Structured Review

Promega rex3 sequences
Karyotypes of Characidium females subjected to fluorescence in situ hybridization (FISH) with TE probes. (a) C. zebra, (b) C. gomesi (PG), (c) C. gomesi (SJ); (d) C. zebra, (e) C. gomesi (SJ). The <t>Rex3</t> probe did not show any hybridization signals in C. gomesi (PG) chromosomes (not shown). The W and Z sex chromosomes of C. gomesi females are highlighted in the box. PG, Paiol Grande Stream population; SJ, São João River population. Scale bar, 10 μm.
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Images

1) Product Images from "Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes"

Article Title: Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes

Journal: Genetics and Molecular Biology

doi: 10.1590/1678-4685-GMB-2017-0121

Karyotypes of Characidium females subjected to fluorescence in situ hybridization (FISH) with TE probes. (a) C. zebra, (b) C. gomesi (PG), (c) C. gomesi (SJ); (d) C. zebra, (e) C. gomesi (SJ). The Rex3 probe did not show any hybridization signals in C. gomesi (PG) chromosomes (not shown). The W and Z sex chromosomes of C. gomesi females are highlighted in the box. PG, Paiol Grande Stream population; SJ, São João River population. Scale bar, 10 μm.
Figure Legend Snippet: Karyotypes of Characidium females subjected to fluorescence in situ hybridization (FISH) with TE probes. (a) C. zebra, (b) C. gomesi (PG), (c) C. gomesi (SJ); (d) C. zebra, (e) C. gomesi (SJ). The Rex3 probe did not show any hybridization signals in C. gomesi (PG) chromosomes (not shown). The W and Z sex chromosomes of C. gomesi females are highlighted in the box. PG, Paiol Grande Stream population; SJ, São João River population. Scale bar, 10 μm.

Techniques Used: Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Hybridization

2) Product Images from "Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis"

Article Title: Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.118.200231

A null ap2m mutation does not disrupt SRKb function and SI. (A) Structure of the AP2M gene and sequence of the CRISPR/Cas9 -targeted site. The AP2M gene structure is depicted with exons representing boxes and lines representing introns. Arrows represent annealing sites of the forward [F] and reverse [R] primers used for AP2M genotyping ( Figure 4B ) and the red line below the first exon shows the location of the targeted site. In the nucleotide sequences of the region containing the targeted site in wild-type ( AP2M ) and mutant ( ap2m ), the sequence of the sgRNA spacer is shown in red characters, the Protospacer Associated Motif (PAM) sequence GGG is underlined, and the Bsa HI restriction site is highlighted in bold red characters. The asterisk shows the cytosine insertion in ap2m that abolishes the BsaH1 restriction site. (B) Genotyping of AP2M , SRKb , and the CRISPR/Cas9 gene cassette in C24 untransformed wild-type C24 ( AP2M ), AP2M [ AtS1 pro :SRKb-FLAG+SCRb ] ( AP2M [ SRKb ]), and ap2m [ AtS1 pro :SRKb-FLAG+SCRb ] ( ap2m [ SRKb ]) plants. The AP2M PCR products (top panel) were digested with Bsa HI enzymes [ AP2M ( Bsa HI cut) panel]. The 162-bp and 81-bp digestion products are indicated by asterisks. Note that the AP2M fragments amplified from ap2m [ AtS1 pro AtS1 pro :SRKb-FLAG+SCRb ] DNA were not digested with Bsa HI. The presence of the AtS1 pro :SRKb-FLAG+SCRb transgenes was assessed by PCR with SRKb -specific primers ( SRKb panel), and the AP2MsgRNA-Cas9 gene cassette was detected by PCR using DNA of the AP2MsgRNA-Cas9 transformation plasmid as positive control (“P” lane in the sgRNA-Cas9 panel). (C) Pollination phenotypes of ap2m [ AtS1 pro :SRKb-FLAG+SCRb] ( ap2m [ SRKb ]) mutant plants and AP2M [ AtS1 pro :SRKb-FLAG+SCRb ] ( AP2M [ SRKb ]) control plants. The images show the pollination responses toward pollen from untransformed wild-type C24 plants (upper panel) and SCRb pollen (lower panel). The genotype of stigmas used for pollination is indicated in each panel. Note that the ap2m [ AtS1 pro :SRKb-FLAG+SCRb ] stigma shows a compatible response toward wild-type pollen and an incompatible response toward SCRb pollen. Bar = 100 μm.
Figure Legend Snippet: A null ap2m mutation does not disrupt SRKb function and SI. (A) Structure of the AP2M gene and sequence of the CRISPR/Cas9 -targeted site. The AP2M gene structure is depicted with exons representing boxes and lines representing introns. Arrows represent annealing sites of the forward [F] and reverse [R] primers used for AP2M genotyping ( Figure 4B ) and the red line below the first exon shows the location of the targeted site. In the nucleotide sequences of the region containing the targeted site in wild-type ( AP2M ) and mutant ( ap2m ), the sequence of the sgRNA spacer is shown in red characters, the Protospacer Associated Motif (PAM) sequence GGG is underlined, and the Bsa HI restriction site is highlighted in bold red characters. The asterisk shows the cytosine insertion in ap2m that abolishes the BsaH1 restriction site. (B) Genotyping of AP2M , SRKb , and the CRISPR/Cas9 gene cassette in C24 untransformed wild-type C24 ( AP2M ), AP2M [ AtS1 pro :SRKb-FLAG+SCRb ] ( AP2M [ SRKb ]), and ap2m [ AtS1 pro :SRKb-FLAG+SCRb ] ( ap2m [ SRKb ]) plants. The AP2M PCR products (top panel) were digested with Bsa HI enzymes [ AP2M ( Bsa HI cut) panel]. The 162-bp and 81-bp digestion products are indicated by asterisks. Note that the AP2M fragments amplified from ap2m [ AtS1 pro AtS1 pro :SRKb-FLAG+SCRb ] DNA were not digested with Bsa HI. The presence of the AtS1 pro :SRKb-FLAG+SCRb transgenes was assessed by PCR with SRKb -specific primers ( SRKb panel), and the AP2MsgRNA-Cas9 gene cassette was detected by PCR using DNA of the AP2MsgRNA-Cas9 transformation plasmid as positive control (“P” lane in the sgRNA-Cas9 panel). (C) Pollination phenotypes of ap2m [ AtS1 pro :SRKb-FLAG+SCRb] ( ap2m [ SRKb ]) mutant plants and AP2M [ AtS1 pro :SRKb-FLAG+SCRb ] ( AP2M [ SRKb ]) control plants. The images show the pollination responses toward pollen from untransformed wild-type C24 plants (upper panel) and SCRb pollen (lower panel). The genotype of stigmas used for pollination is indicated in each panel. Note that the ap2m [ AtS1 pro :SRKb-FLAG+SCRb ] stigma shows a compatible response toward wild-type pollen and an incompatible response toward SCRb pollen. Bar = 100 μm.

Techniques Used: Mutagenesis, Sequencing, CRISPR, Polymerase Chain Reaction, Amplification, Transformation Assay, Plasmid Preparation, Positive Control

3) Product Images from "Lutzomyia longipalpis TGF-β Has a Role in Leishmania infantum chagasi Survival in the Vector"

Article Title: Lutzomyia longipalpis TGF-β Has a Role in Leishmania infantum chagasi Survival in the Vector

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2019.00071

Gene expression in sand flies infected by L. i. chagasi after abrogation of the LlTGF-β signaling pathway. (A,C,E) Gene expression of LlTGF-β silenced insects. (B,D,F) Gene expression of insects fed with TGF-β receptor inhibitor. (A,B) Quantification of L. i. chagasi in sand flies. (C,D) Relative expression of cecropin. (E,F) Relative expression of defensin 2. Samples were collected at 24, 72, and 144 h post infection. Dotted lines indicate gene expression of β-galactosidase dsRNA injected (A,C,E) or DMSO fed flies (B,D,F) control groups, or. Both test and control groups were infected by L. i. chagasi . Comparisons were done between silenced vs. non-silenced, or inhibitor treated vs. non-treated sand fly groups. Bars represent mean with standard error of fold change in gene expression relative to control groups of 3 independent experiments. Significant differences were evaluated by t -test and Mann–Whitney post-test (* p
Figure Legend Snippet: Gene expression in sand flies infected by L. i. chagasi after abrogation of the LlTGF-β signaling pathway. (A,C,E) Gene expression of LlTGF-β silenced insects. (B,D,F) Gene expression of insects fed with TGF-β receptor inhibitor. (A,B) Quantification of L. i. chagasi in sand flies. (C,D) Relative expression of cecropin. (E,F) Relative expression of defensin 2. Samples were collected at 24, 72, and 144 h post infection. Dotted lines indicate gene expression of β-galactosidase dsRNA injected (A,C,E) or DMSO fed flies (B,D,F) control groups, or. Both test and control groups were infected by L. i. chagasi . Comparisons were done between silenced vs. non-silenced, or inhibitor treated vs. non-treated sand fly groups. Bars represent mean with standard error of fold change in gene expression relative to control groups of 3 independent experiments. Significant differences were evaluated by t -test and Mann–Whitney post-test (* p

Techniques Used: Expressing, Infection, Injection, MANN-WHITNEY

Nitric oxide production in sand flies abrogated for the LlTGF-β signaling pathway. (A) iNOS gene expression of LlTGF-β silenced insects, (B) fed with blood containing TGF-β receptor inhibitor, both (A,B) infected by L. i. chagasi . Samples were collected at 24, 72, and 144 h post infection. Dotted line indicates gene expression of control group, β-gal dsRNA injected or blood containing DMSO fed flies. Bars represent mean with standard error of fold change in gene expression relative to control groups of three independent experiments. Significant differences were evaluated by t -test and Mann-Whitney post-test. (C) NO measurement in LlTGF-β silenced sand flies fed on blood. (D) Nitrite measurement in flies fed on blood containing TGF-β receptor inhibitor. Bars represent mean with standard error of nitrite measurements in test and control groups of 3 independent experiments. Significant differences were evaluated by t -test and Mann-Whitney post-test (* p
Figure Legend Snippet: Nitric oxide production in sand flies abrogated for the LlTGF-β signaling pathway. (A) iNOS gene expression of LlTGF-β silenced insects, (B) fed with blood containing TGF-β receptor inhibitor, both (A,B) infected by L. i. chagasi . Samples were collected at 24, 72, and 144 h post infection. Dotted line indicates gene expression of control group, β-gal dsRNA injected or blood containing DMSO fed flies. Bars represent mean with standard error of fold change in gene expression relative to control groups of three independent experiments. Significant differences were evaluated by t -test and Mann-Whitney post-test. (C) NO measurement in LlTGF-β silenced sand flies fed on blood. (D) Nitrite measurement in flies fed on blood containing TGF-β receptor inhibitor. Bars represent mean with standard error of nitrite measurements in test and control groups of 3 independent experiments. Significant differences were evaluated by t -test and Mann-Whitney post-test (* p

Techniques Used: Expressing, Infection, Injection, MANN-WHITNEY

4) Product Images from "The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis"

Article Title: The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0037001

ISKNV ORF111L-induced apoptosis was mediated by caspase 8. (A) The caspase 8 expression was tested by RT-qPCR assay. The expression level of β-actin was set as 1, and values were normalized to the corresponding β-actin values to determine the relative copy number. All data are presented as means from three individual injection experiments. (B) The caspase 8 activity was tested in wild type, pdsRed2-C1- and pRFP-111L-injected embryos. (C) Knockdown efficiency of caspase 8 Spl-MO in zebrafish embryos. Schematic representation of normal transcription of caspase 8 and morpholino splicing blocking of caspase 8 was shown. The caspase 8 Spl-MO interferes with splicing junction at exon 3/intron 3, resulting in retention of the intron 3. The retention of intron 3 resulted in a frame-shift and the truncation of protein translation. Knockdown effect of caspase 8 Spl-MO was tested in by RT-PCR. Forward primer is located in exon 2, and the reverse primer is located in exon 4. Result showed that caspase 8 Spl-MO strongly depletes the wild type caspase 8 mRNA and had a high gene knockdown effect. (D) Caspase 8 knockdown effectively blocked ISKNV ORF111L-induced apoptosis. The caspase 8 Spl-MO and pRFP-111L were co-injected into 1–2 cell stage embryos. The yolk sac of embryo was healthily developed (panel a). The expression of RFP-111L fusion proteins were clear (panel b), and no obvious apoptosis signal were found in TUNEL assay (panel c), indicating that knockdown the expression of caspase 8 could effectively block ISKNV ORF111L-induced apoptosis. Embryos shown above are the typical phenotype in three independent experiments.
Figure Legend Snippet: ISKNV ORF111L-induced apoptosis was mediated by caspase 8. (A) The caspase 8 expression was tested by RT-qPCR assay. The expression level of β-actin was set as 1, and values were normalized to the corresponding β-actin values to determine the relative copy number. All data are presented as means from three individual injection experiments. (B) The caspase 8 activity was tested in wild type, pdsRed2-C1- and pRFP-111L-injected embryos. (C) Knockdown efficiency of caspase 8 Spl-MO in zebrafish embryos. Schematic representation of normal transcription of caspase 8 and morpholino splicing blocking of caspase 8 was shown. The caspase 8 Spl-MO interferes with splicing junction at exon 3/intron 3, resulting in retention of the intron 3. The retention of intron 3 resulted in a frame-shift and the truncation of protein translation. Knockdown effect of caspase 8 Spl-MO was tested in by RT-PCR. Forward primer is located in exon 2, and the reverse primer is located in exon 4. Result showed that caspase 8 Spl-MO strongly depletes the wild type caspase 8 mRNA and had a high gene knockdown effect. (D) Caspase 8 knockdown effectively blocked ISKNV ORF111L-induced apoptosis. The caspase 8 Spl-MO and pRFP-111L were co-injected into 1–2 cell stage embryos. The yolk sac of embryo was healthily developed (panel a). The expression of RFP-111L fusion proteins were clear (panel b), and no obvious apoptosis signal were found in TUNEL assay (panel c), indicating that knockdown the expression of caspase 8 could effectively block ISKNV ORF111L-induced apoptosis. Embryos shown above are the typical phenotype in three independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR, Injection, Activity Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, TUNEL Assay

ISKNV ORF111L and zebrafish TRAF2 overexpression resulted in caspase 8 and caspase 3 upregulation. (A–B) The caspase 8 or caspase 3 expression was shown in wild type embryos by whole mount RNA in situ hybridization assay. (C–D) ISKNV ORF111L overexpression resulted in significant caspase 8 (C, arrow) and caspase 3 (D, arrow head) upregulation. (E–F) Zebrafish TRAF2 overexpression induced modest caspase 8 (E, arrow) and caspase 3 (F, arrow head) upregulation compared with those of ORF111L. All embryos shown represent the typical staining and are lateral views with anterior to the left at 3 dpf.
Figure Legend Snippet: ISKNV ORF111L and zebrafish TRAF2 overexpression resulted in caspase 8 and caspase 3 upregulation. (A–B) The caspase 8 or caspase 3 expression was shown in wild type embryos by whole mount RNA in situ hybridization assay. (C–D) ISKNV ORF111L overexpression resulted in significant caspase 8 (C, arrow) and caspase 3 (D, arrow head) upregulation. (E–F) Zebrafish TRAF2 overexpression induced modest caspase 8 (E, arrow) and caspase 3 (F, arrow head) upregulation compared with those of ORF111L. All embryos shown represent the typical staining and are lateral views with anterior to the left at 3 dpf.

Techniques Used: Over Expression, Expressing, RNA In Situ Hybridization, Staining

5) Product Images from "An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases"

Article Title: An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-13-6

The activity of siRNAs targeting SNP sites in the SCA3 model . (A) The target sites and nucleotide sequences of siRNAs directed at rs12895357 SNP in the ATXN3 gene. The nucleotide in the antisense strand of siRNA that directly targets the SNP site is marked in bold. The mismatches introduced into siRNA duplex are in gray boxes. RNA is in lowercase; DNA is in uppercase. (B) The RT-PCR analysis of ATXN3 expression at the transcript level in the SCA3 fibroblasts (GM06153) at 72 h after transfection with 50 nM siRNAs. (C) The western blot analysis of the ataxin-3 levels for the experiment described in (B). (D) The western blot analysis of the ataxin-3 levels in the SCA3 fibroblasts at 72 h after transfection with 2, 10, 50 or 200 nM siRNA G9. (E) The western blot analysis as in (D) for siRNA G10. C - the reference bar indicates the ATXN3 expression level in the cells transfected with control siRNA. In the graphs, the signal intensities were normalized to GAPDH mRNA or protein levels and compared using a one-sample t -test. The error bars represent the standard deviations. The P -value is indicated by asterisks (* p
Figure Legend Snippet: The activity of siRNAs targeting SNP sites in the SCA3 model . (A) The target sites and nucleotide sequences of siRNAs directed at rs12895357 SNP in the ATXN3 gene. The nucleotide in the antisense strand of siRNA that directly targets the SNP site is marked in bold. The mismatches introduced into siRNA duplex are in gray boxes. RNA is in lowercase; DNA is in uppercase. (B) The RT-PCR analysis of ATXN3 expression at the transcript level in the SCA3 fibroblasts (GM06153) at 72 h after transfection with 50 nM siRNAs. (C) The western blot analysis of the ataxin-3 levels for the experiment described in (B). (D) The western blot analysis of the ataxin-3 levels in the SCA3 fibroblasts at 72 h after transfection with 2, 10, 50 or 200 nM siRNA G9. (E) The western blot analysis as in (D) for siRNA G10. C - the reference bar indicates the ATXN3 expression level in the cells transfected with control siRNA. In the graphs, the signal intensities were normalized to GAPDH mRNA or protein levels and compared using a one-sample t -test. The error bars represent the standard deviations. The P -value is indicated by asterisks (* p

Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot

The replacement strategy tested for ATXN3 . (A) The target site and nucleotide sequence of siRNA directed at the ATXN3 transcript. (B) The western blot analysis of the ataxin-3 levels in the HeLa cells at 72 h after transfection with 100 nM siRNA SCA3 and/or ATXN3-8Q plasmid. C - the reference line shows the ATXN3 expression level in the cells transfected with control siRNA. The GAPDH protein level was used as a loading control.
Figure Legend Snippet: The replacement strategy tested for ATXN3 . (A) The target site and nucleotide sequence of siRNA directed at the ATXN3 transcript. (B) The western blot analysis of the ataxin-3 levels in the HeLa cells at 72 h after transfection with 100 nM siRNA SCA3 and/or ATXN3-8Q plasmid. C - the reference line shows the ATXN3 expression level in the cells transfected with control siRNA. The GAPDH protein level was used as a loading control.

