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    Thermo Fisher superscript iii
    Mm-VSP mRNA and protein are expressed and developmentally regulated in mouse brain. ( A ) Mm-VSP mRNA is expressed and developmentally regulated in the brain. <t>RT-PCR</t> amplicons were visualized in an agarose gel using ethidium bromide. Left-hand labels indicate the basepairs of Mm-VSP’s mRNA sequence amplified by each primer pair, where 0 is the beginning of the open reading frame. Primers against MAP2 mRNA were used as a positive control. Top labels indicate the postnatal age (in days), gender, and tissue of the mice from which RNA was collected. Each gel is representative of <t>three</t> replicates. ( B ) A novel Mm-VSP variant lacks exon 9. In dark gray ( top row ) is the cDNA sequence for Mm-VSP spanning exon 9 and flanking bases. In the second row, the sequence of the splice variant is shown with exon 9 indicated by dashes. The amino acids encoded by the cDNA are indicated below in blue. The alternate splice variant identified in ( A ) was found to be lacking exon 9 after sequencing of the amplicon. Shown below in black are the amino acid sequences of other VSP family members, aligned with Mm-VSP using COBALT. Note that the Mm-VSP amino terminus is much longer than that of other VSPs and lacks obvious sequence similarity to other orthologs in the region encoded by exon 9. ( C ) Mm-VSP protein is expressed and developmentally regulated in the brain. Whole-brain homogenates were probed in a western blot to detect the presence of Mm-VSP protein. MAP2, probed for on the same blot, served as a loading control. Tissue samples from two separate mice are shown for each age. The blot is representative of three technical replicates. The predicted mass of Mm-VSP is 77 kDa. Two bands near this size are seen to be present in whole-brain homogenate, and their intensity increases with the age of the mice. To see this figure in color, go online.
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    Images

    1) Product Images from "Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase"

    Article Title: Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2015.11.004

    Mm-VSP mRNA and protein are expressed and developmentally regulated in mouse brain. ( A ) Mm-VSP mRNA is expressed and developmentally regulated in the brain. RT-PCR amplicons were visualized in an agarose gel using ethidium bromide. Left-hand labels indicate the basepairs of Mm-VSP’s mRNA sequence amplified by each primer pair, where 0 is the beginning of the open reading frame. Primers against MAP2 mRNA were used as a positive control. Top labels indicate the postnatal age (in days), gender, and tissue of the mice from which RNA was collected. Each gel is representative of three replicates. ( B ) A novel Mm-VSP variant lacks exon 9. In dark gray ( top row ) is the cDNA sequence for Mm-VSP spanning exon 9 and flanking bases. In the second row, the sequence of the splice variant is shown with exon 9 indicated by dashes. The amino acids encoded by the cDNA are indicated below in blue. The alternate splice variant identified in ( A ) was found to be lacking exon 9 after sequencing of the amplicon. Shown below in black are the amino acid sequences of other VSP family members, aligned with Mm-VSP using COBALT. Note that the Mm-VSP amino terminus is much longer than that of other VSPs and lacks obvious sequence similarity to other orthologs in the region encoded by exon 9. ( C ) Mm-VSP protein is expressed and developmentally regulated in the brain. Whole-brain homogenates were probed in a western blot to detect the presence of Mm-VSP protein. MAP2, probed for on the same blot, served as a loading control. Tissue samples from two separate mice are shown for each age. The blot is representative of three technical replicates. The predicted mass of Mm-VSP is 77 kDa. Two bands near this size are seen to be present in whole-brain homogenate, and their intensity increases with the age of the mice. To see this figure in color, go online.
    Figure Legend Snippet: Mm-VSP mRNA and protein are expressed and developmentally regulated in mouse brain. ( A ) Mm-VSP mRNA is expressed and developmentally regulated in the brain. RT-PCR amplicons were visualized in an agarose gel using ethidium bromide. Left-hand labels indicate the basepairs of Mm-VSP’s mRNA sequence amplified by each primer pair, where 0 is the beginning of the open reading frame. Primers against MAP2 mRNA were used as a positive control. Top labels indicate the postnatal age (in days), gender, and tissue of the mice from which RNA was collected. Each gel is representative of three replicates. ( B ) A novel Mm-VSP variant lacks exon 9. In dark gray ( top row ) is the cDNA sequence for Mm-VSP spanning exon 9 and flanking bases. In the second row, the sequence of the splice variant is shown with exon 9 indicated by dashes. The amino acids encoded by the cDNA are indicated below in blue. The alternate splice variant identified in ( A ) was found to be lacking exon 9 after sequencing of the amplicon. Shown below in black are the amino acid sequences of other VSP family members, aligned with Mm-VSP using COBALT. Note that the Mm-VSP amino terminus is much longer than that of other VSPs and lacks obvious sequence similarity to other orthologs in the region encoded by exon 9. ( C ) Mm-VSP protein is expressed and developmentally regulated in the brain. Whole-brain homogenates were probed in a western blot to detect the presence of Mm-VSP protein. MAP2, probed for on the same blot, served as a loading control. Tissue samples from two separate mice are shown for each age. The blot is representative of three technical replicates. The predicted mass of Mm-VSP is 77 kDa. Two bands near this size are seen to be present in whole-brain homogenate, and their intensity increases with the age of the mice. To see this figure in color, go online.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing, Amplification, Positive Control, Mouse Assay, Variant Assay, Western Blot

    2) Product Images from "Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection"

