corresponding to fragments in unmethylated HpyCH4IV loci. Comparison of signals from different samples enabled identification of differentially methylated regions ( lower panel). HpyCH4IV loci overlapping with regions yielding differential signals were defined as T-DMRtags. ( B ) Agarose gel electrophoresis of undigested (lane 2 ), HpyCH4IV-digested (lane 3 ), and HpyCH4IV–TaqI-digested (lane 4 ) mouse liver DNA. Positions corresponding to 0.1, 0.5, 1.0, and 2.0 kbp (lanes 1 , 5 ) are indicated on one side of the gel image. ( C ) Venn diagram of DNA methylation status at HpyCH4IV sites in mouse liver and cerebrum. Numbers without parentheses represent numbers of HpyCH4IV sites, while Ensembl transcripts IDs are in parentheses. Outer and inner rectangles represent whole mouse genome and regions covered by the promoter tiling array, respectively. Ovals indicate unmethylated HpyCH4IV sites of liver and cerebrum identified by D-REAM. ( D ) Correlation of microarray probe intensities in duplicate mouse liver experiments, plotted on logarithmic axes (base 2). ( E ) MATscore distribution of array regions corresponding to the TaqI–TaqI fragments (gray) and HpyCH4IV-digested fragments (black). The dotted line represents the MATscore cutoff value. ( F ) Reliability of comparative MAT analysis. Bar-plots of MATscores of the hypomethylated regions identified by MAT ( P

Techniques Used: DNA Methylation Assay, Methylation, Amplification, Modification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Microarray

20) Product Images from "Glucose‐6‐Phosphate Regulates Hepatic Bile Acid Synthesis in Mice"

Article Title: Glucose‐6‐Phosphate Regulates Hepatic Bile Acid Synthesis in Mice

Journal: Hepatology (Baltimore, Md.)

doi: 10.1002/hep.30778

ChREBP does not directly regulate hepatic Cyp8b1 transcription. (A) Schematic presentation of putative consensus and alternative ChREBP response elements within the murine Cyp8b1 promoter. (B) Luciferase activity for the murine and human CYP8B1 promoter reporter and minimal promoter ACC/chore after transfection with Hnf4α, ChREBPα, and ChREBPβ plasmids (n = 5‐6). (C) In vivo ChIP analysis of the putative ChREBP response elements in the hepatic Cyp8b1 and L‐pk gene and (D) of acetylated histone H4 around the hepatic Cyp8b1 gene in mice treated with either shChREBP or scrambled shRNA and infused with S4048 or vehicle (n = 7). (E) Hepatic mRNA levels of Acly in C57BL/6 mice treated with either shChREBP or scrambled shRNA, infused with S4048 or vehicle (n = 7‐8). Data are represented as means ± SEM. *** P
Figure Legend Snippet: ChREBP does not directly regulate hepatic Cyp8b1 transcription. (A) Schematic presentation of putative consensus and alternative ChREBP response elements within the murine Cyp8b1 promoter. (B) Luciferase activity for the murine and human CYP8B1 promoter reporter and minimal promoter ACC/chore after transfection with Hnf4α, ChREBPα, and ChREBPβ plasmids (n = 5‐6). (C) In vivo ChIP analysis of the putative ChREBP response elements in the hepatic Cyp8b1 and L‐pk gene and (D) of acetylated histone H4 around the hepatic Cyp8b1 gene in mice treated with either shChREBP or scrambled shRNA and infused with S4048 or vehicle (n = 7). (E) Hepatic mRNA levels of Acly in C57BL/6 mice treated with either shChREBP or scrambled shRNA, infused with S4048 or vehicle (n = 7‐8). Data are represented as means ± SEM. *** P

Techniques Used: Luciferase, Activity Assay, Transfection, In Vivo, Chromatin Immunoprecipitation, Mouse Assay, shRNA