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

6) Product Images from "In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5? splice sites"

Article Title: In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5? splice sites

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm647

( A ) The U6 snRNA base pairs with nucleotides at positions +2, +5 and +6. ( B ) RT–PCR analysis of minigene constructs transfected into HEK293 cells with artificial U1 or U6 snRNA. A single Cp-nuc is introduced into U1 or U6 snRNA while retaining the mismatch at the mutation. Wt, wild-type construct; Mt, mutation observed in a patient. For example, +6U1 indicates that a nucleotide on U1 snRNA corresponding to position +6 is substituted to match the 5′ splice site. Circles and squares represent nucleotides that become complementary to the artificial U1 and U6 snRNAs, respectively. The open arrowhead indicates a normally spliced fragment, whereas the closed arrowhead indicates an exon-skipped fragment. The rightmost column shows the densitometric ratio of the exon-skipped fragment. The asterisk indicates a mixture of fragments due to activated 5′ splice sites four and 13 nucleotides downstream of the native 5′ splice site at the 5′ exon of pSPL3. The dagger indicates a heteroduplex formed by normally spliced and exon-skipped products. Uppercase nucleotides are complementary to U1 snRNA, whereas lowercase nucleotides are not.
Figure Legend Snippet: ( A ) The U6 snRNA base pairs with nucleotides at positions +2, +5 and +6. ( B ) RT–PCR analysis of minigene constructs transfected into HEK293 cells with artificial U1 or U6 snRNA. A single Cp-nuc is introduced into U1 or U6 snRNA while retaining the mismatch at the mutation. Wt, wild-type construct; Mt, mutation observed in a patient. For example, +6U1 indicates that a nucleotide on U1 snRNA corresponding to position +6 is substituted to match the 5′ splice site. Circles and squares represent nucleotides that become complementary to the artificial U1 and U6 snRNAs, respectively. The open arrowhead indicates a normally spliced fragment, whereas the closed arrowhead indicates an exon-skipped fragment. The rightmost column shows the densitometric ratio of the exon-skipped fragment. The asterisk indicates a mixture of fragments due to activated 5′ splice sites four and 13 nucleotides downstream of the native 5′ splice site at the 5′ exon of pSPL3. The dagger indicates a heteroduplex formed by normally spliced and exon-skipped products. Uppercase nucleotides are complementary to U1 snRNA, whereas lowercase nucleotides are not.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Construct, Transfection, Mutagenesis

7) Product Images from "Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae)"

Article Title: Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae)

Journal: BMC Microbiology

doi: 10.1186/1471-2180-12-S1-S4

Relative abundance of G fp-Asaia within the whole Asaia populations. The relative abundance of the tagged strain in total Asaia community is calculated by the ratio between the number of gfp gene copies per sample and the number of Asaia cells (which is Asaia 16S rRNA gene copies divided by four, assuming that four rRNA gene copies per cell are present in Asaia , as reported in Crotti et al. [ 4 ]) per sample. In each graph white columns represent S. titanus individuals, and grey columns represent diets. The “donors” columns refer to average values of donor insects in all trials. “24h”, “48h”, “72h”, and “96h” indicate the time of exposure to co-feeding or the time of incubation after mating with infected individuals. The Gfp-tagged Asaia to total Asaia ratio is indicated in insects and diets submitted to co-feeding trials (A), and to venereal transmission experiments, from male to female (B) and from female to male (C), respectively. The bars on each column represent the standard error.
Figure Legend Snippet: Relative abundance of G fp-Asaia within the whole Asaia populations. The relative abundance of the tagged strain in total Asaia community is calculated by the ratio between the number of gfp gene copies per sample and the number of Asaia cells (which is Asaia 16S rRNA gene copies divided by four, assuming that four rRNA gene copies per cell are present in Asaia , as reported in Crotti et al. [ 4 ]) per sample. In each graph white columns represent S. titanus individuals, and grey columns represent diets. The “donors” columns refer to average values of donor insects in all trials. “24h”, “48h”, “72h”, and “96h” indicate the time of exposure to co-feeding or the time of incubation after mating with infected individuals. The Gfp-tagged Asaia to total Asaia ratio is indicated in insects and diets submitted to co-feeding trials (A), and to venereal transmission experiments, from male to female (B) and from female to male (C), respectively. The bars on each column represent the standard error.

Techniques Used: Incubation, Infection, Transmission Assay

Gfp-Asaia infection rates and density within infected samples. White columns represent S. titanus individuals, and grey columns represent diets. The “donors” columns refer to the average values of donor insects in all of the trials. “24h”, “48h”, “72h”, and “96h” indicate the time of exposure to co-feeding or the time of incubation after mating with infected individuals. The “control” columns represent the values obtained from insects fed on sterile sugar diets, as well as those obtained from individuals co-housed with Gfp Asaia -infected specimens of the same sex. A-C) Percentage of insects and diets colonized by Gfp-tagged Asaia . D-F) Transformed (10 + log) number of gfp gene copies per positive sample. Bars represent the standard error of transformed data. Different letters (black for insect and grey for diet samples) indicate significantly different values (ANOVA, P
Figure Legend Snippet: Gfp-Asaia infection rates and density within infected samples. White columns represent S. titanus individuals, and grey columns represent diets. The “donors” columns refer to the average values of donor insects in all of the trials. “24h”, “48h”, “72h”, and “96h” indicate the time of exposure to co-feeding or the time of incubation after mating with infected individuals. The “control” columns represent the values obtained from insects fed on sterile sugar diets, as well as those obtained from individuals co-housed with Gfp Asaia -infected specimens of the same sex. A-C) Percentage of insects and diets colonized by Gfp-tagged Asaia . D-F) Transformed (10 + log) number of gfp gene copies per positive sample. Bars represent the standard error of transformed data. Different letters (black for insect and grey for diet samples) indicate significantly different values (ANOVA, P

Techniques Used: Infection, Incubation, Transformation Assay

Positive and negative controls for FISH experiments targeting the gfp gene. The presence and distribution of Gfp-tagged Asaia in tissues of donor insects (positive controls) and of individuals submitted to transmission trials in absence of the tagged strain (negative controls) have been evaluated by FISH with the FITC-labelled Eu338 eubacterial probe (green), the Cy3-labelled Asaia -specific probes (magenta) and the Cy5.5-labeled probes specific for the gfp gene (yellow). A) Superposition of a CLSM image after staining with DAPI over the interferential contrast microscopy picture of a salivary gland lobe of an individual used as donor during co-feeding trials (bar = 50 µm). B,C) CLSM images after hybridization with the Cy3-tagged probes targeting the whole Asaia population (B), or with the Cy5.5-marked probes specific for the Gfp strain (C). In D-G) an ovariole of a female mated with a male which was not previously fed with the Gfp-tagged Asaia is shown. D) Interferential contrast micrograph showing the ovariole (bar = 150 µm). E-G) CLSM images of FISH with the FITC-labeled eubacterial probe (E), the Cy3-tagged probes targeting the whole Asaia population (F), and the Cy5.5-marked probes specific for the gfp gene (G). While the occurrence of bacteria (and Asaia in particular) is shown, no hybridization signal was observed with the gfp gene-specific probes.
Figure Legend Snippet: Positive and negative controls for FISH experiments targeting the gfp gene. The presence and distribution of Gfp-tagged Asaia in tissues of donor insects (positive controls) and of individuals submitted to transmission trials in absence of the tagged strain (negative controls) have been evaluated by FISH with the FITC-labelled Eu338 eubacterial probe (green), the Cy3-labelled Asaia -specific probes (magenta) and the Cy5.5-labeled probes specific for the gfp gene (yellow). A) Superposition of a CLSM image after staining with DAPI over the interferential contrast microscopy picture of a salivary gland lobe of an individual used as donor during co-feeding trials (bar = 50 µm). B,C) CLSM images after hybridization with the Cy3-tagged probes targeting the whole Asaia population (B), or with the Cy5.5-marked probes specific for the Gfp strain (C). In D-G) an ovariole of a female mated with a male which was not previously fed with the Gfp-tagged Asaia is shown. D) Interferential contrast micrograph showing the ovariole (bar = 150 µm). E-G) CLSM images of FISH with the FITC-labeled eubacterial probe (E), the Cy3-tagged probes targeting the whole Asaia population (F), and the Cy5.5-marked probes specific for the gfp gene (G). While the occurrence of bacteria (and Asaia in particular) is shown, no hybridization signal was observed with the gfp gene-specific probes.

Techniques Used: Fluorescence In Situ Hybridization, Transmission Assay, Labeling, Confocal Laser Scanning Microscopy, Staining, Microscopy, Hybridization

Localization of horizontally-transmitted Gfp Asaia in organs of S. titanus individuals. Images of insect tissues after hybridization with the Cy3-labeled Asaia -specific probes (magenta) and the Cy5.5-labeled probes specific for the gfp gene (yellow) showing the distribution of the symbiont within the gut, the ovaries and testes of specimens after acquisition of the tagged bacterium via co-feeding or venereal transmission. A-C) Midgut portion of an individual after 48-hour acquisition during the co-feeding trial, observed by interferential contrast microscopy (A) and CLSM after hybridization with the Cy3-tagged probes targeting the whole Asaia population (B), or with the Cy5.5-marked probes specific for the gfp gene(C). D-F) Testis portion of an individual after co-feeding trial observed by interferential contrast microscopy (D), and by CLSM after hybridization with the Cy3-tagged probes targeting the whole Asaia population (E) and the Cy5.5-marked probes specific for the gfp gene (F). In G-I) ovaries of a S. titanus individual after the acquisition in venereal transmission experiments are shown. G) Interferential contrast micrograph showing a group of ovarioles. H, I) CLSM images of FISH with the Cy3-tagged probes targeting the whole Asaia population (H) and the Cy5.5-marked probes specific for the gfp gene (I). Bars = 150 µm.
Figure Legend Snippet: Localization of horizontally-transmitted Gfp Asaia in organs of S. titanus individuals. Images of insect tissues after hybridization with the Cy3-labeled Asaia -specific probes (magenta) and the Cy5.5-labeled probes specific for the gfp gene (yellow) showing the distribution of the symbiont within the gut, the ovaries and testes of specimens after acquisition of the tagged bacterium via co-feeding or venereal transmission. A-C) Midgut portion of an individual after 48-hour acquisition during the co-feeding trial, observed by interferential contrast microscopy (A) and CLSM after hybridization with the Cy3-tagged probes targeting the whole Asaia population (B), or with the Cy5.5-marked probes specific for the gfp gene(C). D-F) Testis portion of an individual after co-feeding trial observed by interferential contrast microscopy (D), and by CLSM after hybridization with the Cy3-tagged probes targeting the whole Asaia population (E) and the Cy5.5-marked probes specific for the gfp gene (F). In G-I) ovaries of a S. titanus individual after the acquisition in venereal transmission experiments are shown. G) Interferential contrast micrograph showing a group of ovarioles. H, I) CLSM images of FISH with the Cy3-tagged probes targeting the whole Asaia population (H) and the Cy5.5-marked probes specific for the gfp gene (I). Bars = 150 µm.

Techniques Used: Hybridization, Labeling, Transmission Assay, Microscopy, Confocal Laser Scanning Microscopy, Fluorescence In Situ Hybridization

8) Product Images from "Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane"

Article Title: Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2015.00197

Diagrams of ALA6 and ALA7 showing locations of T-DNA disruptions . Filled boxes represent exons and open boxes represent introns. T-DNA insertions are represented with triangles and identified by ala allele numbers and T-DNA allele accessions. Primers used for PCR genotyping are represented by arrows and point in the 5′ to 3′ direction. Primer 1343 corresponds to the T-DNA left border. The left-border junctions are as follows: ala6-1 , TGGGACTCCGGCTCAAGCACGCACCgatcgccttaatcgccttaatccgt; and ala7-2 LB: atttgtttacaccacaatatatcctGAACTATCAAATGTGAAGATCCCAA. Capital letters represent ALA genomic DNA and lowercase letters represent T-DNA .
Figure Legend Snippet: Diagrams of ALA6 and ALA7 showing locations of T-DNA disruptions . Filled boxes represent exons and open boxes represent introns. T-DNA insertions are represented with triangles and identified by ala allele numbers and T-DNA allele accessions. Primers used for PCR genotyping are represented by arrows and point in the 5′ to 3′ direction. Primer 1343 corresponds to the T-DNA left border. The left-border junctions are as follows: ala6-1 , TGGGACTCCGGCTCAAGCACGCACCgatcgccttaatcgccttaatccgt; and ala7-2 LB: atttgtttacaccacaatatatcctGAACTATCAAATGTGAAGATCCCAA. Capital letters represent ALA genomic DNA and lowercase letters represent T-DNA .

Techniques Used: Polymerase Chain Reaction

9) Product Images from "Post-transcriptional regulation of cellulose synthase genes by small RNAs derived from CESA antisense transcripts"

Article Title: Post-transcriptional regulation of cellulose synthase genes by small RNAs derived from CESA antisense transcripts

Journal: bioRxiv

doi: 10.1101/2020.04.30.070854

Detection of HvCesA1 antisense transcripts by SS-RT-PCR. (A) Tagged SS-RT-PCR was performed to detect HvCesA1 antisense transcripts over the course of third leaf development (10-16 days post imbibition). First-strand cDNAs were prepared using HvA1-sense-Tag1 GSP (antisense; NRT control), oligo dT (sense; Tag control), or no primer at all (NPC). The HvA1-antisense GSP and the Tag1 primer were used for amplification of the antisense, Tag, NPC, and NRT samples. For sense amplification, HvA1-sense and HvA1-antisense GSPs were used with oligo dT primed cDNAs. PCR products were confirmed by DNA sequencing. (B) Gel densitometry was performed to estimate HvCESA1 sense and antisense transcript abundances. Data were normalized to RNA loadings and expressed relative to the first day of collection (=1). Values are representative of multiple technical replicates (n ≥ 3). Overlaid are the average leaf blade lengths (mm) ± SD (n ≥ 3).
Figure Legend Snippet: Detection of HvCesA1 antisense transcripts by SS-RT-PCR. (A) Tagged SS-RT-PCR was performed to detect HvCesA1 antisense transcripts over the course of third leaf development (10-16 days post imbibition). First-strand cDNAs were prepared using HvA1-sense-Tag1 GSP (antisense; NRT control), oligo dT (sense; Tag control), or no primer at all (NPC). The HvA1-antisense GSP and the Tag1 primer were used for amplification of the antisense, Tag, NPC, and NRT samples. For sense amplification, HvA1-sense and HvA1-antisense GSPs were used with oligo dT primed cDNAs. PCR products were confirmed by DNA sequencing. (B) Gel densitometry was performed to estimate HvCESA1 sense and antisense transcript abundances. Data were normalized to RNA loadings and expressed relative to the first day of collection (=1). Values are representative of multiple technical replicates (n ≥ 3). Overlaid are the average leaf blade lengths (mm) ± SD (n ≥ 3).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, DNA Sequencing

Additional time course study for HvCESA1 antisense. SS-RT-PCR was performed to determine the expression of HvCESA1 antisense transcripts over the course of barley third leaf development. Band intensities after agarose gel electrophoresis were determined by densitometry and expressed relative to day 10 of the time course. Values are averages of three technical replicates (gel images shown below each time point). Error bars represent SD.
Figure Legend Snippet: Additional time course study for HvCESA1 antisense. SS-RT-PCR was performed to determine the expression of HvCESA1 antisense transcripts over the course of barley third leaf development. Band intensities after agarose gel electrophoresis were determined by densitometry and expressed relative to day 10 of the time course. Values are averages of three technical replicates (gel images shown below each time point). Error bars represent SD.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis

Detection of HvCesA1 sRNAs by Ribonuclease Protection assay. (A) Ribonuclease protection assays were performed to detect HvCESA1 -derived sRNAs across barley leaf development (11-16dpi). A sense RNA probe was used to specifically protect HvCESA1 antisense RNAs. HvCESA1 sRNAs (∼21-24-nts) were detected with size estimation by Decade Ladder (Ambion). (B) Densitometry was performed to evaluate the change in HvCESA1 derived sRNA abundances. Data were normalized to RNA loadings and are expressed relative to the first day of collection (=1). Values are representative of multiple technical replicates (n ≥ 3). Overlaid are the average leaf blade lengths (mm) ± SD (n ≥ 3).
Figure Legend Snippet: Detection of HvCesA1 sRNAs by Ribonuclease Protection assay. (A) Ribonuclease protection assays were performed to detect HvCESA1 -derived sRNAs across barley leaf development (11-16dpi). A sense RNA probe was used to specifically protect HvCESA1 antisense RNAs. HvCESA1 sRNAs (∼21-24-nts) were detected with size estimation by Decade Ladder (Ambion). (B) Densitometry was performed to evaluate the change in HvCESA1 derived sRNA abundances. Data were normalized to RNA loadings and are expressed relative to the first day of collection (=1). Values are representative of multiple technical replicates (n ≥ 3). Overlaid are the average leaf blade lengths (mm) ± SD (n ≥ 3).

Techniques Used: Derivative Assay

HvCESA1-associated sRNAs. Ribonuclease protection assays were performed to detect HvCESA1 -derived sRNAs across barley leaf development 11-16dpi (Lanes 1-6). A sense RNA probe was used to protect HvCESA1 antisense RNAs. Band intensities for lanes 1-6 are shown in individual panels to evaluate the resolution and abundance of 21-24nt bands. Smaller bands (
Figure Legend Snippet: HvCESA1-associated sRNAs. Ribonuclease protection assays were performed to detect HvCESA1 -derived sRNAs across barley leaf development 11-16dpi (Lanes 1-6). A sense RNA probe was used to protect HvCESA1 antisense RNAs. Band intensities for lanes 1-6 are shown in individual panels to evaluate the resolution and abundance of 21-24nt bands. Smaller bands (

Techniques Used: Derivative Assay

Map to scale of HvCESA1 RPA probe and HvCESA1 antisense transcripts. PCR amplicons and RPA probes were designed internal to the coding region of HvCESA1 . The untranslated regions (UTR) at the 5’ and 3’ ends are indicated in green, with the coding sequence (CDS) indicated in tan. The region amplified to detect HvCESA1 antisense transcript is in purple, and the sequence region used to probe for antisense HvCESA1 sRNAs is indicated in red.
Figure Legend Snippet: Map to scale of HvCESA1 RPA probe and HvCESA1 antisense transcripts. PCR amplicons and RPA probes were designed internal to the coding region of HvCESA1 . The untranslated regions (UTR) at the 5’ and 3’ ends are indicated in green, with the coding sequence (CDS) indicated in tan. The region amplified to detect HvCESA1 antisense transcript is in purple, and the sequence region used to probe for antisense HvCESA1 sRNAs is indicated in red.