    Article Title: Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection

    Journal: Genome Biology

    doi: 10.1186/s13059-017-1329-5

    Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top
    Figure Legend Snippet: Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top

    Techniques Used: Isolation, Infection, Transformation Assay, Expressing

    3) Product Images from "Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus"

    Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1713705

    DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).
    Figure Legend Snippet: DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

    Techniques Used: Binding Assay, Infection, Expressing, Mutagenesis, Negative Control, Incubation, Polymerase Chain Reaction

    4) Product Images from "The eIF2-alpha kinase HRI is a novel therapeutic target in multiple myeloma"

    Article Title: The eIF2-alpha kinase HRI is a novel therapeutic target in multiple myeloma

    Journal: Leukemia research

    doi: 10.1016/j.leukres.2017.01.007

    MM patient cells are sensitive to BTdCPU (A) Bone marrow purified CD138+ cells from three relapsed/refractory MM patients (MM 1,2,3) were treated with 1 μM Dex, 10 μM BTdCPU or control for 24 hours and relative survival was assessed. (B) Three healthy donor bone marrow mononuclear control samples (ctrl 1,2,3) were treated with increasing doses of BTdCPU and relative survival was assessed. (C) MM patient cells or healthy donor marrow mononuclear cells (MNC) were treated with 10 μM BTdCPU for 0, 4 or 8 hours. Total RNA expression levels of CHOP relative to GAPDH were quantitated. (D) Bone marrow biopsies from healthy donors (left panel) or MM patients (right panel) were stained for eIF2α expression by immunohistochemistry. Normal hematopoietic elements show weak staining, while MM patient cells demonstrate uniformly strong staining for eIF2α. Images are 630X magnification, taken on a Leica DM3000 microscope.
    Figure Legend Snippet: MM patient cells are sensitive to BTdCPU (A) Bone marrow purified CD138+ cells from three relapsed/refractory MM patients (MM 1,2,3) were treated with 1 μM Dex, 10 μM BTdCPU or control for 24 hours and relative survival was assessed. (B) Three healthy donor bone marrow mononuclear control samples (ctrl 1,2,3) were treated with increasing doses of BTdCPU and relative survival was assessed. (C) MM patient cells or healthy donor marrow mononuclear cells (MNC) were treated with 10 μM BTdCPU for 0, 4 or 8 hours. Total RNA expression levels of CHOP relative to GAPDH were quantitated. (D) Bone marrow biopsies from healthy donors (left panel) or MM patients (right panel) were stained for eIF2α expression by immunohistochemistry. Normal hematopoietic elements show weak staining, while MM patient cells demonstrate uniformly strong staining for eIF2α. Images are 630X magnification, taken on a Leica DM3000 microscope.

    Techniques Used: Purification, RNA Expression, Staining, Expressing, Immunohistochemistry, Microscopy

    5) Product Images from "A Rapid Progressor-Specific Variant Clone of Simian Immunodeficiency Virus Replicates Efficiently In Vivo Only in the Absence of Immune Reponses ▿"

    Article Title: A Rapid Progressor-Specific Variant Clone of Simian Immunodeficiency Virus Replicates Efficiently In Vivo Only in the Absence of Immune Reponses ▿

    Journal:

    doi: 10.1128/JVI.00614-07

    Differentiation of viruses in three macaques coinfected with SIVsmE543-3 and SIVsmH635FC. The env V1-V5 region was amplified by RT-PCR from viral RNA isolated from plasma samples of these macaques at 10 days (10d), 8 weeks (8w), 20 weeks, and 26 weeks
    Figure Legend Snippet: Differentiation of viruses in three macaques coinfected with SIVsmE543-3 and SIVsmH635FC. The env V1-V5 region was amplified by RT-PCR from viral RNA isolated from plasma samples of these macaques at 10 days (10d), 8 weeks (8w), 20 weeks, and 26 weeks

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Isolation

    Viral RNA loads in the plasma (A) and CSF (B) of three rhesus macaques (H711, H712, and H713) that were coinoculated with SIVsmE543-3 and SIVsmH635FC.
    Figure Legend Snippet: Viral RNA loads in the plasma (A) and CSF (B) of three rhesus macaques (H711, H712, and H713) that were coinoculated with SIVsmE543-3 and SIVsmH635FC.

    Techniques Used:

    6) Product Images from "Inducible Expression of CXCL1 within the Central Nervous System Amplifies Viral-Induced Demyelination"

    Article Title: Inducible Expression of CXCL1 within the Central Nervous System Amplifies Viral-Induced Demyelination