Working model of the mechanism by which intrahepatic glucose controls bile acid synthesis and intestinal cholesterol handling in mice. Intrahepatic glucose (G6P) controls bile acid synthesis through a ChREBP‐dependent induction of Cyp8b1 by H4 acetylation, whereas hepatic Cyp7a1 expression is regulated by blood glucose levels. Hepatic G6P‐ChREBP ‐CYP8B1 hence induces corresponding shifts in bile composition, which subsequently promotes intestinal cholesterol absorption.
Figure Legend Snippet: Working model of the mechanism by which intrahepatic glucose controls bile acid synthesis and intestinal cholesterol handling in mice. Intrahepatic glucose (G6P) controls bile acid synthesis through a ChREBP‐dependent induction of Cyp8b1 by H4 acetylation, whereas hepatic Cyp7a1 expression is regulated by blood glucose levels. Hepatic G6P‐ChREBP ‐CYP8B1 hence induces corresponding shifts in bile composition, which subsequently promotes intestinal cholesterol absorption.

Techniques Used: Mouse Assay, Expressing

21) Product Images from "Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides New Insights into Sex Chromosome Evolution in Paradoxical Frogs"

Article Title: Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides New Insights into Sex Chromosome Evolution in Paradoxical Frogs

Journal: Genes

doi: 10.3390/genes10020160

Phylogenetic relationships of Pseudis and Lysapsus inferred by Bayesian analysis of the mitochondrial H1 and cytb fragments. Numbers at the nodes indicate posterior probability values. Pseudis fusca from Carlos Chagas – MG is in bold.
Figure Legend Snippet: Phylogenetic relationships of Pseudis and Lysapsus inferred by Bayesian analysis of the mitochondrial H1 and cytb fragments. Numbers at the nodes indicate posterior probability values. Pseudis fusca from Carlos Chagas – MG is in bold.

Techniques Used:

22) Product Images from "A Novel Multiplex PCR-RFLP Method for Simultaneous Genotyping of CYP3A4*4 A>G, CYP3A4*18B G>A and CYP3A4*22 C>T"

Article Title: A Novel Multiplex PCR-RFLP Method for Simultaneous Genotyping of CYP3A4*4 A>G, CYP3A4*18B G>A and CYP3A4*22 C>T

Journal: The Malaysian Journal of Medical Sciences : MJMS

doi: 10.21315/mjms2018.25.4.7

A 4% agarose gel of multiplex PCR-RFLP analysis of CYP3A4*4 , CYP3A4*18B and CYP3A4*22 . L1 contained GeneRuler 50bp DNA ladder (Thermo Fisher Scientific Inc, Massachusetts, USA). L2 contained wild type CYP3A4*4 allele (88 bp, and 141 bp) together with 331 bp for CYP3A4*18B and 153 bp and 525 bp from CYP3A4*22 digestions. L3, L4 and L5 contain wild type (216 bp and 115 bp), homozygous (undigested 331 bp) and heterozygous (115 bp, 216 bp and 331 bp) variants, respectively for CYP3A4*18B . They also contain 112 bp (except for L4 in which it is not shown) and 681 bp from CYP3A4*22 as well as 244 bp for CYP3A4*4 . L6 contained wild type CYP3A4*22 (219 bp and 574 bp) as well as 244 bp and 331 bp for CYP3A4*4 and CYP3A4*18B, respectively. L6 contained negative control
Figure Legend Snippet: A 4% agarose gel of multiplex PCR-RFLP analysis of CYP3A4*4 , CYP3A4*18B and CYP3A4*22 . L1 contained GeneRuler 50bp DNA ladder (Thermo Fisher Scientific Inc, Massachusetts, USA). L2 contained wild type CYP3A4*4 allele (88 bp, and 141 bp) together with 331 bp for CYP3A4*18B and 153 bp and 525 bp from CYP3A4*22 digestions. L3, L4 and L5 contain wild type (216 bp and 115 bp), homozygous (undigested 331 bp) and heterozygous (115 bp, 216 bp and 331 bp) variants, respectively for CYP3A4*18B . They also contain 112 bp (except for L4 in which it is not shown) and 681 bp from CYP3A4*22 as well as 244 bp for CYP3A4*4 . L6 contained wild type CYP3A4*22 (219 bp and 574 bp) as well as 244 bp and 331 bp for CYP3A4*4 and CYP3A4*18B, respectively. L6 contained negative control