Techniques Used: Recombinase Polymerase Amplification, Polymerase Chain Reaction, Sequencing, Amplification

CESA Phylogenetic Tree. Multiple sequence alignment of Arabidopsis (At), barley (Hv), and Brachypodium (Bd) CESA proteins was performed using the Clustal Omega tool with default settings. Alignment file was loaded into Dendroscope 3 to generate an unrooted radial dendrogram. CESAs expressing antisense transcripts are highlighted in red. Protein sequences for AtCESA1 (At4g32410), AtCESA2 (At4g39350), AtCESA3 (At5g05170), AtCESA4 (At5g44030), AtCESA5 (At5g09870), AtCESA6 (At5g64740), AtCESA7 (At5g17420), AtCESA8 (At4g18780), AtCESA9 (At2g21770), and AtCESA10 (At2g25540) were collected from TAIR ( https://www.arabidopsis.org/ ). Protein sequences for HvCESA1 (MLOC_55153.1), HvCESA2 (AK366571), HvCESA3 (MLOC_61930.2), HvCESA4 (MLOC_66568.3), HvCESA5/7 (AK365079), HvCESA6 (MLOC_64555.1), HvCESA8 (MLOC_68431.4), BdCESA1 (Bradi2g34240), BdCESA2 (Bradi1g04597), BdCESA4 (Bradi2g49912), BdCESA5 (Bradi1g02510), BdCESA6 (Bradi1g53207), BdCESA7 (Bradi3g28350), BdCESA8 (Bradi1g54250), and BdCESA9 (Bradi4g30540) were collected from PGSB ( http://pgsb.helmholtz-muenchen.de/plant/barley/index.jsp ).
Figure Legend Snippet: CESA Phylogenetic Tree. Multiple sequence alignment of Arabidopsis (At), barley (Hv), and Brachypodium (Bd) CESA proteins was performed using the Clustal Omega tool with default settings. Alignment file was loaded into Dendroscope 3 to generate an unrooted radial dendrogram. CESAs expressing antisense transcripts are highlighted in red. Protein sequences for AtCESA1 (At4g32410), AtCESA2 (At4g39350), AtCESA3 (At5g05170), AtCESA4 (At5g44030), AtCESA5 (At5g09870), AtCESA6 (At5g64740), AtCESA7 (At5g17420), AtCESA8 (At4g18780), AtCESA9 (At2g21770), and AtCESA10 (At2g25540) were collected from TAIR ( https://www.arabidopsis.org/ ). Protein sequences for HvCESA1 (MLOC_55153.1), HvCESA2 (AK366571), HvCESA3 (MLOC_61930.2), HvCESA4 (MLOC_66568.3), HvCESA5/7 (AK365079), HvCESA6 (MLOC_64555.1), HvCESA8 (MLOC_68431.4), BdCESA1 (Bradi2g34240), BdCESA2 (Bradi1g04597), BdCESA4 (Bradi2g49912), BdCESA5 (Bradi1g02510), BdCESA6 (Bradi1g53207), BdCESA7 (Bradi3g28350), BdCESA8 (Bradi1g54250), and BdCESA9 (Bradi4g30540) were collected from PGSB ( http://pgsb.helmholtz-muenchen.de/plant/barley/index.jsp ).

Techniques Used: Sequencing, Expressing

10) Product Images from "Lactobacillus brevis Strains from Fermented Aloe vera Survive Gastroduodenal Environment and Suppress Common Food Borne Enteropathogens"

Article Title: Lactobacillus brevis Strains from Fermented Aloe vera Survive Gastroduodenal Environment and Suppress Common Food Borne Enteropathogens

Journal: PLoS ONE

doi: 10.1371/journal.pone.0090866

Base changes in the 16S rRNA sequences of L. brevis strains.
Figure Legend Snippet: Base changes in the 16S rRNA sequences of L. brevis strains.

Techniques Used:

11) Product Images from "UmuDAb: An Error-Prone Polymerase Accessory Homolog Whose N-Terminal Domain Is Required for Repression of DNA Damage Inducible Gene Expression in Acinetobacter baylyi"

Article Title: UmuDAb: An Error-Prone Polymerase Accessory Homolog Whose N-Terminal Domain Is Required for Repression of DNA Damage Inducible Gene Expression in Acinetobacter baylyi

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152013

DNA damage-induced mutagenesis experiments suggest that UmuDAb does not perform UmuD polymerase accessory function. (A) DNA damage-induced mutagenesis (measured by, and represented as, the ratio of increased rifampin resistance observed in UV-treated vs untreated cells) was performed to assess whether umuDAb could complement an E . coli ΔumuD mutant. The E . coli Δ umuD772 ::kan strain 315 carried either: pIX3.0 (containing no DNA insert), pIX2b (pIX3.0 carrying A . baylyi umuDAb [ 20 ]), pIX2bˊ (pIX3.0 carrying A . baylyi umuDAb ˊ ( umuDAbΔ2–83 ), or pIXUDEC (pIX3.0 carrying E . coli umuD ). Differences among strains were analyzed with a two-tailed, one-way Kruskal-Wallis ANOVA, followed by Dunn’s multiple comparisons post-test indicating significance denoted by * (p
Figure Legend Snippet: DNA damage-induced mutagenesis experiments suggest that UmuDAb does not perform UmuD polymerase accessory function. (A) DNA damage-induced mutagenesis (measured by, and represented as, the ratio of increased rifampin resistance observed in UV-treated vs untreated cells) was performed to assess whether umuDAb could complement an E . coli ΔumuD mutant. The E . coli Δ umuD772 ::kan strain 315 carried either: pIX3.0 (containing no DNA insert), pIX2b (pIX3.0 carrying A . baylyi umuDAb [ 20 ]), pIX2bˊ (pIX3.0 carrying A . baylyi umuDAb ˊ ( umuDAbΔ2–83 ), or pIXUDEC (pIX3.0 carrying E . coli umuD ). Differences among strains were analyzed with a two-tailed, one-way Kruskal-Wallis ANOVA, followed by Dunn’s multiple comparisons post-test indicating significance denoted by * (p

Techniques Used: Mutagenesis, Two Tailed Test

Modeling of the N-terminal domains of LexA and UmuDAb monomers. (A) The N-terminal 60 amino acids of E . coli LexA and A . baylyi UmuDAb, showing underlined alpha-helical regions predicted by the Predict Protein server [ 28 ]. In LexA, helices α1–3 span amino acids 8–20, 28–35, and 41–55 [ 25 ]. For UmuDAb, helices are predicted to form from amino acids 3–9, 22–29, and 36–46. (B) Predicted secondary structures of LexA and UmuDAb, showing alpha-helices 1–3 represented by red thin bars and beta sheets represented by thick yellow boxes; predicted by the Predict Protein server. (C) I-TASSER modeling of LexA and UmuDAb, oriented to align the NTDs (in blue shading) and showing the wing of the wHTH structures. The interdomain linker between the LexA NTD and CTD is extremely flexible [ 4 ] and is likely responsible for the variation between the two proteins’ total orientations. Arrows point to some of the amino acids in the LexA α2 helix and α3 recognition helix that are required for DNA binding [ 4 ], and the similarly located sites of directed mutations in UmuDAb.
Figure Legend Snippet: Modeling of the N-terminal domains of LexA and UmuDAb monomers. (A) The N-terminal 60 amino acids of E . coli LexA and A . baylyi UmuDAb, showing underlined alpha-helical regions predicted by the Predict Protein server [ 28 ]. In LexA, helices α1–3 span amino acids 8–20, 28–35, and 41–55 [ 25 ]. For UmuDAb, helices are predicted to form from amino acids 3–9, 22–29, and 36–46. (B) Predicted secondary structures of LexA and UmuDAb, showing alpha-helices 1–3 represented by red thin bars and beta sheets represented by thick yellow boxes; predicted by the Predict Protein server. (C) I-TASSER modeling of LexA and UmuDAb, oriented to align the NTDs (in blue shading) and showing the wing of the wHTH structures. The interdomain linker between the LexA NTD and CTD is extremely flexible [ 4 ] and is likely responsible for the variation between the two proteins’ total orientations. Arrows point to some of the amino acids in the LexA α2 helix and α3 recognition helix that are required for DNA binding [ 4 ], and the similarly located sites of directed mutations in UmuDAb.

Techniques Used: Binding Assay

A . baylyi UmuDAb is required for repression of a sub-set of DNA damage-induced genes. RT-qPCR experiments measured expression of the umuDAb -regulated ddrR gene ( ACIAD2730 ), and the non- umuDAb regulated genes gst ( ACIAD0445 ) and nrdA ( ACIAD0724 ) in the Δ umuDAb mutant strain ACIAD2729. The transcription of each gene was induced by DNA damage incurred by growth in 2 μg/mL MMC-containing medium. Each gene was assayed in one RT-qPCR experiment (plate), with error bars indicating standard error of the mean from technical triplicates of biological triplicates. Each gene was significantly induced in the wild type ADP1 strain (p
Figure Legend Snippet: A . baylyi UmuDAb is required for repression of a sub-set of DNA damage-induced genes. RT-qPCR experiments measured expression of the umuDAb -regulated ddrR gene ( ACIAD2730 ), and the non- umuDAb regulated genes gst ( ACIAD0445 ) and nrdA ( ACIAD0724 ) in the Δ umuDAb mutant strain ACIAD2729. The transcription of each gene was induced by DNA damage incurred by growth in 2 μg/mL MMC-containing medium. Each gene was assayed in one RT-qPCR experiment (plate), with error bars indicating standard error of the mean from technical triplicates of biological triplicates. Each gene was significantly induced in the wild type ADP1 strain (p

Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis

12) Product Images from "Detection of an Abundant Plant-Based Small RNA in Healthy Consumers"

Article Title: Detection of an Abundant Plant-Based Small RNA in Healthy Consumers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0137516

Serum and urine MIR2911 levels in mice fed various herb- or flower-containing diets. (A) Detection of MIR2911 in sera from mice fed various herb and flower diets. (B) Detection of MIR2911 in urine from mice fed various herb and flower diets. For (A) and (B), mice were fed diets for 7 days before RNA was isolated form serum and urine samples and analyzed. N = 5. Asterisk: p
Figure Legend Snippet: Serum and urine MIR2911 levels in mice fed various herb- or flower-containing diets. (A) Detection of MIR2911 in sera from mice fed various herb and flower diets. (B) Detection of MIR2911 in urine from mice fed various herb and flower diets. For (A) and (B), mice were fed diets for 7 days before RNA was isolated form serum and urine samples and analyzed. N = 5. Asterisk: p

Techniques Used: Mouse Assay, Isolation

Serum MIR2911 is not associated primarily with AGO2. (A) Quantification of MIR2911 in AGO2-associated immunoprecipitates and unbound fractions of serum from animals fed an herb-based diet or a standard chow diet. (B) miR-16 immunoprecipitation was analyzed as an endogenous control for AGO2 precipitation. Levels of MIR2911 and miR-16 were each normalized to spiked-in exogenous MIR161. This experiment is representative of more than five different experiments done with herbal-fed mice (honeysuckle, chamomile).
Figure Legend Snippet: Serum MIR2911 is not associated primarily with AGO2. (A) Quantification of MIR2911 in AGO2-associated immunoprecipitates and unbound fractions of serum from animals fed an herb-based diet or a standard chow diet. (B) miR-16 immunoprecipitation was analyzed as an endogenous control for AGO2 precipitation. Levels of MIR2911 and miR-16 were each normalized to spiked-in exogenous MIR161. This experiment is representative of more than five different experiments done with herbal-fed mice (honeysuckle, chamomile).

Techniques Used: Immunoprecipitation, Mouse Assay

Time course analysis of absorption of gavage fed synthetic MIR2911 Time course analysis of serum MIR2911 levels in mice gavage fed 400 pmols synthetic MIR2911 at 0.5 hour, 1 hour, 3 hours after gavage feeding. Mice were pre-fed chow diet. N = 5. Experiment replicated three times.
Figure Legend Snippet: Time course analysis of absorption of gavage fed synthetic MIR2911 Time course analysis of serum MIR2911 levels in mice gavage fed 400 pmols synthetic MIR2911 at 0.5 hour, 1 hour, 3 hours after gavage feeding. Mice were pre-fed chow diet. N = 5. Experiment replicated three times.

Techniques Used: Mouse Assay

Antibiotic treatments do not appear to impact MIR2911 serum detection in honeysuckle fed mice. Analysis of serum MIR2911 levels in mice fed honeysuckle (HS), mice fed honeysuckle and treated with antibiotics (HS+Ab), and mice fed chow. Mice were fed the diets for 7 days before analysis of serum MIR2911 levels. N = 5. Experiment replicated more than three times with each diet and condition.
Figure Legend Snippet: Antibiotic treatments do not appear to impact MIR2911 serum detection in honeysuckle fed mice. Analysis of serum MIR2911 levels in mice fed honeysuckle (HS), mice fed honeysuckle and treated with antibiotics (HS+Ab), and mice fed chow. Mice were fed the diets for 7 days before analysis of serum MIR2911 levels. N = 5. Experiment replicated more than three times with each diet and condition.

Techniques Used: Mouse Assay

13) Product Images from "Regulation of Human Formyl Peptide Receptor 1 Synthesis: Role of Single Nucleotide Polymorphisms, Transcription Factors, and Inflammatory Mediators"

Article Title: Regulation of Human Formyl Peptide Receptor 1 Synthesis: Role of Single Nucleotide Polymorphisms, Transcription Factors, and Inflammatory Mediators

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028712

Four FPR1 variants show similar expression levels. FPR1 haplotypes 8A, 11A, 12D and 16A were expressed as fusion proteins with firefly luciferase in U937 cells. Cells were electroporated with various amounts of the firefly luciferase reporter plasmid (as shown) and 300 ng pRL-TK Renilla luciferase control reporter plasmid. 24 h post-transfection cell extracts were analyzed using the Promega dual luciferase assay kit. The graphs show the mean ratios of firefly and Renilla luciferase from five separate experiments ± S.E.M. One-way analysis of variance showed no statistical differences between the haplotypes.
Figure Legend Snippet: Four FPR1 variants show similar expression levels. FPR1 haplotypes 8A, 11A, 12D and 16A were expressed as fusion proteins with firefly luciferase in U937 cells. Cells were electroporated with various amounts of the firefly luciferase reporter plasmid (as shown) and 300 ng pRL-TK Renilla luciferase control reporter plasmid. 24 h post-transfection cell extracts were analyzed using the Promega dual luciferase assay kit. The graphs show the mean ratios of firefly and Renilla luciferase from five separate experiments ± S.E.M. One-way analysis of variance showed no statistical differences between the haplotypes.

Techniques Used: Expressing, Luciferase, Plasmid Preparation, Transfection

14) Product Images from "Characterization of Three New Glutaredoxin Genes in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis: Putative Role of RiGRX4 and RiGRX5 in Iron Homeostasis"

Article Title: Characterization of Three New Glutaredoxin Genes in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis: Putative Role of RiGRX4 and RiGRX5 in Iron Homeostasis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0149606

Complementation of the sensitivity to external oxidants of the grx yeast mutants by the R . irregularis GRX genes. A. Effect of RiGRX4 , RiGRX5 and RiGRX6 expression on the sensitivity of the Δgrx3Δgrx4 strain to 1 mM hydrogen peroxide (H 2 O 2 ). B. Effect of RiGRX4 , RiGRX5 and RiGRX6 expression on the sensitivity of the Δgrx5 strain to 0.1 mM menadione (Md). The photographs were taken after 3 days of growth at 30°C. C. Effect of RiGRX6 expression on the sensitivity of Δgrx6Δgrx7 to 500 mM CaCl 2 (40 h). Data are means of three independent experiments +/- standard error and represent the growth yield ratio between treated and untreated cultures and then made relative to this ratio in cells expressing the S . cerevisiae Grx6 . Asterisks show statistically significant differences (p
Figure Legend Snippet: Complementation of the sensitivity to external oxidants of the grx yeast mutants by the R . irregularis GRX genes. A. Effect of RiGRX4 , RiGRX5 and RiGRX6 expression on the sensitivity of the Δgrx3Δgrx4 strain to 1 mM hydrogen peroxide (H 2 O 2 ). B. Effect of RiGRX4 , RiGRX5 and RiGRX6 expression on the sensitivity of the Δgrx5 strain to 0.1 mM menadione (Md). The photographs were taken after 3 days of growth at 30°C. C. Effect of RiGRX6 expression on the sensitivity of Δgrx6Δgrx7 to 500 mM CaCl 2 (40 h). Data are means of three independent experiments +/- standard error and represent the growth yield ratio between treated and untreated cultures and then made relative to this ratio in cells expressing the S . cerevisiae Grx6 . Asterisks show statistically significant differences (p

Techniques Used: Expressing

A. Domain organization of the R . irregularis GRXs. Glutaredoxin domains are represented by black boxes. The thioredoxin-like (Trx) domain of RiGRX4, the mitochondrial location signal (MLS) of RiGRX5 and the domain of unknown function of RiGRX6 (white box) are also indicated. Numbers correspond to the position of the first cysteine in the active site in the GRX domains, the first glycine of the glutathione binding domains and the total length of the proteins. The position of the cysteine in the Trx domain of RiGRX4 is also indicated. B. Unrooted Nieghbor-Joining tree of the GRX family in fungi. Organisms: An, Aspergillus niger ; Bc, Botrytis cinerea ; Cc, Coprinopsis cinerea ; Cn, Cryptococcus neoformans ; Lb, Laccaria bicolor; Mg, Magnaporthe grisea ; Nc, Neurospora crassa ; Pc, Phanerochaete chrysosporium ; Pg, Puccinia graminis; Ri, Rhizophagus irregularis ; Ro, Rhizopus oryzae ; Sc, Saccharomyces cerevisiae ; Sp, Schizosaccharomyces pombe ; Tm, Tuber melanosporum ; Um, Ustilago maydis . R . irregularis GRXs are emphasized in bold. Protein JGI identification numbers are indicated. R . oryzae sequences were retrieved from the Broad Institute databases ( http://www.broad.mit.edu/annotation/ ). Bootstrap values above 70 and supporting a node are indicated.
Figure Legend Snippet: A. Domain organization of the R . irregularis GRXs. Glutaredoxin domains are represented by black boxes. The thioredoxin-like (Trx) domain of RiGRX4, the mitochondrial location signal (MLS) of RiGRX5 and the domain of unknown function of RiGRX6 (white box) are also indicated. Numbers correspond to the position of the first cysteine in the active site in the GRX domains, the first glycine of the glutathione binding domains and the total length of the proteins. The position of the cysteine in the Trx domain of RiGRX4 is also indicated. B. Unrooted Nieghbor-Joining tree of the GRX family in fungi. Organisms: An, Aspergillus niger ; Bc, Botrytis cinerea ; Cc, Coprinopsis cinerea ; Cn, Cryptococcus neoformans ; Lb, Laccaria bicolor; Mg, Magnaporthe grisea ; Nc, Neurospora crassa ; Pc, Phanerochaete chrysosporium ; Pg, Puccinia graminis; Ri, Rhizophagus irregularis ; Ro, Rhizopus oryzae ; Sc, Saccharomyces cerevisiae ; Sp, Schizosaccharomyces pombe ; Tm, Tuber melanosporum ; Um, Ustilago maydis . R . irregularis GRXs are emphasized in bold. Protein JGI identification numbers are indicated. R . oryzae sequences were retrieved from the Broad Institute databases ( http://www.broad.mit.edu/annotation/ ). Bootstrap values above 70 and supporting a node are indicated.