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1501802

    CXCL1 is induced in vivo following administration of Dox into double-tg mice during acute JHMV infection. ( A ) To test the ability of Dox to induce CXCL1 overproduction in vivo, double-tg and single-tg mice were infected with 250 PFU JHMV and treated daily with 50 mg/kg Dox between days 2 and 12 p.i. ( B ) Administration of Dox to double-tg mice resulted in a significant increase in the expression of CXCL1 mRNA compared with Dox-treated single-tg mice within the brain at days 4, 7, and 12 p.i. as measured by quantitative real-time PCR. ( C ) Within the spinal cord, dox-treated double-tg mice had statistically significant increases in CXCL1 mRNA expression over Dox-treated single-tg mice at days 7 and 12 p.i. ( D ) CXCL1 transgene expression within the brain and spinal cord did not impact endogenous CXCL1 production within Dox-treated double-tg mice compared with Dox-treated single-tg mice. Overproduction of CXCL1 protein was observed within the spinal cord at day 7 p.i. within double-tg mice ( E ), and this correlated with an increase in CXCL1 protein within the blood serum ( F ) at day 7 p.i. as measured by ELISA. ( G ) Immunofluorscence analysis (original magnification ×40) of spinal cord tissue from Dox-treated double-tg mice confirmed that astrocytes were the source of CXCL1 production at day 7 p.i. ( H ) No significant changes in proinflammatory gene expression between double-tg and single-tg mice were observed within the spinal cord at day 12 p.i. using an RNA superarray. Data from (B) represent two experiments with a minimum of four mice per group. All quantitative real-time PCR samples were run in triplicate. (D) and (E) represent a minimum of three mice per group. Superarray data were compiled used the average value of two mice per group run in duplicate. Data are presented as average ± SEM; statistical significance was measured using an unpaired two-tailed Student t test. * p
    Figure Legend Snippet: CXCL1 is induced in vivo following administration of Dox into double-tg mice during acute JHMV infection. ( A ) To test the ability of Dox to induce CXCL1 overproduction in vivo, double-tg and single-tg mice were infected with 250 PFU JHMV and treated daily with 50 mg/kg Dox between days 2 and 12 p.i. ( B ) Administration of Dox to double-tg mice resulted in a significant increase in the expression of CXCL1 mRNA compared with Dox-treated single-tg mice within the brain at days 4, 7, and 12 p.i. as measured by quantitative real-time PCR. ( C ) Within the spinal cord, dox-treated double-tg mice had statistically significant increases in CXCL1 mRNA expression over Dox-treated single-tg mice at days 7 and 12 p.i. ( D ) CXCL1 transgene expression within the brain and spinal cord did not impact endogenous CXCL1 production within Dox-treated double-tg mice compared with Dox-treated single-tg mice. Overproduction of CXCL1 protein was observed within the spinal cord at day 7 p.i. within double-tg mice ( E ), and this correlated with an increase in CXCL1 protein within the blood serum ( F ) at day 7 p.i. as measured by ELISA. ( G ) Immunofluorscence analysis (original magnification ×40) of spinal cord tissue from Dox-treated double-tg mice confirmed that astrocytes were the source of CXCL1 production at day 7 p.i. ( H ) No significant changes in proinflammatory gene expression between double-tg and single-tg mice were observed within the spinal cord at day 12 p.i. using an RNA superarray. Data from (B) represent two experiments with a minimum of four mice per group. All quantitative real-time PCR samples were run in triplicate. (D) and (E) represent a minimum of three mice per group. Superarray data were compiled used the average value of two mice per group run in duplicate. Data are presented as average ± SEM; statistical significance was measured using an unpaired two-tailed Student t test. * p

    Techniques Used: In Vivo, Mouse Assay, Infection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    CXCL1 overproduction from astrocytes mobilizes neutrophils and directs them to the CNS. Blood from Dox-treated, JHMV-infected double-tg and single-tg mice was isolated at day 4 and day 7 p.i. and used for flow cytometric analysis. ( A ) The frequency of CD11b + Ly6G + neutrophils was significantly higher within the blood at days 4 and 7 p.i. Neutrophil migration to the brains ( B ) and spinal cords ( C ) of both groups was assessed by flow cytometry at days 4 and 7 p.i. A significant increase in neutrophil frequency within the brain and spinal cord was observed in Dox-treated double-tg mice at both time points. ( D ) Immunofluorescence analysis indicated that Ly6B.2 + neutrophils were primarily located at the ependymal lining and perivascular space at the spinal cord, with minimal neutrophil presence within the parenchyma. Scale bars, 50 μM. ( E ) At day 4 p.i., brains and spinal cords from Dox-treated single-tg and double-tg mice treated with NaFl were removed and total NaFl uptake was measured. No differences in the frequency of CD11b + Ly6C + Ly6G − cells were detected within either the blood ( F ) or brain and spinal cords ( G ) between Dox-treated double-tg or single-tg mice at defined times after infection with JHMV. (A)–(C) represent three independent experiments with a minimum of three mice per group per experiment at each time point analyzed; (F) and (G) represent two independent experiments with a minimum of three mice per group per experiment at each time point analyzed. Data are presented as average ± SEM; statistical significance was measured using an unpaired two-tailed Student t test. * p
    Figure Legend Snippet: CXCL1 overproduction from astrocytes mobilizes neutrophils and directs them to the CNS. Blood from Dox-treated, JHMV-infected double-tg and single-tg mice was isolated at day 4 and day 7 p.i. and used for flow cytometric analysis. ( A ) The frequency of CD11b + Ly6G + neutrophils was significantly higher within the blood at days 4 and 7 p.i. Neutrophil migration to the brains ( B ) and spinal cords ( C ) of both groups was assessed by flow cytometry at days 4 and 7 p.i. A significant increase in neutrophil frequency within the brain and spinal cord was observed in Dox-treated double-tg mice at both time points. ( D ) Immunofluorescence analysis indicated that Ly6B.2 + neutrophils were primarily located at the ependymal lining and perivascular space at the spinal cord, with minimal neutrophil presence within the parenchyma. Scale bars, 50 μM. ( E ) At day 4 p.i., brains and spinal cords from Dox-treated single-tg and double-tg mice treated with NaFl were removed and total NaFl uptake was measured. No differences in the frequency of CD11b + Ly6C + Ly6G − cells were detected within either the blood ( F ) or brain and spinal cords ( G ) between Dox-treated double-tg or single-tg mice at defined times after infection with JHMV. (A)–(C) represent three independent experiments with a minimum of three mice per group per experiment at each time point analyzed; (F) and (G) represent two independent experiments with a minimum of three mice per group per experiment at each time point analyzed. Data are presented as average ± SEM; statistical significance was measured using an unpaired two-tailed Student t test. * p