Techniques Used: Agarose Gel Electrophoresis, Multiplex Assay, Polymerase Chain Reaction, Negative Control

A 2% agarose gel showing PCR products from multiplex and uniplex PCR for CYP3A4*4 , CYP3A4*18B and CYP3A4*22 . L1: Quick-Load 100bp DNA ladder (NEB® inc, Massachusetts, USA). L2: multiplex pcr with band sizes of 244 bp, 331 bp and 793 bp. L3, L4 and L5 contain positive controls for CYP3A4*4 with 244 bp, CYP3A4*18B with 331 bp and CYP3A4*22 with 793 bp respectively. L6 is a negative control
Figure Legend Snippet: A 2% agarose gel showing PCR products from multiplex and uniplex PCR for CYP3A4*4 , CYP3A4*18B and CYP3A4*22 . L1: Quick-Load 100bp DNA ladder (NEB® inc, Massachusetts, USA). L2: multiplex pcr with band sizes of 244 bp, 331 bp and 793 bp. L3, L4 and L5 contain positive controls for CYP3A4*4 with 244 bp, CYP3A4*18B with 331 bp and CYP3A4*22 with 793 bp respectively. L6 is a negative control

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Multiplex Assay, Negative Control

23) Product Images from "Construction of the Coding Sequence of the Transcription Variant 2 of the Human Renalase Gene and Its Expression in the Prokaryotic System"

Article Title: Construction of the Coding Sequence of the Transcription Variant 2 of the Human Renalase Gene and Its Expression in the Prokaryotic System

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms140612764

Expression of hRenalase1 and hRenalase2 in E. coli cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of expression of polyHis hRenalase1 (RenI—39 kDa) and hRenalase2 (RenII—36 kDa) in E. coli Rosetta (DE3) cells transformed by pET-hRenI and pET-hRenII vectors. Tracks 1 and 4—polyHis hRenalase2 (RenII—36 kDa) and hRenalase1 (RenI—39 kDa), respectively, purified on Ni-Sepharose; Tracks 2 and 5—lysates of cells transformed pET-hRenI и pET-hRenII induced with 1.0 mM IPTG; Tracks 3 and 6—lysates of cells transformed pET-hRenI and pET-hRenII, without IPTG; Track M—molecular mass markers. Mass values are shown on the right.
Figure Legend Snippet: Expression of hRenalase1 and hRenalase2 in E. coli cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of expression of polyHis hRenalase1 (RenI—39 kDa) and hRenalase2 (RenII—36 kDa) in E. coli Rosetta (DE3) cells transformed by pET-hRenI and pET-hRenII vectors. Tracks 1 and 4—polyHis hRenalase2 (RenII—36 kDa) and hRenalase1 (RenI—39 kDa), respectively, purified on Ni-Sepharose; Tracks 2 and 5—lysates of cells transformed pET-hRenI и pET-hRenII induced with 1.0 mM IPTG; Tracks 3 and 6—lysates of cells transformed pET-hRenI and pET-hRenII, without IPTG; Track M—molecular mass markers. Mass values are shown on the right.

Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, SDS Page, Transformation Assay, Positron Emission Tomography, Purification

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Article Title: Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC
Article Snippet: .. Twenty microliters of the sample (with a concentration of 50 µg/mL) were added into a tube containing a 100 μg Cy3 mono-reactive dye pack carrying only one reactive group on each dye molecule for accurate labeling of amine groups (GE Healthcare, Little Chalfont, UK), which was then mixed and centrifuged. .. Excess free-Cy3 was removed by a desalting spin column (0.5 mL, Thermo Fisher Scientific, Waltham, USA) according to the instructions of the manufacturer.