Techniques Used: Binding Assay

Effect of hydrogen peroxide on the expression of the R . irregularis GRX genes. R . irregularis ERM grown in M-C medium was exposed for different periods of time to 0.1 mM H 2 O 2 (grey columns) or 1 mM H 2 O 2 (black columns). RiGRX1 (A), RiGRX4 (B), RiGRX5 (C), RiGRX6 (D) and GintPDX1 (E) gene expression. Data were normalized using the housekeeping gene RiTEF . Relative expression levels were calculated by the 2 -ΔΔCT method. Data are means +/- standard error. Asterisks show statistically significant differences (p
Figure Legend Snippet: Effect of hydrogen peroxide on the expression of the R . irregularis GRX genes. R . irregularis ERM grown in M-C medium was exposed for different periods of time to 0.1 mM H 2 O 2 (grey columns) or 1 mM H 2 O 2 (black columns). RiGRX1 (A), RiGRX4 (B), RiGRX5 (C), RiGRX6 (D) and GintPDX1 (E) gene expression. Data were normalized using the housekeeping gene RiTEF . Relative expression levels were calculated by the 2 -ΔΔCT method. Data are means +/- standard error. Asterisks show statistically significant differences (p

Techniques Used: Expressing

Localization of R . irregularis GRXs in S . cerevisiae . Soluble GFP (A-C) and C-terminal GFP-tagged versions of RiGRX4 (D-F) and RiGRX5 (G-J) were expressed in the Δgrx3Δgrx4 cells. C-terminal GFP-tagged version of RiGRX6 (K-M) was expressed in the Δgrx6Δgrx7 yeast mutant cells. Cells were grown to mid-logarithmic phase and localization of fusion proteins was visualized by fluorescence microscopy (A, D, G and K). Mitochondria in the RiGRX6 -GF P expressing cells were stained with MitoTracker ® Red and visualized by fluorescence microscopy (H). Bright field (B, E, J and L) and merged (C, F, I and M) images.
Figure Legend Snippet: Localization of R . irregularis GRXs in S . cerevisiae . Soluble GFP (A-C) and C-terminal GFP-tagged versions of RiGRX4 (D-F) and RiGRX5 (G-J) were expressed in the Δgrx3Δgrx4 cells. C-terminal GFP-tagged version of RiGRX6 (K-M) was expressed in the Δgrx6Δgrx7 yeast mutant cells. Cells were grown to mid-logarithmic phase and localization of fusion proteins was visualized by fluorescence microscopy (A, D, G and K). Mitochondria in the RiGRX6 -GF P expressing cells were stained with MitoTracker ® Red and visualized by fluorescence microscopy (H). Bright field (B, E, J and L) and merged (C, F, I and M) images.

Techniques Used: Mutagenesis, Fluorescence, Microscopy, Expressing, Staining

Effect of iron on the expression of the R . irregularis GRX genes. R . irregularis was grown in M-C media containing 45 μM Fe (control) or supplemented with 4.5 mM Fe or 45 mM Fe medium for 2 weeks. RiGRX1 (A), RiGRX4 (B), RiGRX5 (C) and RiGRX6 (D) gene expression. Data were normalized using the housekeeping gene RiTEF . Relative expression levels were calculated by the 2 -ΔΔCT method. Data are means +/- standard error. Asterisks show statistically significant differences (p
Figure Legend Snippet: Effect of iron on the expression of the R . irregularis GRX genes. R . irregularis was grown in M-C media containing 45 μM Fe (control) or supplemented with 4.5 mM Fe or 45 mM Fe medium for 2 weeks. RiGRX1 (A), RiGRX4 (B), RiGRX5 (C) and RiGRX6 (D) gene expression. Data were normalized using the housekeeping gene RiTEF . Relative expression levels were calculated by the 2 -ΔΔCT method. Data are means +/- standard error. Asterisks show statistically significant differences (p

Techniques Used: Expressing

15) Product Images from "Impact of voluntary exercise and housing conditions on hippocampal glucocorticoid receptor, miR-124 and anxiety"

Article Title: Impact of voluntary exercise and housing conditions on hippocampal glucocorticoid receptor, miR-124 and anxiety

Journal: Molecular Brain

doi: 10.1186/s13041-015-0128-8

Impact of exercise and housing conditions on Nr3c1 and Nr3c1-1F expression at the hippocampus. a Nr3c1 and c Nr3c1-1F expression for single-housed and pair-housed mice that exercised or remained sedentary. Pearson’s correlation between weight gain and hippocampal expression of b Nr3c1 or d Nr3c1-1F . **** p
Figure Legend Snippet: Impact of exercise and housing conditions on Nr3c1 and Nr3c1-1F expression at the hippocampus. a Nr3c1 and c Nr3c1-1F expression for single-housed and pair-housed mice that exercised or remained sedentary. Pearson’s correlation between weight gain and hippocampal expression of b Nr3c1 or d Nr3c1-1F . **** p

Techniques Used: Expressing, Mouse Assay

Impact of exercise and housing conditions on DNA methylation at the promoter region of Nr3c1-1F . a Schematic representation of the multiple first exons of the Nr3c1 gene. +1 indicates the translational start site. The promoter region of Nr3c1-1F has been enlarged and the 18 CpGs it contains highlighted. b Methylation rates of Nr3c1-1F promoter are shown for single-housed mice and pair-housed that either exercised or remained sedentary. Each row represents a clone and each column indicates a CpG (empty square = not methylated CpG and full square = methylated CpG)
Figure Legend Snippet: Impact of exercise and housing conditions on DNA methylation at the promoter region of Nr3c1-1F . a Schematic representation of the multiple first exons of the Nr3c1 gene. +1 indicates the translational start site. The promoter region of Nr3c1-1F has been enlarged and the 18 CpGs it contains highlighted. b Methylation rates of Nr3c1-1F promoter are shown for single-housed mice and pair-housed that either exercised or remained sedentary. Each row represents a clone and each column indicates a CpG (empty square = not methylated CpG and full square = methylated CpG)

Techniques Used: DNA Methylation Assay, Methylation, Mouse Assay

16) Product Images from "Citrobacter rodentium, the Causative Agent of Transmissible Murine Colonic Hyperplasia, Exhibits Clonality: Synonymy of C. rodentium and Mouse-Pathogenic Escherichia coli"

Article Title: Citrobacter rodentium, the Causative Agent of Transmissible Murine Colonic Hyperplasia, Exhibits Clonality: Synonymy of C. rodentium and Mouse-Pathogenic Escherichia coli

Journal: Journal of Clinical Microbiology

doi:

Transmission electron micrographs showing AE pedestal induction on mouse colonic epithelial cells by (A) CDC1843-73 T (original magnification, ×12,000; inset, ×20,000) and (B) MPEC (original magnification, ×6,000; inset, ×25,000). Bars, 1 μm.
Figure Legend Snippet: Transmission electron micrographs showing AE pedestal induction on mouse colonic epithelial cells by (A) CDC1843-73 T (original magnification, ×12,000; inset, ×20,000) and (B) MPEC (original magnification, ×6,000; inset, ×25,000). Bars, 1 μm.

Techniques Used: Transmission Assay

17) Product Images from "The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator"

Article Title: The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator

Journal: Genes & Development

doi: 10.1101/gad.1416106

Sonic hedgehog induces the expression of Evf, Dlx-2, and Dlx-5 in vivo. ( a ) Diagram of retroviral backbone used to express the human sonic hedgehog protein (pwtShhCLE) or control (pCLE). Alkaline phosphatase is bicistronic with the Shh cDNA, allowing detection of virally infected cells. ( b ) Lysates of C17 cells infected with wtShh virus (lane 3 ), pCLE control virus (lane 2 ), or 7 ng of purified recombinant-unmodified Shh (lane 1, uShhN) were Western-blotted and probed with anti-Shh antibody (Santa Cruz Biotechnology). ( c ) In utero UBM-guided entry of viruses into E9.5 mouse forebrain. Sections of E12.5 mouse brains 3 d after infection with control pCLE virus ( d–h ), littermate control ( i–n ), or wtShh virus ( o–t ). ( d,e ) pCLE virus-infected clusters. ( o,p ) wtshh-infected viral clusters visualized by alkaline phosphatase staining in the dorsal midline of the telencephalon. ( i,j ) Uninfected littermate control does not contain alkaline phosphatase-expressing clusters of cells. In situ hybridization of adjacent sections probed for ectopic expression of ventral genes Dlx-2 ( f,k,q ), Dlx-5 ( g,l,r ), Evf c ( h ), Dlx-6 ( m,s ), and Evf-2 ( n,t ). Orientation of the section is indicated in the top right box.
Figure Legend Snippet: Sonic hedgehog induces the expression of Evf, Dlx-2, and Dlx-5 in vivo. ( a ) Diagram of retroviral backbone used to express the human sonic hedgehog protein (pwtShhCLE) or control (pCLE). Alkaline phosphatase is bicistronic with the Shh cDNA, allowing detection of virally infected cells. ( b ) Lysates of C17 cells infected with wtShh virus (lane 3 ), pCLE control virus (lane 2 ), or 7 ng of purified recombinant-unmodified Shh (lane 1, uShhN) were Western-blotted and probed with anti-Shh antibody (Santa Cruz Biotechnology). ( c ) In utero UBM-guided entry of viruses into E9.5 mouse forebrain. Sections of E12.5 mouse brains 3 d after infection with control pCLE virus ( d–h ), littermate control ( i–n ), or wtShh virus ( o–t ). ( d,e ) pCLE virus-infected clusters. ( o,p ) wtshh-infected viral clusters visualized by alkaline phosphatase staining in the dorsal midline of the telencephalon. ( i,j ) Uninfected littermate control does not contain alkaline phosphatase-expressing clusters of cells. In situ hybridization of adjacent sections probed for ectopic expression of ventral genes Dlx-2 ( f,k,q ), Dlx-5 ( g,l,r ), Evf c ( h ), Dlx-6 ( m,s ), and Evf-2 ( n,t ). Orientation of the section is indicated in the top right box.

Techniques Used: Expressing, In Vivo, Infection, Purification, Recombinant, Western Blot, In Utero, Staining, In Situ Hybridization

Evf-2 and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll, anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using Evf-2 antisense RNA probe and anti- dll antibody. DAPI staining (blue) reveals nuclei. Evf-2 RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of Evf-2 and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.
Figure Legend Snippet: Evf-2 and Dlx-2 form a complex in vivo. ( a ) Nuclear extracts made from C17 neural cells transfected with Flag-tagged Emx-1, Flag-tagged Dlx-2, or pcDNA control were analyzed for the presence of Evf-2 –Dlx-2 complexes by immunoprecipitation with anti-Flag antibody, followed by RT–PCR against Evf-2 -specific primers and S17 control primers. Western analysis shows that both Flag-Emx-1 and Flag-Dlx-2 are present in nuclear extracts transfected with constructs expressing these proteins. ( b ) Nuclear extracts made from rat E11.5 BAs were analyzed for the presence of Evf-2 /Dlx-2 complexes by immunoprecipitation with anti- dll, anti-Islet 1/2, or anti-IgG antibody, followed by RT–PCR against Evf-2 -specific primers or GAPDH. ( c ) Single-cell suspensions made from mouse E12.5 dorsal and ventral telencephalon were dissected as shown in the schematic, centrifuged onto slides, and processed for fluorescent in situ/immunolocalization using Evf-2 antisense RNA probe and anti- dll antibody. DAPI staining (blue) reveals nuclei. Evf-2 RNA (Alexa fluor 568) is in red, Dlx (Alexa fluor 488) is in green, and regions of overlap are in yellow. ( d ) A model proposing that a complex of Evf-2 and Dlx-2 affects ei activity, ultimately affecting transcription of the Dlx-5 and Dlx-6 genes.

Techniques Used: In Vivo, Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Construct, Expressing, In Situ, Staining, Activity Assay

18) Product Images from "Demonstration of a Glycoprotein Derived From the Ceacam10 Gene in Mouse Seminal Vesicle Secretions 1"

Article Title: Demonstration of a Glycoprotein Derived From the Ceacam10 Gene in Mouse Seminal Vesicle Secretions 1

Journal: Biology of Reproduction

doi: 10.1095/biolreprod.105.039651

Ceacam10 expression in the luminal epithelium of the seminal vesicle. A ) Immunolocalization of CEACAM10 to the luminal epithelium of the seminal vesicle. Tissue slices were histochemically stained for CEACAM10 with antibody against the protein, biotin-conjugate goat anti-rabbit IgG, and alkaline phosphatase-conjugated streptavidin a . The specimens were stained as in a except that the antibody was replaced by normal serum ( b ). For contrast, the specimens were further stained with Nuclear Fast Red. Photographs were taken with brightfield illumination. MF, mucosal fold; SM, smooth muscle; LF, luminal fluid. The staining of Nuclear Fast Red is in pink and the signals of CEACAM10 protein, demonstrated by staining of alkaline phosphatase activity, are in dark blue. Bar = 20 μm. B ) Demonstration of Ceacam10 mRNA in the luminal epithelial cells of the seminal vesicle. The epithelial cells of MF or SM cells in a tissue slice (8 μm) of mouse seminal vesicle were selectively captured and transferred to films by LCM. The tissue slides before and after LCM were stained and observed (see text for details). A Ceacam10 cDNA fragment (237 bp) or a Gapd cDNA fragment (557 bp) was amplified from the total RNA of MF or SM by reverse transcription-polymerase chain reaction. The level of Gapd mRNA was used as an internal control. Bar = 100 μm
Figure Legend Snippet: Ceacam10 expression in the luminal epithelium of the seminal vesicle. A ) Immunolocalization of CEACAM10 to the luminal epithelium of the seminal vesicle. Tissue slices were histochemically stained for CEACAM10 with antibody against the protein, biotin-conjugate goat anti-rabbit IgG, and alkaline phosphatase-conjugated streptavidin a . The specimens were stained as in a except that the antibody was replaced by normal serum ( b ). For contrast, the specimens were further stained with Nuclear Fast Red. Photographs were taken with brightfield illumination. MF, mucosal fold; SM, smooth muscle; LF, luminal fluid. The staining of Nuclear Fast Red is in pink and the signals of CEACAM10 protein, demonstrated by staining of alkaline phosphatase activity, are in dark blue. Bar = 20 μm. B ) Demonstration of Ceacam10 mRNA in the luminal epithelial cells of the seminal vesicle. The epithelial cells of MF or SM cells in a tissue slice (8 μm) of mouse seminal vesicle were selectively captured and transferred to films by LCM. The tissue slides before and after LCM were stained and observed (see text for details). A Ceacam10 cDNA fragment (237 bp) or a Gapd cDNA fragment (557 bp) was amplified from the total RNA of MF or SM by reverse transcription-polymerase chain reaction. The level of Gapd mRNA was used as an internal control. Bar = 100 μm

Techniques Used: Expressing, Staining, Activity Assay, Laser Capture Microdissection, Amplification, Reverse Transcription Polymerase Chain Reaction

19) Product Images from "Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants"

Article Title: Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants

Journal: Molecular Immunology

doi: 10.1016/j.molimm.2015.06.022

Expression of IL-26 alternative splice variants in different tissues of dromedary camel tissues and in-vitro mitogen stimulated PBMCs (A) Total mRNA was isolated from camel tissues were reverse transcribed and the cDNA was used as template for amplification with IL26XF1 IL26XR1 primers (B) Camel PBMCs were stimulated in vitro with PWM or Con A (10 μg/ml) for 16 hrs. Unstimulated PBMCs under identical culture conditions were also kept as control for 16 h. RNA isolated from stimulated samples and control (3 different camels) was used for cDNA synthesis. IL26 expression profile was analyzed with IL26XF1 and IL26XR1 primers on an Agilent 2100 Bioanalyzer using a DNA 1000 kit. Size standards for IL-26 transcript variants were made by PCR amplification of the previously cloned camel IL-26 Tv1 to Tv5 variants using IL26XF1 and IL26XR1. Equal mixture of these amplicons was used to create the size standards loaded in lane marked IL26Tv Mix.
Figure Legend Snippet: Expression of IL-26 alternative splice variants in different tissues of dromedary camel tissues and in-vitro mitogen stimulated PBMCs (A) Total mRNA was isolated from camel tissues were reverse transcribed and the cDNA was used as template for amplification with IL26XF1 IL26XR1 primers (B) Camel PBMCs were stimulated in vitro with PWM or Con A (10 μg/ml) for 16 hrs. Unstimulated PBMCs under identical culture conditions were also kept as control for 16 h. RNA isolated from stimulated samples and control (3 different camels) was used for cDNA synthesis. IL26 expression profile was analyzed with IL26XF1 and IL26XR1 primers on an Agilent 2100 Bioanalyzer using a DNA 1000 kit. Size standards for IL-26 transcript variants were made by PCR amplification of the previously cloned camel IL-26 Tv1 to Tv5 variants using IL26XF1 and IL26XR1. Equal mixture of these amplicons was used to create the size standards loaded in lane marked IL26Tv Mix.

Techniques Used: Expressing, In Vitro, Isolation, Amplification, Polymerase Chain Reaction, Clone Assay

Analysis of Expression of camel IL-26 alternative splice variants in camel ovary fibroblasts cells infected with Camel pox virus: Camel ovary fibroblasts were infected with Camel Pox virus in-vitro and RNA isolated post 48 hrs after infection from virus infected and uninfected cells. IL-26 transcript variants were amplified by RT-PCR using IL26XF1 /IL26XR1 primers and analyzed on Agilent 2100 Bioanalyzer using a DNA 1000 kit. Size standards for IL-26 transcript variants were made by PCR amplification of the cloned camel IL-26 Tv1 to Tv5 variants using IL26XF1 and IL26XR1. Equal mixture of these amplicons was used to create the size standards and analyzed along with the samples. First panel in each set represents the virtual image for the fragments separated on the Agilent 2100 Bioanalyzer. The second and third panel represent the electropherograms of IL-26 transcript variants of control/infected (blue) overlaid with the IL-26Tv standard mix (red) for size comparison. The induction of IL-26 transcript variant 2 in virus infected samples is marked by an arrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Analysis of Expression of camel IL-26 alternative splice variants in camel ovary fibroblasts cells infected with Camel pox virus: Camel ovary fibroblasts were infected with Camel Pox virus in-vitro and RNA isolated post 48 hrs after infection from virus infected and uninfected cells. IL-26 transcript variants were amplified by RT-PCR using IL26XF1 /IL26XR1 primers and analyzed on Agilent 2100 Bioanalyzer using a DNA 1000 kit. Size standards for IL-26 transcript variants were made by PCR amplification of the cloned camel IL-26 Tv1 to Tv5 variants using IL26XF1 and IL26XR1. Equal mixture of these amplicons was used to create the size standards and analyzed along with the samples. First panel in each set represents the virtual image for the fragments separated on the Agilent 2100 Bioanalyzer. The second and third panel represent the electropherograms of IL-26 transcript variants of control/infected (blue) overlaid with the IL-26Tv standard mix (red) for size comparison. The induction of IL-26 transcript variant 2 in virus infected samples is marked by an arrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Expressing, Infection, In Vitro, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Clone Assay, Variant Assay

20) Product Images from "The genetic architecture of a host shift: An adaptive walk protected an aphid and its endosymbiont from plant chemical defenses"

Article Title: The genetic architecture of a host shift: An adaptive walk protected an aphid and its endosymbiont from plant chemical defenses

Journal: Science Advances

doi: 10.1126/sciadv.aba1070

Further amplification of CYP6CY3 is associated with the insertion of transposable elements that are highly expressed in the aphid gut. ( A ) Copy number of CYP6CY3 , CYP6CY4 , and CYP6CY23 in M. p. nicotianae clones Mn1–6 compared to M. persicae clone Mp3 as determined by qPCR. Error bars indicate 95% confidence intervals ( n = 4). ( B ) Schematic of the CYP6CY3 amplicon obtained from BAC sequencing. The insertion sites of a Tc1/Mariner (Tc1/Mar), hAT, and TTAA3 transposable element are illustrated. ( C ) Colocalization of the BAC clone bearing a copy of CYP6CY3 on scaffold 15, with scaffold 16 on metaphase chromosomes of Mp1 and Mn6. The scaffold 16 probe was detected with tyramide–fluorescein isothiocyanate (green, arrows), and the BAC was directly labeled with Cy3 (red, arrowheads). Note that only one scaffold 16 locus was detected; this was due to a limitation of TSA-FISH to provide balanced signals, rather than its absence in the homologous chromosome. X, sex chromosome. Scale bar, 5 μm. ( D ) Additional long single-molecule sequencing identified copies of CYP6CY3 at the scaffold 16 locus in combination with the hAT element or in combination with the TTAA3, HAT, and Mutator-like (MULE) elements. ( E ) Features of the four transposons found in association with CYP6CY3 . ORF, open reading frame; aa, amino acid. ( F ) Expression of hAT, TTAA3, Tc1/Mariner, and MULE in carcass, gut, or head tissue of M. persicae clone Mp1 and M. p. nicotianae clone Mn6. Error bars indicate 95% confidence limits ( n = 4).
Figure Legend Snippet: Further amplification of CYP6CY3 is associated with the insertion of transposable elements that are highly expressed in the aphid gut. ( A ) Copy number of CYP6CY3 , CYP6CY4 , and CYP6CY23 in M. p. nicotianae clones Mn1–6 compared to M. persicae clone Mp3 as determined by qPCR. Error bars indicate 95% confidence intervals ( n = 4). ( B ) Schematic of the CYP6CY3 amplicon obtained from BAC sequencing. The insertion sites of a Tc1/Mariner (Tc1/Mar), hAT, and TTAA3 transposable element are illustrated. ( C ) Colocalization of the BAC clone bearing a copy of CYP6CY3 on scaffold 15, with scaffold 16 on metaphase chromosomes of Mp1 and Mn6. The scaffold 16 probe was detected with tyramide–fluorescein isothiocyanate (green, arrows), and the BAC was directly labeled with Cy3 (red, arrowheads). Note that only one scaffold 16 locus was detected; this was due to a limitation of TSA-FISH to provide balanced signals, rather than its absence in the homologous chromosome. X, sex chromosome. Scale bar, 5 μm. ( D ) Additional long single-molecule sequencing identified copies of CYP6CY3 at the scaffold 16 locus in combination with the hAT element or in combination with the TTAA3, HAT, and Mutator-like (MULE) elements. ( E ) Features of the four transposons found in association with CYP6CY3 . ORF, open reading frame; aa, amino acid. ( F ) Expression of hAT, TTAA3, Tc1/Mariner, and MULE in carcass, gut, or head tissue of M. persicae clone Mp1 and M. p. nicotianae clone Mn6. Error bars indicate 95% confidence limits ( n = 4).