    Techniques Used: Infection, Mouse Assay, Isolation, Flow Cytometry, Migration, Cytometry, Immunofluorescence, Two Tailed Test

    Neutrophils amplify the severity of demyelination. ( A ) Dox-treated JHMV-infected double-tg mice were treated with either anti-Ly6g Ab or isotype-matched control starting between days 3 and 4 p.i. and continuing every other day until day 15 p.i. Representative flow analysis of spinal cords confirmed anti-Ly6G treatment successfully depleted neutrophils. ( B ) Representative Luxol fast blue–stained spinal cord sections from JHMV-infected double-tg mice treated with either control IgG2a or anti-Ly6G Ab between days 3 and 15 p.i. Quantification of the severity of demyelination revealed reduced white matter damage in mice treated with anti-Ly6G Ab compared with mice treated with isogenic IgG2a control Ab; data are derived from two independent experiments with a minimum of three mice per group per experiment. Data are presented as average ± SEM; statistical significance was measured using an unpaired two-tailed Student t test. * p
    Figure Legend Snippet: Neutrophils amplify the severity of demyelination. ( A ) Dox-treated JHMV-infected double-tg mice were treated with either anti-Ly6g Ab or isotype-matched control starting between days 3 and 4 p.i. and continuing every other day until day 15 p.i. Representative flow analysis of spinal cords confirmed anti-Ly6G treatment successfully depleted neutrophils. ( B ) Representative Luxol fast blue–stained spinal cord sections from JHMV-infected double-tg mice treated with either control IgG2a or anti-Ly6G Ab between days 3 and 15 p.i. Quantification of the severity of demyelination revealed reduced white matter damage in mice treated with anti-Ly6G Ab compared with mice treated with isogenic IgG2a control Ab; data are derived from two independent experiments with a minimum of three mice per group per experiment. Data are presented as average ± SEM; statistical significance was measured using an unpaired two-tailed Student t test. * p

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Staining, Derivative Assay, Two Tailed Test

    7) Product Images from "Lariat capping as a tool to manipulate the 5′ end of individual yeast mRNA species in vivo"

    Article Title: Lariat capping as a tool to manipulate the 5′ end of individual yeast mRNA species in vivo

    Journal: RNA

    doi: 10.1261/rna.059337.116

    Transcripts are lariat-capped cotranscriptionally and processed to have oligo(A) tails. ( A ) Outline of the RNase H assay applied to measure poly(A) tail lengths of GFP mRNAs. Relative positions of the oligo used to cleave the GFP mRNA, the d(T)NN oligo used to trim the poly(A) tail, and the Northern hybridization probe oligo are indicated. ( B ) RNase H/Northern assay of whole cell RNA extracted from wt cells expressing m 7 G GFP-, LC GFP-, and LCmutGFP-mRNA as indicated. The mobility shift between samples treated without (−) and with (+) oligo d(T)NN indicates the length of the poly(A) tail. ( C ) RNase H/Northern assay of whole cell RNA from cells expressing m 7 G GFP- or LC GFP-mRNA in the ccr4 Δ and ccr4 Δ pan2 Δ mutant cell backgrounds as indicated. The gel was run at identical conditions and to the same length as in B as judged by the xylene cyanol and bromophenol blue dye markers. ( D ) Box-plot showing lengths of poly(A) tails from individual clones of PCR products derived from 3′ RACE experiments of RNA from B and C . ( E ) Map of amplicons used for nascent RNA analysis by qRT-PCR. Three primer sets were used to generate amplicon a located upstream of the IPS (the LCrz processing site), amplicon b spanning the IPS, and amplicon c targeting the GFP part of the transcript downstream from the IPS. ( F ) qRT-PCR of nascent RNA. All amplicon signals were normalized to that of amplicon c and plotted as the ratio between LC- and LCmut-RNA (uncleaved). The error bars indicate the standard error of the mean (SEM), n = 3.
    Figure Legend Snippet: Transcripts are lariat-capped cotranscriptionally and processed to have oligo(A) tails. ( A ) Outline of the RNase H assay applied to measure poly(A) tail lengths of GFP mRNAs. Relative positions of the oligo used to cleave the GFP mRNA, the d(T)NN oligo used to trim the poly(A) tail, and the Northern hybridization probe oligo are indicated. ( B ) RNase H/Northern assay of whole cell RNA extracted from wt cells expressing m 7 G GFP-, LC GFP-, and LCmutGFP-mRNA as indicated. The mobility shift between samples treated without (−) and with (+) oligo d(T)NN indicates the length of the poly(A) tail. ( C ) RNase H/Northern assay of whole cell RNA from cells expressing m 7 G GFP- or LC GFP-mRNA in the ccr4 Δ and ccr4 Δ pan2 Δ mutant cell backgrounds as indicated. The gel was run at identical conditions and to the same length as in B as judged by the xylene cyanol and bromophenol blue dye markers. ( D ) Box-plot showing lengths of poly(A) tails from individual clones of PCR products derived from 3′ RACE experiments of RNA from B and C . ( E ) Map of amplicons used for nascent RNA analysis by qRT-PCR. Three primer sets were used to generate amplicon a located upstream of the IPS (the LCrz processing site), amplicon b spanning the IPS, and amplicon c targeting the GFP part of the transcript downstream from the IPS. ( F ) qRT-PCR of nascent RNA. All amplicon signals were normalized to that of amplicon c and plotted as the ratio between LC- and LCmut-RNA (uncleaved). The error bars indicate the standard error of the mean (SEM), n = 3.