Article Title: Effects of Hemagglutination Activity in the Serum of a Deep-Sea Vent Endemic Crab, Shinkaia Crosnieri, on Non-Symbiotic and Symbiotic Bacteria
Article Snippet: .. Serum components were fluorescently labeled using a Cy3 Mono-reactive Dye kit (GE healthcare, Buckinghamshire, UK), and dissolved at a concentration of 5% (v/v) in a buffer (25 mM Tris-HCl, pH 7.4 containing 0.8% [w/v] NaCl, 1% [v/v] Triton-X, 1 mM MnCl2 , 1 mM CaCl2 ). .. The labeled serum was applied to a glycoconjugate microarray.

Incubation:

Article Title: Using Single-Particle Tracking to Study Nuclear Trafficking of Viral Genes
Article Snippet: .. Purified vRNPs were then incubated with amine reactive Cy3 (PA23001; Amersham Biosciences, Piscataway, NJ) in a carbonate buffer (pH 9.3) with occasional mixing for 1 h at room temperature. ..

Purification:

Article Title: Using Single-Particle Tracking to Study Nuclear Trafficking of Viral Genes
Article Snippet: .. Purified vRNPs were then incubated with amine reactive Cy3 (PA23001; Amersham Biosciences, Piscataway, NJ) in a carbonate buffer (pH 9.3) with occasional mixing for 1 h at room temperature. ..

Labeling:

Article Title: PTP1B regulates Eph receptor function and trafficking
Article Snippet: .. Cy5-FG6 and Cy3.5-PY72 ( ) were labeled using a α-Cy5 or Cy3.5 mono-Reactive Dye Pack (GE Healthcare). ..

Article Title: Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC
Article Snippet: .. Twenty microliters of the sample (with a concentration of 50 µg/mL) were added into a tube containing a 100 μg Cy3 mono-reactive dye pack carrying only one reactive group on each dye molecule for accurate labeling of amine groups (GE Healthcare, Little Chalfont, UK), which was then mixed and centrifuged. .. Excess free-Cy3 was removed by a desalting spin column (0.5 mL, Thermo Fisher Scientific, Waltham, USA) according to the instructions of the manufacturer.

Article Title: Effects of Hemagglutination Activity in the Serum of a Deep-Sea Vent Endemic Crab, Shinkaia Crosnieri, on Non-Symbiotic and Symbiotic Bacteria
Article Snippet: .. Serum components were fluorescently labeled using a Cy3 Mono-reactive Dye kit (GE healthcare, Buckinghamshire, UK), and dissolved at a concentration of 5% (v/v) in a buffer (25 mM Tris-HCl, pH 7.4 containing 0.8% [w/v] NaCl, 1% [v/v] Triton-X, 1 mM MnCl2 , 1 mM CaCl2 ). .. The labeled serum was applied to a glycoconjugate microarray.

Article Title: Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain
Article Snippet: .. For several immunocytochemical experiments, anti-CLRN1 IgG was labeled with Cy3 by Amersham Biosciences Cy3 Mono-Reactive Dye Pack (Amersham Biosciences) following the manufacturer's instructions. .. Goat anti-actin polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).

Conjugation Assay:

Article Title: Inhibiting ACAT1/SOAT1 in Microglia Stimulates Autophagy-Mediated Lysosomal Proteolysis and Increases Aβ1–42 Clearance
Article Snippet: .. Conjugation of Aβ1–42 to Cy3 was performed as previously described ( ) by using Cy3 Mono-Reactive Dye Pack (GE Healthcare Life Sciences) according to the manufacturer's protocol. ..

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