Techniques Used: Amplification, Clone Assay, Real-time Polymerase Chain Reaction, BAC Assay, Sequencing, HAT Assay, Labeling, Fluorescence In Situ Hybridization, Expressing

A large segmental duplication in M. p. nicotianae led to the amplification and overexpression of multiple genes. ( A ) Gene expression heat map showing genes consistently DE in 36 comparisons of M. p. nicotianae with M. persicae s.s. [6 M. p. nicotianae clones (Mn1 to Mn6) compared to 6 M. persicae s.s. clones (Mp1 to Mp6) are shown]; cell color indicates log 2 fold change. Four of these genes localize to scaffold 16 [indicated by the blue lines linking (A) and (B)]. ( B ) Sliding window analysis of CNV between M. p. nicotianae and M. persicae across scaffold 16. In this representative plot, clone Mn3 was compared with clone Mp2; see data file S2 for the results of all 36 comparisons. ( C ) The region of elevated copy number includes some or all of the coding sequence of the genes, RPS11 , Src42A (SRC), T-type calcium channel (CaCh), CYP6CY23 (CY23), CYP6CY4 (CY4), CYP6CY3 (CY3), pseudogene of unknown function (Un_Pro) and ADAMTS9 , and has been tandemly duplicated as a series of direct repeats. ( D ) Precise determination of copy number of the region amplified by qPCR in all M. p. nicotianae clones compared to M. persicae clone Mp3. Error bars indicate 95% confidence limits ( n = 4). ( E ) Localization of CYP6CY3 detected with tyramide-Cy3 (red, arrowheads) on metaphase chromosomes of Mp1 and Mn6 counterstained with 4′,6-diamidino-2-phenylindole (blue) by means of TSA-FISH. X, sex chromosome. Scale bar, 5 μm.
Figure Legend Snippet: A large segmental duplication in M. p. nicotianae led to the amplification and overexpression of multiple genes. ( A ) Gene expression heat map showing genes consistently DE in 36 comparisons of M. p. nicotianae with M. persicae s.s. [6 M. p. nicotianae clones (Mn1 to Mn6) compared to 6 M. persicae s.s. clones (Mp1 to Mp6) are shown]; cell color indicates log 2 fold change. Four of these genes localize to scaffold 16 [indicated by the blue lines linking (A) and (B)]. ( B ) Sliding window analysis of CNV between M. p. nicotianae and M. persicae across scaffold 16. In this representative plot, clone Mn3 was compared with clone Mp2; see data file S2 for the results of all 36 comparisons. ( C ) The region of elevated copy number includes some or all of the coding sequence of the genes, RPS11 , Src42A (SRC), T-type calcium channel (CaCh), CYP6CY23 (CY23), CYP6CY4 (CY4), CYP6CY3 (CY3), pseudogene of unknown function (Un_Pro) and ADAMTS9 , and has been tandemly duplicated as a series of direct repeats. ( D ) Precise determination of copy number of the region amplified by qPCR in all M. p. nicotianae clones compared to M. persicae clone Mp3. Error bars indicate 95% confidence limits ( n = 4). ( E ) Localization of CYP6CY3 detected with tyramide-Cy3 (red, arrowheads) on metaphase chromosomes of Mp1 and Mn6 counterstained with 4′,6-diamidino-2-phenylindole (blue) by means of TSA-FISH. X, sex chromosome. Scale bar, 5 μm.

Techniques Used: Amplification, Over Expression, Expressing, Clone Assay, Sequencing, Real-time Polymerase Chain Reaction, Fluorescence In Situ Hybridization

Molecular, bioinformatic, and functional characterization of candidate genes within the segmental duplication. ( A and B ) qPCR analysis of copy number (A) and mRNA expression (B) of genes within the segmental duplication. In each case, data are shown as fold change between the six M. p. nicotianae clones and M. persicae clone Mp3. Error bars indicate 95% confidence limits ( n = 4). ( C ) Nonsense mutations are observed in exon 1 of certain copies of Src42A (light blue, left), and in the T-type calcium channel gene (green, right) caused by an internal duplication (duplicated region indicated by white boxes). In each case, the wild-type sequence is shown on top, and those carrying mutations are shown underneath. ( D ) Metabolism of nicotine by recombinant CYP6CY3, CYP6CY4, and CYP6CY23. Recovery of the nicotine metabolite cotinine over time in the presence (blue lines) or absence of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate; black lines) is shown. Error bars display SD ( n = 3). ( E ) Sensitivity of transgenic flies expressing ADAMTS9 or RPS11/ADAMTS9 to nicotine compared to a fly line of the same genetic background without a transgene (control). Sensitivity was measured by calculating lethal concentration 50 (LC 50 ) values for each line. Error bars indicate 95% confidence limits ( n = 5).
Figure Legend Snippet: Molecular, bioinformatic, and functional characterization of candidate genes within the segmental duplication. ( A and B ) qPCR analysis of copy number (A) and mRNA expression (B) of genes within the segmental duplication. In each case, data are shown as fold change between the six M. p. nicotianae clones and M. persicae clone Mp3. Error bars indicate 95% confidence limits ( n = 4). ( C ) Nonsense mutations are observed in exon 1 of certain copies of Src42A (light blue, left), and in the T-type calcium channel gene (green, right) caused by an internal duplication (duplicated region indicated by white boxes). In each case, the wild-type sequence is shown on top, and those carrying mutations are shown underneath. ( D ) Metabolism of nicotine by recombinant CYP6CY3, CYP6CY4, and CYP6CY23. Recovery of the nicotine metabolite cotinine over time in the presence (blue lines) or absence of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate; black lines) is shown. Error bars display SD ( n = 3). ( E ) Sensitivity of transgenic flies expressing ADAMTS9 or RPS11/ADAMTS9 to nicotine compared to a fly line of the same genetic background without a transgene (control). Sensitivity was measured by calculating lethal concentration 50 (LC 50 ) values for each line. Error bars indicate 95% confidence limits ( n = 5).

Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Clone Assay, Sequencing, Recombinant, Transgenic Assay, Concentration Assay

Overexpression of CYP6CY3 in the bacteriocyte and the gut of M. p. nicotianae protects this aphid subspecies and its obligate endosymbiont Buchnera aphidicola from nicotine. ( A to D ) Immunohistochemical localization of CYP6CY3 (green signal) in the bacteriocytes of M. persicae [(A) and (B)] and M. p. nicotianae (C) and (D). Nucleic acids stained with To-PRO 3-Iodide (red signal). ( E ) Expression of CYP6CY3 and CYP6CY4 in the carcass (CAR), gut, bacteriocyte (BAC), and head of the M. persicae clone Mp1 and M. p. nicotianae clone Mn6 as determined by qPCR. Error bars indicate 95% confidence limits ( n = 4). ( F ) Titer of B. aphidicola in two clones of M. persicae (blue bars) and two clones of M. p. nicotianae (red bars) after feeding on a diet with [2 or 8 parts per million (ppm)] or without nicotine (control). Error bars indicate 95% confidence limits ( n = 4). Significant differences ( P
Figure Legend Snippet: Overexpression of CYP6CY3 in the bacteriocyte and the gut of M. p. nicotianae protects this aphid subspecies and its obligate endosymbiont Buchnera aphidicola from nicotine. ( A to D ) Immunohistochemical localization of CYP6CY3 (green signal) in the bacteriocytes of M. persicae [(A) and (B)] and M. p. nicotianae (C) and (D). Nucleic acids stained with To-PRO 3-Iodide (red signal). ( E ) Expression of CYP6CY3 and CYP6CY4 in the carcass (CAR), gut, bacteriocyte (BAC), and head of the M. persicae clone Mp1 and M. p. nicotianae clone Mn6 as determined by qPCR. Error bars indicate 95% confidence limits ( n = 4). ( F ) Titer of B. aphidicola in two clones of M. persicae (blue bars) and two clones of M. p. nicotianae (red bars) after feeding on a diet with [2 or 8 parts per million (ppm)] or without nicotine (control). Error bars indicate 95% confidence limits ( n = 4). Significant differences ( P

Techniques Used: Over Expression, Immunohistochemistry, Staining, Expressing, BAC Assay, Real-time Polymerase Chain Reaction, Clone Assay

Schematic of the evolution of the molecular innovations in M. p. nicotianae that provide protection from toxic nicotine. Nicotine (1) is taken up via the gut during feeding and causes toxicity to M. persicae via its action at the nicotinic acetylcholine receptor (nAChR) (2) and on the obligate endosymbiont B. aphidicola in aphid bacteriocytes (3). In M. p. nicotianae , chromosomal rearrangements result in the increased expression of CYP6CY3 and CYP6CY4, which detoxify nicotine. In the case of CYP6CY3, expression is significantly enhanced in bacteriocytes (4) and reprogrammed to include the aphid gut (5) providing two lines of defense that protect M. p. nicotianae and its symbiont from this secondary metabolite. CNS, central nervous system.
Figure Legend Snippet: Schematic of the evolution of the molecular innovations in M. p. nicotianae that provide protection from toxic nicotine. Nicotine (1) is taken up via the gut during feeding and causes toxicity to M. persicae via its action at the nicotinic acetylcholine receptor (nAChR) (2) and on the obligate endosymbiont B. aphidicola in aphid bacteriocytes (3). In M. p. nicotianae , chromosomal rearrangements result in the increased expression of CYP6CY3 and CYP6CY4, which detoxify nicotine. In the case of CYP6CY3, expression is significantly enhanced in bacteriocytes (4) and reprogrammed to include the aphid gut (5) providing two lines of defense that protect M. p. nicotianae and its symbiont from this secondary metabolite. CNS, central nervous system.

Techniques Used: Expressing

21) Product Images from "Differential polysaccharide utilization is the basis for a nanohaloarchaeon : haloarchaeon symbiosis"

Article Title: Differential polysaccharide utilization is the basis for a nanohaloarchaeon : haloarchaeon symbiosis

Journal: bioRxiv

doi: 10.1101/794461

Reconstruction of central metabolic and homeostatic functions of ‘ Ca . Nanohalobium constans’ LC1Nh based on genomic, proteomic, targeted metabolomics and physiological analyses. Enzymes involved in energy production and in reactive oxygen species (ROS) homeostasis/redox regulation are highlighted in yellow. Chitin degradation enabled by seven extracellularly expressed GH18 endochitinases and one GH20 chitodextrinase of the host, Halomicrobium sp. LC1Hm, is shown by grey arrow in the bottom-right part of the figure. Depolymerization of (1→4)- and (1→6)-α-D-glucans by two experimentally confirmed extracellularly expressed glucoamylases of ‘ Ca . Nanohalobium constans’ LC1Nh is shown with green arrow in the upper-left part of the figure. The “mutualistic” uptake of formed sugars is shown by red arrow. Details on genes and systems abbreviations are provided in the Supplementary Table 5. CoA, coenzyme A; Glc-6P, glucose-6-phosphate; Glc-1P, glucose-1-phosphate; GlcNAc, N-acetyl-glucosamine; GlcNAc-6P, N-acetyl-glucosamine-6-phosphate; GlcN-6P, N-glucosamine-6-phosphate; Glu, glutamate; Gln, glutamine; F-6P, fructose-6-phosphate; F1,6BP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 1,3BPG, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenol pyruvate; Prx, peroxiredoxin; Trx, thioredoxin.
Figure Legend Snippet: Reconstruction of central metabolic and homeostatic functions of ‘ Ca . Nanohalobium constans’ LC1Nh based on genomic, proteomic, targeted metabolomics and physiological analyses. Enzymes involved in energy production and in reactive oxygen species (ROS) homeostasis/redox regulation are highlighted in yellow. Chitin degradation enabled by seven extracellularly expressed GH18 endochitinases and one GH20 chitodextrinase of the host, Halomicrobium sp. LC1Hm, is shown by grey arrow in the bottom-right part of the figure. Depolymerization of (1→4)- and (1→6)-α-D-glucans by two experimentally confirmed extracellularly expressed glucoamylases of ‘ Ca . Nanohalobium constans’ LC1Nh is shown with green arrow in the upper-left part of the figure. The “mutualistic” uptake of formed sugars is shown by red arrow. Details on genes and systems abbreviations are provided in the Supplementary Table 5. CoA, coenzyme A; Glc-6P, glucose-6-phosphate; Glc-1P, glucose-1-phosphate; GlcNAc, N-acetyl-glucosamine; GlcNAc-6P, N-acetyl-glucosamine-6-phosphate; GlcN-6P, N-glucosamine-6-phosphate; Glu, glutamate; Gln, glutamine; F-6P, fructose-6-phosphate; F1,6BP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 1,3BPG, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenol pyruvate; Prx, peroxiredoxin; Trx, thioredoxin.

Techniques Used:

Growth of Halomicrobium sp. LC1Hm in pure (axenic) culture and in co-culture with Ca . Nanohalobium constans LC1Nh. Panel a : growth on chitin. Panel d : growth on starch. Insertions ( b, e ) indicate the production of acetate and reduced sugars, N-acetylglucosamine and glucose during growth on chitin and starch, respectively. Insert ( c ) indicates the oxygen consumption of axenic LC1Hm and LC1Hm+LC1Nh cultures during growth on chitin. The overall significance level p
Figure Legend Snippet: Growth of Halomicrobium sp. LC1Hm in pure (axenic) culture and in co-culture with Ca . Nanohalobium constans LC1Nh. Panel a : growth on chitin. Panel d : growth on starch. Insertions ( b, e ) indicate the production of acetate and reduced sugars, N-acetylglucosamine and glucose during growth on chitin and starch, respectively. Insert ( c ) indicates the oxygen consumption of axenic LC1Hm and LC1Hm+LC1Nh cultures during growth on chitin. The overall significance level p

Techniques Used: Co-Culture Assay

TEM microscopy of chitin-growing co-culture of Halomicrobium sp. LC1Hm and Ca . Nanohalobium constans LC1Nh. ( a ) symbiont-free Halomicrobium sp. LC1Hm cell; ( b ) pronounced membrane stretching and ( c ) exfoliation of a thick electron-dense external layer of the host cell; (c-f) various stages of cells fusion suggestive of a shared lipid membrane and extracellular matrix. Bars represent 500 nm.
Figure Legend Snippet: TEM microscopy of chitin-growing co-culture of Halomicrobium sp. LC1Hm and Ca . Nanohalobium constans LC1Nh. ( a ) symbiont-free Halomicrobium sp. LC1Hm cell; ( b ) pronounced membrane stretching and ( c ) exfoliation of a thick electron-dense external layer of the host cell; (c-f) various stages of cells fusion suggestive of a shared lipid membrane and extracellular matrix. Bars represent 500 nm.

Techniques Used: Transmission Electron Microscopy, Microscopy, Co-Culture Assay

Fluorescence, TEM and FESEM micrographs of nanohaloarchaeote Ca . Nanohalobium constans LC1Nh and its chitinotrophic host Halomicrobium sp. LC1Hm cells. DAPI ( a ) and CARD-FISH ( b ) staining shows tiny coccoidal nanohaloarchaeal cells (285 ± 50 nm in diameter) either detached or adhering to the host haloarchaeal cells contact with host haloarchaea (tyramide Alexa488 and Alexa594 were used for nanohaloarchaeal and haloarchaeal cells, respectively); FESEM image depicts the coccoidal shape of detached nanohaloarchaeota cells ( c ); up to 17 nanohaloarchaeota cells can closely interact with the host Halomicrobium sp. LC1Hm cell ( d-f ); some nanohaloarchaeal cells can be attached to more than one host ( g , h ); a series of TEM ultrathin sections depict the intimate contact of nanohaloarchaeota cells with its chitinolytic host resulting in the formation of membrane depression (stretching) on the host cell surface ( g-i ); nanohaloarchaeal cells express long archaella ( j , k ). Bars represent 5 µm in ( a-b ), 0.2 µm in ( c-I, k ) and 0.5 µm in ( j ).
Figure Legend Snippet: Fluorescence, TEM and FESEM micrographs of nanohaloarchaeote Ca . Nanohalobium constans LC1Nh and its chitinotrophic host Halomicrobium sp. LC1Hm cells. DAPI ( a ) and CARD-FISH ( b ) staining shows tiny coccoidal nanohaloarchaeal cells (285 ± 50 nm in diameter) either detached or adhering to the host haloarchaeal cells contact with host haloarchaea (tyramide Alexa488 and Alexa594 were used for nanohaloarchaeal and haloarchaeal cells, respectively); FESEM image depicts the coccoidal shape of detached nanohaloarchaeota cells ( c ); up to 17 nanohaloarchaeota cells can closely interact with the host Halomicrobium sp. LC1Hm cell ( d-f ); some nanohaloarchaeal cells can be attached to more than one host ( g , h ); a series of TEM ultrathin sections depict the intimate contact of nanohaloarchaeota cells with its chitinolytic host resulting in the formation of membrane depression (stretching) on the host cell surface ( g-i ); nanohaloarchaeal cells express long archaella ( j , k ). Bars represent 5 µm in ( a-b ), 0.2 µm in ( c-I, k ) and 0.5 µm in ( j ).