    Techniques Used: Rnase H Assay, Northern Blot, Hybridization, Expressing, Mobility Shift, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Amplification

    8) Product Images from "BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity"

    Article Title: BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07927-y

    Genetic determinants of cellular survival in BRCA2-depleted KBM-7 cells. a KBM-7 cells were stably transduced with indicated doxycyline-inducible shRNA vectors. Cells were treated with doxycycline for 3 or 5 days and lysates were immunoblotted for BRCA2 and Actin. b Quantification of the percentage of cells with ≥10 RAD51 foci after 5 Gy irradiation. KBM-7 cells harboring indicated shRNAs were treated with doxycycline for 96 h prior to irradiation. Approximately 100 cells were scored per condition per replicate. Error bars indicate s.d. of two independent experiments. P values were calculated using two-tailed Student’s t -test. c KBM-7 cells expressing shBRCA2 #2 were processed for DNA fiber analysis after treatment with doxycycline for 96 h. Cells were then incubated with CldU (25 μM) for 40 min to label replication tracks and subsequently treated with HU (2 mM) for 4 h. CldU track lengths are plotted for ±500 fibers per condition. Median values are indicated and error bars indicate s.d. P values were calculated using two-tailed Student’s t -test. d Indicated KBM-7 cells were plated in the presence or absence of doxycycline. At indicated time points, cell numbers were assessed. Error bars indicate s.d. of three independent experiments. e Workflow of genetic screen in near-haploid KBM-7 cells. f Insertions sites identified in gene-trap mutagenized KBM-7 cells which survived doxycycline-induced BRCA2 inactivation (shBRCA2 #2). Dots represent individual genes. The frequency of insertions mapped to a specific gene is plotted on the x -axis. The ratio of gene-traps inserted in the sense orientation over total insertions are plotted on the y -axis. Genes that are neutral in conferring a survival advantage in BRCA2-depleted cells have a sense/total insertion ratio of 0.5 (indicated by the red dashed line). Insertion site ratios > 0.5 represent genes that when mutated confer survival benefit to BRCA2-depleted cells. Throughout the figure, * P
    Figure Legend Snippet: Genetic determinants of cellular survival in BRCA2-depleted KBM-7 cells. a KBM-7 cells were stably transduced with indicated doxycyline-inducible shRNA vectors. Cells were treated with doxycycline for 3 or 5 days and lysates were immunoblotted for BRCA2 and Actin. b Quantification of the percentage of cells with ≥10 RAD51 foci after 5 Gy irradiation. KBM-7 cells harboring indicated shRNAs were treated with doxycycline for 96 h prior to irradiation. Approximately 100 cells were scored per condition per replicate. Error bars indicate s.d. of two independent experiments. P values were calculated using two-tailed Student’s t -test. c KBM-7 cells expressing shBRCA2 #2 were processed for DNA fiber analysis after treatment with doxycycline for 96 h. Cells were then incubated with CldU (25 μM) for 40 min to label replication tracks and subsequently treated with HU (2 mM) for 4 h. CldU track lengths are plotted for ±500 fibers per condition. Median values are indicated and error bars indicate s.d. P values were calculated using two-tailed Student’s t -test. d Indicated KBM-7 cells were plated in the presence or absence of doxycycline. At indicated time points, cell numbers were assessed. Error bars indicate s.d. of three independent experiments. e Workflow of genetic screen in near-haploid KBM-7 cells. f Insertions sites identified in gene-trap mutagenized KBM-7 cells which survived doxycycline-induced BRCA2 inactivation (shBRCA2 #2). Dots represent individual genes. The frequency of insertions mapped to a specific gene is plotted on the x -axis. The ratio of gene-traps inserted in the sense orientation over total insertions are plotted on the y -axis. Genes that are neutral in conferring a survival advantage in BRCA2-depleted cells have a sense/total insertion ratio of 0.5 (indicated by the red dashed line). Insertion site ratios > 0.5 represent genes that when mutated confer survival benefit to BRCA2-depleted cells. Throughout the figure, * P

    Techniques Used: Stable Transfection, Transduction, shRNA, Irradiation, Two Tailed Test, Expressing, Incubation

    9) Product Images from "Human teneurin-1 is a direct target of the homeobox transcription factor EMX2 at a novel alternate promoter"

    Article Title: Human teneurin-1 is a direct target of the homeobox transcription factor EMX2 at a novel alternate promoter

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-11-35

    Determination of teneurin-1 transcription start sites . BLAT alignments to the corresponding RefSeq sequences of 2 representative clones per species obtained by 5' rapid amplification of cDNA ends are shown. The sequences of the clones are given in Table 1. The human clone h1_cl_1, the mouse clone m1_cl_1 and the chicken clone c1_cl_1 start with the annotated first exon containing the ATG translation start, whereas clones h1_cl_2, m1_cl_2 and c1_cl_2 contain up to three further non-coding exons. Based on the presence of two different transcription start sites, two alternate promoters (grey shaded areas) are postulated to control the expression of the two types of transcripts. Promoter 1 resides upstream of the first coding exon, whereas Promoter 2 is located about 100-200 kb upstream depending on the species analyzed.
    Figure Legend Snippet: Determination of teneurin-1 transcription start sites . BLAT alignments to the corresponding RefSeq sequences of 2 representative clones per species obtained by 5' rapid amplification of cDNA ends are shown. The sequences of the clones are given in Table 1. The human clone h1_cl_1, the mouse clone m1_cl_1 and the chicken clone c1_cl_1 start with the annotated first exon containing the ATG translation start, whereas clones h1_cl_2, m1_cl_2 and c1_cl_2 contain up to three further non-coding exons. Based on the presence of two different transcription start sites, two alternate promoters (grey shaded areas) are postulated to control the expression of the two types of transcripts. Promoter 1 resides upstream of the first coding exon, whereas Promoter 2 is located about 100-200 kb upstream depending on the species analyzed.

    Techniques Used: Clone Assay, Rapid Amplification of cDNA Ends, Expressing

    10) Product Images from "Butyrate inhibits pro-proliferative miR-92a by diminishing c-Myc-induced miR-17-92a cluster transcription in human colon cancer cells"

    Article Title: Butyrate inhibits pro-proliferative miR-92a by diminishing c-Myc-induced miR-17-92a cluster transcription in human colon cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/s12943-015-0450-x

    c-Myc-induced over-expression of pri-miR-17-92a was attenuated by butyrate treatment. HCT116 human colon cancer cells were infected with adenovirus carrying the c-Myc coding sequence driven by a CMV promoter to overexpress c-Myc (cMyc) or a null virus vehicle (Veh) for 48 h before analysis. Cells without virus infection were analyzed as control (Cont). The effects of treatment with 2 mM butyrate was assessed using three experimental designs: a no butyrate treatment, b 24-h treatment prior to cell harvest, and c ) 56-h treatment starting 8 h prior to virus infection. Untreated cells (UNTD) were assessed as control. Protein levels of c-Myc and β-actin were analyzed by immunoblotting. Images shown are representative of three individual experiments. d The abundance of pri-miR-17-92a was measured using qPCR. Bars represent means ± SEM. * P
    Figure Legend Snippet: c-Myc-induced over-expression of pri-miR-17-92a was attenuated by butyrate treatment. HCT116 human colon cancer cells were infected with adenovirus carrying the c-Myc coding sequence driven by a CMV promoter to overexpress c-Myc (cMyc) or a null virus vehicle (Veh) for 48 h before analysis. Cells without virus infection were analyzed as control (Cont). The effects of treatment with 2 mM butyrate was assessed using three experimental designs: a no butyrate treatment, b 24-h treatment prior to cell harvest, and c ) 56-h treatment starting 8 h prior to virus infection. Untreated cells (UNTD) were assessed as control. Protein levels of c-Myc and β-actin were analyzed by immunoblotting. Images shown are representative of three individual experiments. d The abundance of pri-miR-17-92a was measured using qPCR. Bars represent means ± SEM. * P

    Techniques Used: Over Expression, Infection, Sequencing, Real-time Polymerase Chain Reaction

    Over-expression of miR-92a reverses butyrate-induced effects on colon cancer cell proliferation and apoptosis. HCT116 human colon cancer cells were transfected with miR-92a mimetics or control miRNA (miR-C) using Lipofectamine 2000 and then treated with 2 mM butyrate for 24 h prior to harvest. Cells without butyrate treatment (UNTD) or treated with Lipofectamine 2000 (Lipo) were analyzed as controls. a Protein levels of total caspase-3, cleaved caspase-3 and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. b Normalized densitometric values of cleaved caspase-3 protein levels. c Relative cell count after miR-92a transfection and butyrate treatment. Cells were trypsinized and counted using a hemocytometer. Values shown are relative to the UNTD sample. d Percentage of positive cells in TUNEL staining. e The images of TUNEL staining shown are representative of three individual experiments. Positive cells stain brown. Results shown are means ± SEM. * P
    Figure Legend Snippet: Over-expression of miR-92a reverses butyrate-induced effects on colon cancer cell proliferation and apoptosis. HCT116 human colon cancer cells were transfected with miR-92a mimetics or control miRNA (miR-C) using Lipofectamine 2000 and then treated with 2 mM butyrate for 24 h prior to harvest. Cells without butyrate treatment (UNTD) or treated with Lipofectamine 2000 (Lipo) were analyzed as controls. a Protein levels of total caspase-3, cleaved caspase-3 and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. b Normalized densitometric values of cleaved caspase-3 protein levels. c Relative cell count after miR-92a transfection and butyrate treatment. Cells were trypsinized and counted using a hemocytometer. Values shown are relative to the UNTD sample. d Percentage of positive cells in TUNEL staining. e The images of TUNEL staining shown are representative of three individual experiments. Positive cells stain brown. Results shown are means ± SEM. * P

    Techniques Used: Over Expression, Transfection, Cell Counting, TUNEL Assay, Staining

    HDAC inhibitors, SAHA and valproic acid, suppressed expression of pri-miR-17-92a, miR-92a and c-Myc in colon cancer cells. HCT116 human colon cancer cells were treated with 0.5 uM SAHA or 1 mM valproic acid for 24 h before harvesting. The abundance of a ) pri-miR-17-92a and b ) miR-92a was measured using qPCR. Bars represent means ± SEM. n = 4. c Protein levels of c-Myc and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. d Relative c-Myc protein levels were measured by densitometry. Bars represent means ± SEM. n = 3
    Figure Legend Snippet: HDAC inhibitors, SAHA and valproic acid, suppressed expression of pri-miR-17-92a, miR-92a and c-Myc in colon cancer cells. HCT116 human colon cancer cells were treated with 0.5 uM SAHA or 1 mM valproic acid for 24 h before harvesting. The abundance of a ) pri-miR-17-92a and b ) miR-92a was measured using qPCR. Bars represent means ± SEM. n = 4. c Protein levels of c-Myc and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. d Relative c-Myc protein levels were measured by densitometry. Bars represent means ± SEM. n = 3