Techniques Used: Fluorescence, Transmission Electron Microscopy, Fluorescence In Situ Hybridization, Staining

22) Product Images from "Comparative Analysis of Bacterial Community Composition and Structure in Clinically Symptomatic and Asymptomatic Central Venous Catheters"

Article Title: Comparative Analysis of Bacterial Community Composition and Structure in Clinically Symptomatic and Asymptomatic Central Venous Catheters

Journal: mSphere

doi: 10.1128/mSphere.00146-17

TIVAP catheter collection from patients suspected or not of catheter-related infection from two French hospitals. (A) Study design. Sterile reagents and techniques were used throughout the study, and the protocol was designed to minimize the possibilities for bacterial contamination as much as possible. Furthermore, samples of used reagents (green asterisks), materials (blue asterisks), and blanks (water used as the template in the procedure; red asterisks) as procedure controls were taken at all steps and also submitted to 16S rRNA sequencing for species identification. (B) TIVAP parts. The TIVAP is composed of a subcutaneously implanted chamber/hub (the reservoir) connected to a catheter that is inserted into a central vein (e.g., jugular or subclavian).
Figure Legend Snippet: TIVAP catheter collection from patients suspected or not of catheter-related infection from two French hospitals. (A) Study design. Sterile reagents and techniques were used throughout the study, and the protocol was designed to minimize the possibilities for bacterial contamination as much as possible. Furthermore, samples of used reagents (green asterisks), materials (blue asterisks), and blanks (water used as the template in the procedure; red asterisks) as procedure controls were taken at all steps and also submitted to 16S rRNA sequencing for species identification. (B) TIVAP parts. The TIVAP is composed of a subcutaneously implanted chamber/hub (the reservoir) connected to a catheter that is inserted into a central vein (e.g., jugular or subclavian).

Techniques Used: Infection, Sequencing

23) Product Images from "A combined human case of Dirofilaria ursi infection in dorsal subcutaneous tissue and Anisakis simplex sensu stricto (s.s.) infection in ventral subcutaneous tissue"

Article Title: A combined human case of Dirofilaria ursi infection in dorsal subcutaneous tissue and Anisakis simplex sensu stricto (s.s.) infection in ventral subcutaneous tissue

Journal: Tropical Medicine and Health

doi: 10.1186/s41182-017-0067-4

Phylogenetic analysis of Dirofilaria species based on 5S rRNA sequences (90 bp). Nucleotide sequences were aligned using the Genetyx ver 11 and CLC Sequence Viewer 6 software. Phylogenetic analyses were conducted using the Mega 6 software. Genetic relationships were inferred by the neighbor-joining (NJ) method. D1 and D2 means Dirofilaria ursi worms collected from Japanese bears in Gifu Prefecture in the middle part of Japan
Figure Legend Snippet: Phylogenetic analysis of Dirofilaria species based on 5S rRNA sequences (90 bp). Nucleotide sequences were aligned using the Genetyx ver 11 and CLC Sequence Viewer 6 software. Phylogenetic analyses were conducted using the Mega 6 software. Genetic relationships were inferred by the neighbor-joining (NJ) method. D1 and D2 means Dirofilaria ursi worms collected from Japanese bears in Gifu Prefecture in the middle part of Japan

Techniques Used: Sequencing, Software

24) Product Images from "Decreased Sp1 Expression Mediates Downregulation of SHIP2 in Gastric Cancer Cells"

Article Title: Decreased Sp1 Expression Mediates Downregulation of SHIP2 in Gastric Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18010220

SHIP2 is regulated by transcription factor Sp1 in GC cells. ( A ) Copy number variation of INPPL1 in the normal gastric mucosa epithelial cells GES-1 and a panel of GC cells as quantitated by qPCR analysis of genomic DNA. ( n = 3, mean ± SEM); ( B ) whole cell lysates from GES-1 and a panel of GC cells treated with 5-aza-dC (10 μM) for 96 h or SAHA (5 μM) for 24 h were subjected to Western blot analysis. Data shown are representative of 3 individual experiments; ( C ) a typical CpG island is present at the promoter region of SHIP2. Each vertical bar represents a single CpG site. The transcription start site (TSS) is indicated by a curved arrow. BGS analysis showed no/low promoter methylation in a panel of GC cells; ( D ) a schematic illustration of construction of the luciferase reporter constructs containing a series of 5′-truncted INPPL1 promoters and the Sp1-binding site deletion mutant; ( E ) MGC-803 and SGC-7901 cells were transiently co-transfected with pRL-TK plasmid and the pGL3-basic vector, pGL3- INPPL1 -(−1359/+380) construct and a series of 5′-deletion mutants of pGL3- INPPL1 -(−1359/+380). Luciferase activity was determined 48 h later, and the value was normalized to that of pGL3-basic vector designated as 1 ( n = 3, mean ± SEM, * p
Figure Legend Snippet: SHIP2 is regulated by transcription factor Sp1 in GC cells. ( A ) Copy number variation of INPPL1 in the normal gastric mucosa epithelial cells GES-1 and a panel of GC cells as quantitated by qPCR analysis of genomic DNA. ( n = 3, mean ± SEM); ( B ) whole cell lysates from GES-1 and a panel of GC cells treated with 5-aza-dC (10 μM) for 96 h or SAHA (5 μM) for 24 h were subjected to Western blot analysis. Data shown are representative of 3 individual experiments; ( C ) a typical CpG island is present at the promoter region of SHIP2. Each vertical bar represents a single CpG site. The transcription start site (TSS) is indicated by a curved arrow. BGS analysis showed no/low promoter methylation in a panel of GC cells; ( D ) a schematic illustration of construction of the luciferase reporter constructs containing a series of 5′-truncted INPPL1 promoters and the Sp1-binding site deletion mutant; ( E ) MGC-803 and SGC-7901 cells were transiently co-transfected with pRL-TK plasmid and the pGL3-basic vector, pGL3- INPPL1 -(−1359/+380) construct and a series of 5′-deletion mutants of pGL3- INPPL1 -(−1359/+380). Luciferase activity was determined 48 h later, and the value was normalized to that of pGL3-basic vector designated as 1 ( n = 3, mean ± SEM, * p

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Methylation, Luciferase, Construct, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay

25) Product Images from "The effect of rapamycin, NVP-BEZ235, aspirin, and metformin on PI3K/AKT/mTOR signaling pathway of PIK3CA-related overgrowth spectrum (PROS)"

Article Title: The effect of rapamycin, NVP-BEZ235, aspirin, and metformin on PI3K/AKT/mTOR signaling pathway of PIK3CA-related overgrowth spectrum (PROS)

Journal: Oncotarget

doi: 10.18632/oncotarget.17566

The identification and quantification of the mutation in PIK3CA ( A ) Direct nucleotide sequence analysis of PIK3CA was performed using isolated genomic DNA from blood and six different regions including subcutaneous adipose tissue and skin from the lower limbs. A mosaic mutation (c.3140A > G [p.H1047R]) in exon 20 was identified from affected adipose tissues and skin. The ratios of the mutant allele are different in the patient's affected tissues. Direct nucleotide sequence analysis was performed with plasmids containing normal or mutant PIK3CA fragment as standard reference materials. The right panel shows a standard curve for calculating PIK3CA mutant frequency by direct sequencing. ( B ) Multiplex PCR products of genomic DNA isolated from affected tissues, blood cells, and fibroblast cells were run through a 1.5% agarose gel and stained with ethidium bromide. Plasmids containing PCR products of wild-type and mutant PIK3CA exon 20 were used as standard reference materials. A 370-bp DNA fragment was generated from the mutant allele of PIK3CA (black arrow) and a 480-bp fragment was generated from both the wild-type and mutant PIK3CA alleles, as the internal control (white arrow). The sizes of the DNA markers are indicated on the left side. The upper right panel shows the standard curve for calculating PIK3CA mutant frequency by multiplex PCR. Lane: 1, blood; 2, skin from the perineum; 3, adipose tissue from the perineum; 4, skin from the lower limb; 5, adipose tissue from lower limb–1; 6, adipose tissue from the chest; 7, adipose tissue from lower limb–2; a, control fibroblast C2; b, control fibroblast C3; c, NHDF-c; d, PROS fibroblast from the skin of the lower limb; e, PROS fibroblast from the skin of the perineum. ( C ) PIK3CA mutant allele frequencies at nucleotide position 3140 in the patient's affected tissues and blood lymphocytes were calculated by quantitative multiplex PCR and direct sequencing. The x-axis labels are the same as in (B).
Figure Legend Snippet: The identification and quantification of the mutation in PIK3CA ( A ) Direct nucleotide sequence analysis of PIK3CA was performed using isolated genomic DNA from blood and six different regions including subcutaneous adipose tissue and skin from the lower limbs. A mosaic mutation (c.3140A > G [p.H1047R]) in exon 20 was identified from affected adipose tissues and skin. The ratios of the mutant allele are different in the patient's affected tissues. Direct nucleotide sequence analysis was performed with plasmids containing normal or mutant PIK3CA fragment as standard reference materials. The right panel shows a standard curve for calculating PIK3CA mutant frequency by direct sequencing. ( B ) Multiplex PCR products of genomic DNA isolated from affected tissues, blood cells, and fibroblast cells were run through a 1.5% agarose gel and stained with ethidium bromide. Plasmids containing PCR products of wild-type and mutant PIK3CA exon 20 were used as standard reference materials. A 370-bp DNA fragment was generated from the mutant allele of PIK3CA (black arrow) and a 480-bp fragment was generated from both the wild-type and mutant PIK3CA alleles, as the internal control (white arrow). The sizes of the DNA markers are indicated on the left side. The upper right panel shows the standard curve for calculating PIK3CA mutant frequency by multiplex PCR. Lane: 1, blood; 2, skin from the perineum; 3, adipose tissue from the perineum; 4, skin from the lower limb; 5, adipose tissue from lower limb–1; 6, adipose tissue from the chest; 7, adipose tissue from lower limb–2; a, control fibroblast C2; b, control fibroblast C3; c, NHDF-c; d, PROS fibroblast from the skin of the lower limb; e, PROS fibroblast from the skin of the perineum. ( C ) PIK3CA mutant allele frequencies at nucleotide position 3140 in the patient's affected tissues and blood lymphocytes were calculated by quantitative multiplex PCR and direct sequencing. The x-axis labels are the same as in (B).

Techniques Used: Mutagenesis, Sequencing, Isolation, Multiplex Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Generated

26) Product Images from "Multiplex Detection of Five Canine Viral Pathogens for Dogs as Laboratory Animals by the Luminex xTAG Assay"

Article Title: Multiplex Detection of Five Canine Viral Pathogens for Dogs as Laboratory Animals by the Luminex xTAG Assay

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01783

Specificity analysis of the 5-plex Luminex xTAG assay. Each bar represents the average MFI of duplicate samples. CDV, canine distemper virus; CPV, canine parvovirus; CAV, canine adenovirus; CPIV, canine parainfluenza virus; RV, rabies virus; PRV, pseudorabies virus; CCV, canine coronavirus; CIV, canine influenza virus; NC, negative control (water). The result of the 5-plex Luminex xTAG assay showed high specificity.
Figure Legend Snippet: Specificity analysis of the 5-plex Luminex xTAG assay. Each bar represents the average MFI of duplicate samples. CDV, canine distemper virus; CPV, canine parvovirus; CAV, canine adenovirus; CPIV, canine parainfluenza virus; RV, rabies virus; PRV, pseudorabies virus; CCV, canine coronavirus; CIV, canine influenza virus; NC, negative control (water). The result of the 5-plex Luminex xTAG assay showed high specificity.

Techniques Used: Luminex, Negative Control

Validation of the artificial mixed infection samples. 1, CPV and CAV positive sample; 2, CDV, CPV, and RV positive sample; 3, CAV and CPIV positive samples; 4, CDV and CAV positive sample; 5, CPIV and RV positive sample; 6, CDV, CPV, and CAV positive sample; 7, CPV, CPIV, and RV positive sample; 8, negative control.
Figure Legend Snippet: Validation of the artificial mixed infection samples. 1, CPV and CAV positive sample; 2, CDV, CPV, and RV positive sample; 3, CAV and CPIV positive samples; 4, CDV and CAV positive sample; 5, CPIV and RV positive sample; 6, CDV, CPV, and CAV positive sample; 7, CPV, CPIV, and RV positive sample; 8, negative control.

Techniques Used: Infection, Negative Control

27) Product Images from "An enzymatic ruler modulates Lewis antigen glycosylation of Helicobacter pylori LPS during persistent infection"

Article Title: An enzymatic ruler modulates Lewis antigen glycosylation of Helicobacter pylori LPS during persistent infection

Journal:

doi: 10.1073/pnas.0511119103

Lewis phenotype and fut genotype in isolates from patient I. ( A ) Western blotting was performed on extracted LPS with antibodies detecting Lewis x and Lewis y, respectively. ( B ) PCR amplification of the 3′-repetitive region in futA and futB , respectively.
Figure Legend Snippet: Lewis phenotype and fut genotype in isolates from patient I. ( A ) Western blotting was performed on extracted LPS with antibodies detecting Lewis x and Lewis y, respectively. ( B ) PCR amplification of the 3′-repetitive region in futA and futB , respectively.

Techniques Used: Western Blot, Polymerase Chain Reaction, Amplification

Lewis phenotype and fut genotype in isolates from patient II. ( A ) Western blotting was performed on extracted LPS with antibodies detecting Lewis x and Lewis y, respectively. ( B ) PCR amplification of the 3′-repetitive region in futA and futB ,
Figure Legend Snippet: Lewis phenotype and fut genotype in isolates from patient II. ( A ) Western blotting was performed on extracted LPS with antibodies detecting Lewis x and Lewis y, respectively. ( B ) PCR amplification of the 3′-repetitive region in futA and futB ,

Techniques Used: Western Blot, Polymerase Chain Reaction, Amplification

Model of Lewis x glycosylation in H. pylori . FutA and FutB may form homodimers or heterodimers that differ between isolates, reflecting translational frame and number of C-terminal heptad repeats in the enzymes. In this model, fucosylation occurs at the
Figure Legend Snippet: Model of Lewis x glycosylation in H. pylori . FutA and FutB may form homodimers or heterodimers that differ between isolates, reflecting translational frame and number of C-terminal heptad repeats in the enzymes. In this model, fucosylation occurs at the

Techniques Used:

28) Product Images from "MicroRNA-328 Negatively Regulates the Expression of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Cancer Cells"

Article Title: MicroRNA-328 Negatively Regulates the Expression of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Cancer Cells

Journal:

doi: 10.1124/mol.108.054163

Luciferase reporter assays suggest that human miR-328 directly targets the 3′-UTR of ABCG2. Overexpression of miR-328 led to more than 50% decrease in ABCG2 3′-UTR-luciferase activity in MCF-7/MX100 cells (A). Inhibition of miR-328
Figure Legend Snippet: Luciferase reporter assays suggest that human miR-328 directly targets the 3′-UTR of ABCG2. Overexpression of miR-328 led to more than 50% decrease in ABCG2 3′-UTR-luciferase activity in MCF-7/MX100 cells (A). Inhibition of miR-328

Techniques Used: Luciferase, Over Expression, Activity Assay, Inhibition

29) Product Images from "Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park"

Article Title: Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park

Journal:

doi: 10.1128/AEM.71.10.5943-5950.2005

Gas chromatograms of hydrocarbon components in Rainbow Springs soils RH1 (A) and RH2 (B). Hydrocarbon components: C15 to C30, n- alkanes (the numbers indicate chain lengths); Farn, farnesane; Pris, pristane; Phy, phytane; IS, added internal standard.
Figure Legend Snippet: Gas chromatograms of hydrocarbon components in Rainbow Springs soils RH1 (A) and RH2 (B). Hydrocarbon components: C15 to C30, n- alkanes (the numbers indicate chain lengths); Farn, farnesane; Pris, pristane; Phy, phytane; IS, added internal standard.

Techniques Used:

Mineralization of [1- 14 C]hexadecane in RH1 (circles) and RH2 (triangles) soil-sand mixtures. Solid symbols indicate soil-inoculated samples, and open symbols indicate autoclaved control soil-inoculated samples. Each point represents the average of triplicate
Figure Legend Snippet: Mineralization of [1- 14 C]hexadecane in RH1 (circles) and RH2 (triangles) soil-sand mixtures. Solid symbols indicate soil-inoculated samples, and open symbols indicate autoclaved control soil-inoculated samples. Each point represents the average of triplicate

Techniques Used:

DGGE profiles of 16S rRNA gene fragments during hexadecane mineralization by RH1 and RH2 soil-sand mixtures compared with profiles obtained for the original RH soils and isolate C197. The nucleotide sequences of the labeled bands (bands 1 to 6) were determined
Figure Legend Snippet: DGGE profiles of 16S rRNA gene fragments during hexadecane mineralization by RH1 and RH2 soil-sand mixtures compared with profiles obtained for the original RH soils and isolate C197. The nucleotide sequences of the labeled bands (bands 1 to 6) were determined

Techniques Used: Denaturing Gradient Gel Electrophoresis, Labeling

30) Product Images from "Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale"

Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03123-18

(A) Southern blotting of EcoRI-digested total DNA from FD-C-infected and FD-D-infected and healthy (H) periwinkles probed with a DIG-labeled malG gene amplicon obtained through PCR driven by primer pair malG _F/ malG _R (C+, probe positive control represented by pGEM-T- malG1 plasmid). (B) Electrophoresis separation of amplicons obtained following PCR of total DNA from FD-C-infected and FD-D-infected periwinkles with copy-specific primer pairs (002 and 005), according to the draft genome of FD92, and from healthy periwinkle. *, nonspecific PCR product.
Figure Legend Snippet: (A) Southern blotting of EcoRI-digested total DNA from FD-C-infected and FD-D-infected and healthy (H) periwinkles probed with a DIG-labeled malG gene amplicon obtained through PCR driven by primer pair malG _F/ malG _R (C+, probe positive control represented by pGEM-T- malG1 plasmid). (B) Electrophoresis separation of amplicons obtained following PCR of total DNA from FD-C-infected and FD-D-infected periwinkles with copy-specific primer pairs (002 and 005), according to the draft genome of FD92, and from healthy periwinkle. *, nonspecific PCR product.

Techniques Used: Southern Blot, Infection, Labeling, Amplification, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Electrophoresis

31) Product Images from "A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle [C]A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle [C] [W]A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle [C] [W] [OPEN]"

Article Title: A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle [C]A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle [C] [W]A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle [C] [W] [OPEN]

Journal: The Plant Cell

doi: 10.1105/tpc.113.115154

Differential Conversions of 7-Deoxyloganetic Acid and 7-Deoxyloganetin by Recombinant UGT6, UGT7, and UGT8 to 7-Deoxyloganic Acid and 7-Deoxyloganin.
Figure Legend Snippet: Differential Conversions of 7-Deoxyloganetic Acid and 7-Deoxyloganetin by Recombinant UGT6, UGT7, and UGT8 to 7-Deoxyloganic Acid and 7-Deoxyloganin.

Techniques Used: Recombinant

Differential Expression of UGT8 Metabolite Profiles in Periwinkle Plant Organs.
Figure Legend Snippet: Differential Expression of UGT8 Metabolite Profiles in Periwinkle Plant Organs.

Techniques Used: Expressing

Downregulation of UGT8 , LAMT , and SLS in Periwinkle.
Figure Legend Snippet: Downregulation of UGT8 , LAMT , and SLS in Periwinkle.

Techniques Used:

Localization of UGT8 Cells of Young Developing Leaves of Periwinkle.
Figure Legend Snippet: Localization of UGT8 Cells of Young Developing Leaves of Periwinkle.

Techniques Used:

32) Product Images from "Anopheles gambiae males produce and transfer the vitellogenic steroid hormone 20-hydroxyecdysone to females during mating"

Article Title: Anopheles gambiae males produce and transfer the vitellogenic steroid hormone 20-hydroxyecdysone to females during mating

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0809264105

Ovaries produce 20E after blood meal in A. gambiae . ( A ) RT-PCR analysis of CYP306A1 , CYP302A1 , CYP315A1 , and CYP314A1 expression pattern in ovaries throughout the first gonotrophic cycle (NBF, ovaries from non-blood-fed females; 6, 18, 24, and 48 h, ovaries at different times PBM) and in different body parts of 24 h PBM blood-fed females (GM, gut and Malpighian tubules; Ca, carcass). ( B ) In vitro ecdysteroid secretion by ovaries during the first gonotrophic cycle. (NBF, ovaries from non-blood-fed females; 6, 18, 24, and 48 h, ovaries from blood-fed females at different times PBM.) ( C ) HPLC-EIA analysis of ecdysteroids secreted by ovaries of blood-fed females (24 h PBM). Results are expressed as E (solid line) and 20E (dotted line) equivalents. ( D ) In vitro ecdysteroid secretion by different body parts of blood-fed females (24 h PBM). (He, head; Th, thorax; Gu and Mt, gut and Malpighian tubules; Ov, ovaries; Ca, carcass.)
Figure Legend Snippet: Ovaries produce 20E after blood meal in A. gambiae . ( A ) RT-PCR analysis of CYP306A1 , CYP302A1 , CYP315A1 , and CYP314A1 expression pattern in ovaries throughout the first gonotrophic cycle (NBF, ovaries from non-blood-fed females; 6, 18, 24, and 48 h, ovaries at different times PBM) and in different body parts of 24 h PBM blood-fed females (GM, gut and Malpighian tubules; Ca, carcass). ( B ) In vitro ecdysteroid secretion by ovaries during the first gonotrophic cycle. (NBF, ovaries from non-blood-fed females; 6, 18, 24, and 48 h, ovaries from blood-fed females at different times PBM.) ( C ) HPLC-EIA analysis of ecdysteroids secreted by ovaries of blood-fed females (24 h PBM). Results are expressed as E (solid line) and 20E (dotted line) equivalents. ( D ) In vitro ecdysteroid secretion by different body parts of blood-fed females (24 h PBM). (He, head; Th, thorax; Gu and Mt, gut and Malpighian tubules; Ov, ovaries; Ca, carcass.)

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, In Vitro, High Performance Liquid Chromatography, Enzyme-linked Immunosorbent Assay

Biosynthetic pathway of 20E from cholesterol emphasizing the four terminal steps of steroidogenesis catalyzed by CYP306A1, CYP302A1, CYP315A1, and CYP314A1 (25-, 22-, 2-, and 20-hydroxylases, respectively). (2,22,25dE, 2,22,25-trideoxyecdysone; 2,22dE, 2,22-dideoxyecdysone; 2dE, 2-deoxyecdysone.)
Figure Legend Snippet: Biosynthetic pathway of 20E from cholesterol emphasizing the four terminal steps of steroidogenesis catalyzed by CYP306A1, CYP302A1, CYP315A1, and CYP314A1 (25-, 22-, 2-, and 20-hydroxylases, respectively). (2,22,25dE, 2,22,25-trideoxyecdysone; 2,22dE, 2,22-dideoxyecdysone; 2dE, 2-deoxyecdysone.)

Techniques Used: Two-Dimensional Gel Electrophoresis

Male accessory glands produce 20E in A. gambiae . ( A ) RT-PCR analysis of CYP306A1 , CYP302A1 , CYP315A1 , and CYP314A1 expression pattern in different body parts of 6-day-old males. (Te, testes; GM, gut and Malpighian tubules; Ca, carcass.) In this carcass sample, expression level of the control gene RpL17 was lower than in the other tissues examined; replicate experiments confirmed that expression of the steroidogenic genes was at the detection level limit. ( B and C ) In situ expression pattern of CYP306A1 ( B ) and CYP314A1 ( C ) in MRT from 6-day-old males ( CYP302A1 and CYP315A1 not shown). (Te, testis; Sb, 250 μm.) ( D ) In vitro ecdysteroid secretion by different body parts of 6-day-old males. (He, head; Th, thorax; GM, gut and Malpighian tubules; Ca, carcass.) ( Inset ) In vitro ecdysteroid secretion by separated testes (Te) and MAG of 6-day-old males. ( E ) HPLC-EIA analysis of ecdysteroids secreted by MRT of 6-day-old males. Results are expressed as E (solid line) and 20E (dotted line) equivalents. ( F ) Ecdysteroid titers in MRT (white bars), ecdysteroid titers in whole males (light gray bars), and in vitro ecdysteroid secretion by MRT in 5 h of incubation (dark gray bars) from day 0 (D0) to day 6 (D6) PE (*, P
Figure Legend Snippet: Male accessory glands produce 20E in A. gambiae . ( A ) RT-PCR analysis of CYP306A1 , CYP302A1 , CYP315A1 , and CYP314A1 expression pattern in different body parts of 6-day-old males. (Te, testes; GM, gut and Malpighian tubules; Ca, carcass.) In this carcass sample, expression level of the control gene RpL17 was lower than in the other tissues examined; replicate experiments confirmed that expression of the steroidogenic genes was at the detection level limit. ( B and C ) In situ expression pattern of CYP306A1 ( B ) and CYP314A1 ( C ) in MRT from 6-day-old males ( CYP302A1 and CYP315A1 not shown). (Te, testis; Sb, 250 μm.) ( D ) In vitro ecdysteroid secretion by different body parts of 6-day-old males. (He, head; Th, thorax; GM, gut and Malpighian tubules; Ca, carcass.) ( Inset ) In vitro ecdysteroid secretion by separated testes (Te) and MAG of 6-day-old males. ( E ) HPLC-EIA analysis of ecdysteroids secreted by MRT of 6-day-old males. Results are expressed as E (solid line) and 20E (dotted line) equivalents. ( F ) Ecdysteroid titers in MRT (white bars), ecdysteroid titers in whole males (light gray bars), and in vitro ecdysteroid secretion by MRT in 5 h of incubation (dark gray bars) from day 0 (D0) to day 6 (D6) PE (*, P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, In Situ, In Vitro, High Performance Liquid Chromatography, Enzyme-linked Immunosorbent Assay, Incubation

33) Product Images from "The RNA-binding Protein HuR Stabilizes Cytosolic Phospholipase A2α mRNA under Interleukin-1β Treatment in Non-small Cell Lung Cancer A549 Cells"

Article Title: The RNA-binding Protein HuR Stabilizes Cytosolic Phospholipase A2α mRNA under Interleukin-1β Treatment in Non-small Cell Lung Cancer A549 Cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.263582

The 3′-UTR of cPLA 2 α mRNA binds with protein complexes. A , diagram of various probes used in the following RNA EMSA experiments. These probes span different regions of cPLA 2 α 3′-UTR, which contains different ARE and U-rich sequence. B , probes ( P1–P4 ) were incubated with the cytosolic extracts prepared from A549 cells to perform an RNA EMSA under 5 units and 10 units of RNase T 1 treatment. No protein-RNA complexes are formed on probe P1, but two protein complexes ( C1 and C2 ) are indicated on probes P2–P4.
Figure Legend Snippet: The 3′-UTR of cPLA 2 α mRNA binds with protein complexes. A , diagram of various probes used in the following RNA EMSA experiments. These probes span different regions of cPLA 2 α 3′-UTR, which contains different ARE and U-rich sequence. B , probes ( P1–P4 ) were incubated with the cytosolic extracts prepared from A549 cells to perform an RNA EMSA under 5 units and 10 units of RNase T 1 treatment. No protein-RNA complexes are formed on probe P1, but two protein complexes ( C1 and C2 ) are indicated on probes P2–P4.

Techniques Used: Sequencing, Incubation

34) Product Images from "SOX7 is down-regulated in lung cancer"

Article Title: SOX7 is down-regulated in lung cancer

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-32-17

Methylation analysis of upstream regions of SOX7 gene . ( A ) Schematic illustration of CpG sites spanning 1,500 bp upstream and 350 bp downstream of the transcription start site of SOX7 transcription. Black arrow denotes the transcription start site (+1). Vertical pink bars denotes the CpG sites. Arrowed bars define the regions subjected to bisulfite sequencing (BS). Bars with circles at their end define the regions subjected to methylation specific PCR (MSP). ( B ) Bisulfite sequencing of SOX7 gene in NSCLC cell lines. Each circle in each horizontal row represent the analysis of a single clone of bisulfite-treated DNA encompassing either 20 or 35 CpG sites (-678 to -440, left panels; -71 to +251, right panels, respectively). Open and solid circles represent unmethylated and methylated CpG sites, respectively. ( C ) MPS analysis of upstream region of SOX7 gene in NSCLC cell lines. PCR products in lanes marked “U” and “M” are obtained with unmethylated-specific and methylated-specific primers, respectively. ( D ) Bisulfite sequencing of SOX7 gene in NSCLC samples and their matched normal lung tissues as described for the NSCLC cell lines in Figure 4 B.
Figure Legend Snippet: Methylation analysis of upstream regions of SOX7 gene . ( A ) Schematic illustration of CpG sites spanning 1,500 bp upstream and 350 bp downstream of the transcription start site of SOX7 transcription. Black arrow denotes the transcription start site (+1). Vertical pink bars denotes the CpG sites. Arrowed bars define the regions subjected to bisulfite sequencing (BS). Bars with circles at their end define the regions subjected to methylation specific PCR (MSP). ( B ) Bisulfite sequencing of SOX7 gene in NSCLC cell lines. Each circle in each horizontal row represent the analysis of a single clone of bisulfite-treated DNA encompassing either 20 or 35 CpG sites (-678 to -440, left panels; -71 to +251, right panels, respectively). Open and solid circles represent unmethylated and methylated CpG sites, respectively. ( C ) MPS analysis of upstream region of SOX7 gene in NSCLC cell lines. PCR products in lanes marked “U” and “M” are obtained with unmethylated-specific and methylated-specific primers, respectively. ( D ) Bisulfite sequencing of SOX7 gene in NSCLC samples and their matched normal lung tissues as described for the NSCLC cell lines in Figure 4 B.

Techniques Used: Methylation, Methylation Sequencing, Polymerase Chain Reaction

35) Product Images from "Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA)"

Article Title: Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA)

Journal: Applied Microbiology and Biotechnology

doi: 10.1007/s00253-014-6203-8

HPLC separation profiles of peptide fragments produced by protease digestion of  S -HMGSH dehydrogenase from  Paecilomyces variotii  NBRC 109023.  a  Digestion by  Staphylococcus aureus  protease and  b  digestion by  Achromobacter  protease. HPLC separation of the resulting peptide fragments was performed at a rate of 1.0 ml/min with solvent A (0.1 % ( v / v ) TFA in water) and solvent B (0.1 % ( v / v ) TFA in 60 % ( v / v ) acetonitrile). A TSK gel ODS-120T column was used for the separation. The column was equilibrated with solvent A, and peptide fragments were eluted by a linear gradient of solvent B (from 20 % ( v / v ) to 100 % ( v / v )) over 55 min
Figure Legend Snippet: HPLC separation profiles of peptide fragments produced by protease digestion of S -HMGSH dehydrogenase from Paecilomyces variotii NBRC 109023. a Digestion by Staphylococcus aureus protease and b digestion by Achromobacter protease. HPLC separation of the resulting peptide fragments was performed at a rate of 1.0 ml/min with solvent A (0.1 % ( v / v ) TFA in water) and solvent B (0.1 % ( v / v ) TFA in 60 % ( v / v ) acetonitrile). A TSK gel ODS-120T column was used for the separation. The column was equilibrated with solvent A, and peptide fragments were eluted by a linear gradient of solvent B (from 20 % ( v / v ) to 100 % ( v / v )) over 55 min

Techniques Used: High Performance Liquid Chromatography, Produced

36) Product Images from "Identification of a long non-coding RNA-associated RNP complex regulating metastasis at the translational step"

Article Title: Identification of a long non-coding RNA-associated RNP complex regulating metastasis at the translational step

Journal: The EMBO Journal

doi: 10.1038/emboj.2013.188

treRNA suppresses the expression of epithelial markers at the translation step. ( A ) Immunoblots of epithelial and mesenchymal markers in MCF7 cells expressing treRNA or a vector control or a control long non-coding RNA HAR1A. MCF7 cells expressing treRNA
Figure Legend Snippet: treRNA suppresses the expression of epithelial markers at the translation step. ( A ) Immunoblots of epithelial and mesenchymal markers in MCF7 cells expressing treRNA or a vector control or a control long non-coding RNA HAR1A. MCF7 cells expressing treRNA

Techniques Used: Expressing, Western Blot, Plasmid Preparation

treRNA promotes migration and invasion in vitro and metastasis in vivo . ( A , B ) Quantitative analysis of MCF7 cells expressing a vector control, treRNA, or control long non-coding RNA HAR1A in migration ( A ) and invasion ( B ) assays. Data represent mean
Figure Legend Snippet: treRNA promotes migration and invasion in vitro and metastasis in vivo . ( A , B ) Quantitative analysis of MCF7 cells expressing a vector control, treRNA, or control long non-coding RNA HAR1A in migration ( A ) and invasion ( B ) assays. Data represent mean

Techniques Used: Migration, In Vitro, In Vivo, Expressing, Plasmid Preparation

TreRNA RNP complex binds to the translation initiation factor eIF4G1. ( A ) RNA pull-down was performed by incubating cytoplasmic extracts of MCF7 cells with biotinylated treRNA or antisense treRNA (negative control) followed by detection of the presence
Figure Legend Snippet: TreRNA RNP complex binds to the translation initiation factor eIF4G1. ( A ) RNA pull-down was performed by incubating cytoplasmic extracts of MCF7 cells with biotinylated treRNA or antisense treRNA (negative control) followed by detection of the presence

Techniques Used: Negative Control

37) Product Images from "Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity"

Article Title: Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

Journal: Genome Research

doi: 10.1101/gr.182881.114

Introgression of the BN Shroom3 gene onto FHH background improves glomerular and overall kidney function. ( A ) At 14 wk of age, both homozygous and heterozygous FHH.BN14a congenic animals showed a significantly lower degree of albuminuria compared to FHH ( n = 4, 7, 8, and 3, respectively). ( B ) Both heterozygous and homozygous FHH.BN14a demonstrated significantly improved glomerular permeability (Palb) compared to FHH. ( n ].) ( C ) FHH.BN14a kidney showed a decreased presence of glomerular sclerosis compared to FHH at 14 wk of age. A minimum of 30 glomeruli from three kidneys for each strain were scored for a percentage of sclerosis using a scale from 0 (no sclerosis) to 4 (complete sclerosis). ( D ) Representative trichrome-stained images of glomeruli from FHH and FHH.BN14a are shown. Fibrotic tissues are indicated by blue stain. Scale bars = 50 µm. ( E ) Electron microscopic images of glomeruli showed podocyte foot process fusion (indicated by arrow) in FHH compared to FHH.BN14a animals at 18 wk of age. Scale bars = 500 µm. (CL) Capillary lumen, (*) P
Figure Legend Snippet: Introgression of the BN Shroom3 gene onto FHH background improves glomerular and overall kidney function. ( A ) At 14 wk of age, both homozygous and heterozygous FHH.BN14a congenic animals showed a significantly lower degree of albuminuria compared to FHH ( n = 4, 7, 8, and 3, respectively). ( B ) Both heterozygous and homozygous FHH.BN14a demonstrated significantly improved glomerular permeability (Palb) compared to FHH. ( n ].) ( C ) FHH.BN14a kidney showed a decreased presence of glomerular sclerosis compared to FHH at 14 wk of age. A minimum of 30 glomeruli from three kidneys for each strain were scored for a percentage of sclerosis using a scale from 0 (no sclerosis) to 4 (complete sclerosis). ( D ) Representative trichrome-stained images of glomeruli from FHH and FHH.BN14a are shown. Fibrotic tissues are indicated by blue stain. Scale bars = 50 µm. ( E ) Electron microscopic images of glomeruli showed podocyte foot process fusion (indicated by arrow) in FHH compared to FHH.BN14a animals at 18 wk of age. Scale bars = 500 µm. (CL) Capillary lumen, (*) P

Techniques Used: Permeability, Staining

The G1073S variant decreases the actin-binding affinity of SHROOM3 protein. ( A ) FLAG-tagged BN, FHH, or ∆G1073S SHROOM3 proteins were overexpressed in HEK293 cells, followed by immunoprecipitation against FLAG. Immunoprecipitated lysates were immunoblotted using antibodies against FLAG, ROCK1, and ACTB. A representative Western blot of immunoprecipitated lysate is provided. ( B ) Quantification of the Western blot showed that FHH SHROOM3 and ∆G1073S mutant had significantly reduced actin-binding affinity compared to BN SHROOM3. ( n = 3 per group. [*] P
Figure Legend Snippet: The G1073S variant decreases the actin-binding affinity of SHROOM3 protein. ( A ) FLAG-tagged BN, FHH, or ∆G1073S SHROOM3 proteins were overexpressed in HEK293 cells, followed by immunoprecipitation against FLAG. Immunoprecipitated lysates were immunoblotted using antibodies against FLAG, ROCK1, and ACTB. A representative Western blot of immunoprecipitated lysate is provided. ( B ) Quantification of the Western blot showed that FHH SHROOM3 and ∆G1073S mutant had significantly reduced actin-binding affinity compared to BN SHROOM3. ( n = 3 per group. [*] P

Techniques Used: Variant Assay, Binding Assay, Immunoprecipitation, Western Blot, Mutagenesis