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Over-expression of miR-92a attenuates butyrate-induced p57 expression by blocking p57 translation. a HCT116 human colon cancer cells were treated with 2 mM butyrate for up to 24 h. Cells were harvested for analysis at 1, 2, 4, 8, 16, and 24 h after treatment. Protein levels of p57and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. b HCT116 cells were transfected with miR-92a mimetics or control miRNA (miR-C) using Lipofectamine 2000 then treated with 2 mM butyrate for 24 h prior to harvest. Cells without butyrate treatment (UNTD) or treated with Lipofectamine 2000 (Lipo) were analyzed as controls. The abundance of miR-92a was measured using qPCR. c p57 mRNA levels were measured using qPCR. d Protein levels of p57 and β-actin were analyzed by immunoblotting. The image shown is representative of four individual experiments. e Normalized densitometric values of p57 protein levels. Bars represent means ± SEM. * P
    Figure Legend Snippet: Over-expression of miR-92a attenuates butyrate-induced p57 expression by blocking p57 translation. a HCT116 human colon cancer cells were treated with 2 mM butyrate for up to 24 h. Cells were harvested for analysis at 1, 2, 4, 8, 16, and 24 h after treatment. Protein levels of p57and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. b HCT116 cells were transfected with miR-92a mimetics or control miRNA (miR-C) using Lipofectamine 2000 then treated with 2 mM butyrate for 24 h prior to harvest. Cells without butyrate treatment (UNTD) or treated with Lipofectamine 2000 (Lipo) were analyzed as controls. The abundance of miR-92a was measured using qPCR. c p57 mRNA levels were measured using qPCR. d Protein levels of p57 and β-actin were analyzed by immunoblotting. The image shown is representative of four individual experiments. e Normalized densitometric values of p57 protein levels. Bars represent means ± SEM. * P

    Techniques Used: Over Expression, Expressing, Blocking Assay, Transfection, Real-time Polymerase Chain Reaction

    Time course of changes in expression of miR-92a, c-Myc, Drosha and p57 following butyrate treatment. HCT116 human colon cancer cells were treated with 2 mM butyrate for up to 24 h. Cells were harvested for analysis at 1, 2, 4, 8, 16, and 24 h after treatment. a The abundance of pri-miR-17-92a, pre-miR-92a, and mature miR-92a was measured using qPCR. Bars represent means ± SEM. n = 3. b Protein levels of c-Myc, Drosha and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. c The abundance of c-Myc mRNA was measured using qPCR. Bars represent means ± SEM. n = 3
    Figure Legend Snippet: Time course of changes in expression of miR-92a, c-Myc, Drosha and p57 following butyrate treatment. HCT116 human colon cancer cells were treated with 2 mM butyrate for up to 24 h. Cells were harvested for analysis at 1, 2, 4, 8, 16, and 24 h after treatment. a The abundance of pri-miR-17-92a, pre-miR-92a, and mature miR-92a was measured using qPCR. Bars represent means ± SEM. n = 3. b Protein levels of c-Myc, Drosha and β-actin were analyzed by immunoblotting. The image shown is representative of three individual experiments. c The abundance of c-Myc mRNA was measured using qPCR. Bars represent means ± SEM. n = 3

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    11) Product Images from "Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition"

    Article Title: Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition

    Journal: Genes & Development

    doi: 10.1101/gad.2028911

    Dazl mRNA translation and protein accumulation during maturation. ( A ) qPCR analysis of Dazl mRNA in polysome and subpolysome/RNP fractions and whole-cell lysates during oocyte maturation. Each point is the mean ± SEM of three to five biologically different samples. The microarray data are included for comparison. ( B ) DAZL protein accumulation at the oocyte-to-zygote transition. A representative experiment of the four experiments performed is reported. Accumulation of CPEB1 and α-tubulin was used as a control. ( C ) Quantification of the intensity of the DAZL immunoreactive band from different experiments (mean ± SEM; N = 4).
    Figure Legend Snippet: Dazl mRNA translation and protein accumulation during maturation. ( A ) qPCR analysis of Dazl mRNA in polysome and subpolysome/RNP fractions and whole-cell lysates during oocyte maturation. Each point is the mean ± SEM of three to five biologically different samples. The microarray data are included for comparison. ( B ) DAZL protein accumulation at the oocyte-to-zygote transition. A representative experiment of the four experiments performed is reported. Accumulation of CPEB1 and α-tubulin was used as a control. ( C ) Quantification of the intensity of the DAZL immunoreactive band from different experiments (mean ± SEM; N = 4).