The G1073S variant disrupts the function of the FHH Shroom3 gene. ( A ) Schematic of the different recombinant Shroom3 cDNAs, where a specific region of the BN Shroom3 sequence was replaced by FHH. ( B ) Co-injection of shroom3 + tp53 MO with Shroom3∆641–3044 or Shroom3∆4117–5966 mRNA, but not Shroom3∆3044–4117 mRNA, rescued dextran leakage induced by the MO ( n = 23, 9, 18, 28, and 15, respectively). ( C ) Schematic of Shroom3 single-amino acid mutants created by site-directed mutagenesis. ( D ) Co-injection of ∆Y1291C or ∆A1356V restored normal glomerular permeability, while ∆G1073S failed to exhibit functional rescue ( n = 17, 12, 17, 23, and 21, respectively). (*) P
Figure Legend Snippet: The G1073S variant disrupts the function of the FHH Shroom3 gene. ( A ) Schematic of the different recombinant Shroom3 cDNAs, where a specific region of the BN Shroom3 sequence was replaced by FHH. ( B ) Co-injection of shroom3 + tp53 MO with Shroom3∆641–3044 or Shroom3∆4117–5966 mRNA, but not Shroom3∆3044–4117 mRNA, rescued dextran leakage induced by the MO ( n = 23, 9, 18, 28, and 15, respectively). ( C ) Schematic of Shroom3 single-amino acid mutants created by site-directed mutagenesis. ( D ) Co-injection of ∆Y1291C or ∆A1356V restored normal glomerular permeability, while ∆G1073S failed to exhibit functional rescue ( n = 17, 12, 17, 23, and 21, respectively). (*) P

Techniques Used: Variant Assay, Recombinant, Sequencing, Injection, Mutagenesis, Permeability, Functional Assay

FHH Shroom3 is defective and contributes to glomerular dysfunction. ( A ) Schematic representation of the rat Shroom3 protein is shown. Vertical lines represent the amino acid variants found in FHH Shroom3 . Variants predicted to be damaging by PolyPhen-2 are shown in red. ( B ) Co-injection of shroom3 + tp53 morpholino (MO) with full-length BN, but not FHH, Shroom3 mRNA rescued the edema phenotype. ([***] P
Figure Legend Snippet: FHH Shroom3 is defective and contributes to glomerular dysfunction. ( A ) Schematic representation of the rat Shroom3 protein is shown. Vertical lines represent the amino acid variants found in FHH Shroom3 . Variants predicted to be damaging by PolyPhen-2 are shown in red. ( B ) Co-injection of shroom3 + tp53 morpholino (MO) with full-length BN, but not FHH, Shroom3 mRNA rescued the edema phenotype. ([***] P

Techniques Used: Injection

38) Product Images from "Dendritic transport of tick-borne flavivirus RNA by neuronal granules affects development of neurological disease"

Article Title: Dendritic transport of tick-borne flavivirus RNA by neuronal granules affects development of neurological disease

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1704454114

The 5′ untranslated region of tick-borne encephalitis virus functions as a signal of RNA transport to the neurites of PC12 cells. Differentiated PC12 cells were transfected with plasmids expressing the RNA of luciferase with TBEV sequences ( A – C ) or TBEV/WNV UTRs ( D – F ). Following fixation, the cells were hybridized with a fluorescent RNA probe for the luciferase gene (green), and stained with DAPI (blue) and antibodies against microtubule-associated protein 2 (MAP2; magenta). Fluorescence in situ hybridization signal in the neurites was analyzed from Z -stack images from five independent microscopic fields. ( A ) A coding sequence for luciferase (gray rectangles) was cloned with or without the partial sequence for TBEV replicon RNA. ( B , C , E , and F ) Fluorescent images ( B and E ) and fluorescence intensity ( C and F ) in PC12 neurites. ( D ) A CDS for luciferase was cloned with or without the 5′ and 3′ UTRs of TBEV (black lines) and WNV (striped lines). (Scale bars, 5 μm.) White arrows indicate the FISH signal for luciferase RNA in the neurites. Error bars represent SEM; ** P
Figure Legend Snippet: The 5′ untranslated region of tick-borne encephalitis virus functions as a signal of RNA transport to the neurites of PC12 cells. Differentiated PC12 cells were transfected with plasmids expressing the RNA of luciferase with TBEV sequences ( A – C ) or TBEV/WNV UTRs ( D – F ). Following fixation, the cells were hybridized with a fluorescent RNA probe for the luciferase gene (green), and stained with DAPI (blue) and antibodies against microtubule-associated protein 2 (MAP2; magenta). Fluorescence in situ hybridization signal in the neurites was analyzed from Z -stack images from five independent microscopic fields. ( A ) A coding sequence for luciferase (gray rectangles) was cloned with or without the partial sequence for TBEV replicon RNA. ( B , C , E , and F ) Fluorescent images ( B and E ) and fluorescence intensity ( C and F ) in PC12 neurites. ( D ) A CDS for luciferase was cloned with or without the 5′ and 3′ UTRs of TBEV (black lines) and WNV (striped lines). (Scale bars, 5 μm.) White arrows indicate the FISH signal for luciferase RNA in the neurites. Error bars represent SEM; ** P

Techniques Used: Transfection, Expressing, Luciferase, Staining, Fluorescence, In Situ Hybridization, Sequencing, Clone Assay, Fluorescence In Situ Hybridization

39) Product Images from "The Stem Cell Marker Lgr5 Defines a Subset of Postmitotic Neurons in the Olfactory Bulb"

Article Title: The Stem Cell Marker Lgr5 Defines a Subset of Postmitotic Neurons in the Olfactory Bulb

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0500-17.2017

R-spondin 3, a ligand for Lgr5, is expressed in the olfactory bulb.  A ,  In situ  hybridization of  R-spondin 3 ). Scale bar, 100 μm.  B , Double  in situ  hybridization using  lgr5  (green) and  R-spondin 3  (red) riboprobes showed expression of these two genes in the OB. Arrowheads indicate costained cells.  A ,  B , Results were from 1-month-old mice, but similar results were observed from 2-month-old mice. Scale bar, 20 μm.  C , Bath perfusion of R-spondin 3 did not change the resting membrane potential (RMP), sEPSC frequency, and sEPSC amplitude. Each red line connects the values of preapplication and postapplication of R-spondin 3 from a single cell. The data for the RMP were obtained from perforated patch clamp, whereas those for sEPSCs were from whole-cell patch clamp. An additional two cells in perforated patch-clamp configuration also showed no alteration in sEPSC frequency and amplitude after R-spondin 3 perfusion. The recordings typically lasted 15 min after perfusion onset of R-spondin 3.  p  values: paired  t  test.
Figure Legend Snippet: R-spondin 3, a ligand for Lgr5, is expressed in the olfactory bulb. A , In situ hybridization of R-spondin 3 ). Scale bar, 100 μm. B , Double in situ hybridization using lgr5 (green) and R-spondin 3 (red) riboprobes showed expression of these two genes in the OB. Arrowheads indicate costained cells. A , B , Results were from 1-month-old mice, but similar results were observed from 2-month-old mice. Scale bar, 20 μm. C , Bath perfusion of R-spondin 3 did not change the resting membrane potential (RMP), sEPSC frequency, and sEPSC amplitude. Each red line connects the values of preapplication and postapplication of R-spondin 3 from a single cell. The data for the RMP were obtained from perforated patch clamp, whereas those for sEPSCs were from whole-cell patch clamp. An additional two cells in perforated patch-clamp configuration also showed no alteration in sEPSC frequency and amplitude after R-spondin 3 perfusion. The recordings typically lasted 15 min after perfusion onset of R-spondin 3. p values: paired t test.

Techniques Used: In Situ Hybridization, Expressing, Mouse Assay, Patch Clamp

40) Product Images from "Functional characterization of bitter-taste receptors expressed in mammalian testis"

Article Title: Functional characterization of bitter-taste receptors expressed in mammalian testis

Journal: Molecular Human Reproduction

doi: 10.1093/molehr/gas040

In situ hybridization of mouse testicular sections with Tas2r probes. ( A and D ) Antisense probes of Tas2r105 (A) and Tas2r108 (D) hybridized to subsets of cells in some seminiferous sections (blue staining). ( B and E ) High-magnification images of Tas2r105
Figure Legend Snippet: In situ hybridization of mouse testicular sections with Tas2r probes. ( A and D ) Antisense probes of Tas2r105 (A) and Tas2r108 (D) hybridized to subsets of cells in some seminiferous sections (blue staining). ( B and E ) High-magnification images of Tas2r105

Techniques Used: In Situ Hybridization, Staining

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Article Snippet: .. TEs and multigene family sequences: Sequencing and analyses After the amplification reactions, the PCR products were purified using the GenElute PCR Clean-Up Kit (Sigma Aldrich, St Louis, MO, USA).Rex1 and Rex3 sequences were cloned using pGEM®-T Easy Vector Systems (Promega, Madison, WI, USA). .. The obtained clones were sequenced using an ABI-PRISM Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).

Article Title: Lutzomyia longipalpis TGF-β Has a Role in Leishmania infantum chagasi Survival in the Vector
Article Snippet: .. The control dsRNA cDNA template was obtained from β-galactosidase gene amplified from plasmid pGEM T-easy vector (PROMEGA) (Tinoco-Nunes et al., ). .. The PCR products were used as templates for transcription reactions with the Megascript RNAi Kit (Ambion) according to the manufacturer's instructions and subsequently concentrated to 40 μg/μL using a vacuum microcentrifuge concentrator.

In Vitro:

Article Title: The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis
Article Snippet: .. The partial cDNA (634 base pairs length) sequence of caspase 8 and caspase 3 was PCR amplified, and the fragments were cloned into a pGEM-T-easy vector (Promega, USA) to generate an antisense digoxigenin-labelled RNA probe (DIG RNA Labelling Kit, Roche Applied Science, Germany) in vitro for the WISH assay in zebrafish embryo. ..

Mutagenesis:

Article Title: Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis
Article Snippet: .. To confirm the presence of the ap2m mutation and demonstrate that plants were true ap2m homozygotes and not chimeras, undigested PCR products were cloned into the pGEM-T Easy vector (Promega, Fitchburg, WI), and the inserts of 6 different clones were sequenced using the GenomeLab DTCS Quick Start Kit (SCIEX, Framingham, MA) and a Beckman Coulter CEQ2000XL DNA sequencer (SCIEX, Framingham, MA). .. To confirm the absence of AP2Ms gRNA-Cas9 gene cassette in ap2m [AtS1 pro :SRKb-FLAG+SCRb ] plants, genomic DNA from these plants, along with the AP2Ms gRNA-Cas9 plasmid as positive control, were subjected to PCR using SS42 (5′-TCCCAGGATTAGAATGATTAGG-3′) and SS43 (5′-CGACTAAGGGTTTCTTATATGC-3′) primers ( ).

Purification:

Article Title: Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes
Article Snippet: .. TEs and multigene family sequences: Sequencing and analyses After the amplification reactions, the PCR products were purified using the GenElute PCR Clean-Up Kit (Sigma Aldrich, St Louis, MO, USA).Rex1 and Rex3 sequences were cloned using pGEM®-T Easy Vector Systems (Promega, Madison, WI, USA). .. The obtained clones were sequenced using an ABI-PRISM Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).

Sequencing:

Article Title: SNP and SCAR Markers for Specific Discrimination of Antler-Shaped Ganoderma lucidum
Article Snippet: .. Cloning, Sequencing and Sequence Analysis The PCR products and specific DNA fragments were ligated into pGEM-T easy vector (Promega, Madison, WI, USA), according to the manufacturer’s instruction. .. After ligation, the plasmids were transformed into competent cell (E. coli DH5α; RBC, New Taipei City, Taiwan) by the heat-shock method [ ].

Article Title: The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis
Article Snippet: .. The partial cDNA (634 base pairs length) sequence of caspase 8 and caspase 3 was PCR amplified, and the fragments were cloned into a pGEM-T-easy vector (Promega, USA) to generate an antisense digoxigenin-labelled RNA probe (DIG RNA Labelling Kit, Roche Applied Science, Germany) in vitro for the WISH assay in zebrafish embryo. ..

Article Title: Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes
Article Snippet: .. TEs and multigene family sequences: Sequencing and analyses After the amplification reactions, the PCR products were purified using the GenElute PCR Clean-Up Kit (Sigma Aldrich, St Louis, MO, USA).Rex1 and Rex3 sequences were cloned using pGEM®-T Easy Vector Systems (Promega, Madison, WI, USA). .. The obtained clones were sequenced using an ABI-PRISM Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).

Article Title: An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases
Article Snippet: .. The construction of the ATXN3 plasmid The cDNA sequence of ataxin-3 (~1.8 kb) containing 8 CAG repeats was cloned into a pGEM-T Easy vector (Promega). .. Site-directed mutagenesis of an insert was performed with primers MUT_R and MUT_F (sequences given in Additional file : Table S2) using a Phusion Kit (Finnzymes).

Activity Assay:

Article Title: Altered mRNA Expression Related to the Apoptotic Effect of Three Xanthones on Human Melanoma SK-MEL-28 Cell Line
Article Snippet: .. Caspase-8 and 9 Assay Caspase activity was measured by using Caspase-Glo 8 and 9 assay kits according to the manufacturer's instructions (Promega Corporation, Australia) [ ]. .. Apoptosis Assay To confirm the role of caspase in the apoptosis induced by these xanthones, pan-caspase inhibitor (Z-VAD-FMK) (Promega Corporation, Australia) was used.

Polymerase Chain Reaction:

Article Title: SNP and SCAR Markers for Specific Discrimination of Antler-Shaped Ganoderma lucidum
Article Snippet: .. Cloning, Sequencing and Sequence Analysis The PCR products and specific DNA fragments were ligated into pGEM-T easy vector (Promega, Madison, WI, USA), according to the manufacturer’s instruction. .. After ligation, the plasmids were transformed into competent cell (E. coli DH5α; RBC, New Taipei City, Taiwan) by the heat-shock method [ ].

Article Title: The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis
Article Snippet: .. The partial cDNA (634 base pairs length) sequence of caspase 8 and caspase 3 was PCR amplified, and the fragments were cloned into a pGEM-T-easy vector (Promega, USA) to generate an antisense digoxigenin-labelled RNA probe (DIG RNA Labelling Kit, Roche Applied Science, Germany) in vitro for the WISH assay in zebrafish embryo. ..

Article Title: Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes
Article Snippet: .. TEs and multigene family sequences: Sequencing and analyses After the amplification reactions, the PCR products were purified using the GenElute PCR Clean-Up Kit (Sigma Aldrich, St Louis, MO, USA).Rex1 and Rex3 sequences were cloned using pGEM®-T Easy Vector Systems (Promega, Madison, WI, USA). .. The obtained clones were sequenced using an ABI-PRISM Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).

Article Title: Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis
Article Snippet: .. To confirm the presence of the ap2m mutation and demonstrate that plants were true ap2m homozygotes and not chimeras, undigested PCR products were cloned into the pGEM-T Easy vector (Promega, Fitchburg, WI), and the inserts of 6 different clones were sequenced using the GenomeLab DTCS Quick Start Kit (SCIEX, Framingham, MA) and a Beckman Coulter CEQ2000XL DNA sequencer (SCIEX, Framingham, MA). .. To confirm the absence of AP2Ms gRNA-Cas9 gene cassette in ap2m [AtS1 pro :SRKb-FLAG+SCRb ] plants, genomic DNA from these plants, along with the AP2Ms gRNA-Cas9 plasmid as positive control, were subjected to PCR using SS42 (5′-TCCCAGGATTAGAATGATTAGG-3′) and SS43 (5′-CGACTAAGGGTTTCTTATATGC-3′) primers ( ).

Transformation Assay:

Article Title: P-glycoprotein-9 and macrocyclic lactone resistance status in selected strains of the ovine gastrointestinal nematode, Teladorsagia circumcincta
Article Snippet: .. Amplicons of the expected size (∼3.8 kb) were ligated into pGEM® -T Easy Vector (Promega) and transformed into chemically competent JM109 E. coli (Promega), following the manufacturer's protocols. .. Three clones derived from each UK isolate were sequenced in both sense and antisense directions.

Plasmid Preparation:

Article Title: SNP and SCAR Markers for Specific Discrimination of Antler-Shaped Ganoderma lucidum
Article Snippet: .. Cloning, Sequencing and Sequence Analysis The PCR products and specific DNA fragments were ligated into pGEM-T easy vector (Promega, Madison, WI, USA), according to the manufacturer’s instruction. .. After ligation, the plasmids were transformed into competent cell (E. coli DH5α; RBC, New Taipei City, Taiwan) by the heat-shock method [ ].

Article Title: The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis
Article Snippet: .. The partial cDNA (634 base pairs length) sequence of caspase 8 and caspase 3 was PCR amplified, and the fragments were cloned into a pGEM-T-easy vector (Promega, USA) to generate an antisense digoxigenin-labelled RNA probe (DIG RNA Labelling Kit, Roche Applied Science, Germany) in vitro for the WISH assay in zebrafish embryo. ..

Article Title: Dispersion of transposable elements and multigene families: Microstructural variation in Characidium (Characiformes: Crenuchidae) genomes
Article Snippet: .. TEs and multigene family sequences: Sequencing and analyses After the amplification reactions, the PCR products were purified using the GenElute PCR Clean-Up Kit (Sigma Aldrich, St Louis, MO, USA).Rex1 and Rex3 sequences were cloned using pGEM®-T Easy Vector Systems (Promega, Madison, WI, USA). .. The obtained clones were sequenced using an ABI-PRISM Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).

Article Title: An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases
Article Snippet: .. The construction of the ATXN3 plasmid The cDNA sequence of ataxin-3 (~1.8 kb) containing 8 CAG repeats was cloned into a pGEM-T Easy vector (Promega). .. Site-directed mutagenesis of an insert was performed with primers MUT_R and MUT_F (sequences given in Additional file : Table S2) using a Phusion Kit (Finnzymes).

Article Title: Lutzomyia longipalpis TGF-β Has a Role in Leishmania infantum chagasi Survival in the Vector
Article Snippet: .. The control dsRNA cDNA template was obtained from β-galactosidase gene amplified from plasmid pGEM T-easy vector (PROMEGA) (Tinoco-Nunes et al., ). .. The PCR products were used as templates for transcription reactions with the Megascript RNAi Kit (Ambion) according to the manufacturer's instructions and subsequently concentrated to 40 μg/μL using a vacuum microcentrifuge concentrator.

Article Title: Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis
Article Snippet: .. To confirm the presence of the ap2m mutation and demonstrate that plants were true ap2m homozygotes and not chimeras, undigested PCR products were cloned into the pGEM-T Easy vector (Promega, Fitchburg, WI), and the inserts of 6 different clones were sequenced using the GenomeLab DTCS Quick Start Kit (SCIEX, Framingham, MA) and a Beckman Coulter CEQ2000XL DNA sequencer (SCIEX, Framingham, MA). .. To confirm the absence of AP2Ms gRNA-Cas9 gene cassette in ap2m [AtS1 pro :SRKb-FLAG+SCRb ] plants, genomic DNA from these plants, along with the AP2Ms gRNA-Cas9 plasmid as positive control, were subjected to PCR using SS42 (5′-TCCCAGGATTAGAATGATTAGG-3′) and SS43 (5′-CGACTAAGGGTTTCTTATATGC-3′) primers ( ).

Article Title: P-glycoprotein-9 and macrocyclic lactone resistance status in selected strains of the ovine gastrointestinal nematode, Teladorsagia circumcincta
Article Snippet: .. Amplicons of the expected size (∼3.8 kb) were ligated into pGEM® -T Easy Vector (Promega) and transformed into chemically competent JM109 E. coli (Promega), following the manufacturer's protocols. .. Three clones derived from each UK isolate were sequenced in both sense and antisense directions.

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