    Techniques Used: Real-time Polymerase Chain Reaction, Microarray

    Spindle localization and defective spindle formation in Dazl-deficient oocytes. ( A ) Representative patterns of DNA, tubulin, and DAZL localization in oocytes injected with control MOs, Dazl MOs, or Dazl MOs plus hDAZL protein. ( B ) Quantification of spindle phenotypes in oocytes injected with Dazl MOs in the presence or absence of hDAZL protein. ( C ) Details of spindle localization of DAZL. ( D ) qPCR of Tpx2 mRNA in the polysome and subpolysome/RNP fractions and total transcripts are shown. ( E ) TPX2 expression during meiosis is dependent on Dazl. Oocytes were injected with control MOs or Dazl MOs and incubated in meiotic arresting medium for 20 h. Oocytes were then cultured in medium that allows maturation overnight and collected for Western blot analysis using antibodies against TPX2 and α-tubulin. A representative experiment of the three experiments performed is reported. ( F ) Dazl is required for translation of a RL reporter fused to the 3′UTR of Tpx2 . Luciferase activity was measured in extracts from oocytes coinjected with Tpx2-RL and Dazl MOs or control MOs. The data are mean ± SEM of three independent experiments.
    Figure Legend Snippet: Spindle localization and defective spindle formation in Dazl-deficient oocytes. ( A ) Representative patterns of DNA, tubulin, and DAZL localization in oocytes injected with control MOs, Dazl MOs, or Dazl MOs plus hDAZL protein. ( B ) Quantification of spindle phenotypes in oocytes injected with Dazl MOs in the presence or absence of hDAZL protein. ( C ) Details of spindle localization of DAZL. ( D ) qPCR of Tpx2 mRNA in the polysome and subpolysome/RNP fractions and total transcripts are shown. ( E ) TPX2 expression during meiosis is dependent on Dazl. Oocytes were injected with control MOs or Dazl MOs and incubated in meiotic arresting medium for 20 h. Oocytes were then cultured in medium that allows maturation overnight and collected for Western blot analysis using antibodies against TPX2 and α-tubulin. A representative experiment of the three experiments performed is reported. ( F ) Dazl is required for translation of a RL reporter fused to the 3′UTR of Tpx2 . Luciferase activity was measured in extracts from oocytes coinjected with Tpx2-RL and Dazl MOs or control MOs. The data are mean ± SEM of three independent experiments.

    Techniques Used: Injection, Real-time Polymerase Chain Reaction, Expressing, Incubation, Cell Culture, Western Blot, Luciferase, Activity Assay

    12) Product Images from "The combination of the prodrugs perforin-CEBPD and perforin-granzyme B efficiently enhances the activation of caspase signaling and kills prostate cancer"

    Article Title: The combination of the prodrugs perforin-CEBPD and perforin-granzyme B efficiently enhances the activation of caspase signaling and kills prostate cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.106

    The SUZ12/EZH2-associated histone K27 tri-methylation attenuates DHT-induced CEBPD transcription in PrCa cells. ( a ) The expression of CEBPD and AR in LNCaP 104-S and 104-R1 cells. RT-PCR and western blot assays were conducted to detect the expression of CEBPD and AR. The LNCaP 104-S (androgen sensitive) and 104-R1 (androgen insensitive) cells were cultured in DMEM supplemented with FBS without charcoal treatment. ( b ) The endogenous EZH2, E2F1, SUZ12, DNMT1, DNMT3a, DNMT3b and H3K27me3 expression in LNCaP 104-S and 104-R1 cells. Western blot assays were conducted to detect the expression of protein levels. The LNCaP 104-S (androgen sensitive) and 104-R1 (androgen insensitive) cells were cultured in DMEM supplemented with charcoal-treated FBS. ( c ) The DNA methyltransferase inhibitor 5-AzadC does not reverse the transcription of CEBPD in LNCaP 104-R1 cells. RT-PCR and western blot assays were conducted to detect the expression of CEBPD in response to 5-AzadC in LNCaP 104-S or 104-R1 cells. Two independent experiments were conducted and showed a similar pattern. ( d ) DHT-stimulated CEBPD transcription was enhanced in cells that were treated with the histone methyltransferase inhibitor DZNep. LNCaP 104S and 104-R1 cells incubated with 1 μ M DZNep. After 18 h of treatment, cells were treated with 10 nM DHT and the cell lysates were harvested at the indicated time to perform RT-PCR with specific primers. Three independent experiments were conducted and statistically plotted. Columns, the average of three independent measurements; bars, mean±S.D. (** P
    Figure Legend Snippet: The SUZ12/EZH2-associated histone K27 tri-methylation attenuates DHT-induced CEBPD transcription in PrCa cells. ( a ) The expression of CEBPD and AR in LNCaP 104-S and 104-R1 cells. RT-PCR and western blot assays were conducted to detect the expression of CEBPD and AR. The LNCaP 104-S (androgen sensitive) and 104-R1 (androgen insensitive) cells were cultured in DMEM supplemented with FBS without charcoal treatment. ( b ) The endogenous EZH2, E2F1, SUZ12, DNMT1, DNMT3a, DNMT3b and H3K27me3 expression in LNCaP 104-S and 104-R1 cells. Western blot assays were conducted to detect the expression of protein levels. The LNCaP 104-S (androgen sensitive) and 104-R1 (androgen insensitive) cells were cultured in DMEM supplemented with charcoal-treated FBS. ( c ) The DNA methyltransferase inhibitor 5-AzadC does not reverse the transcription of CEBPD in LNCaP 104-R1 cells. RT-PCR and western blot assays were conducted to detect the expression of CEBPD in response to 5-AzadC in LNCaP 104-S or 104-R1 cells. Two independent experiments were conducted and showed a similar pattern. ( d ) DHT-stimulated CEBPD transcription was enhanced in cells that were treated with the histone methyltransferase inhibitor DZNep. LNCaP 104S and 104-R1 cells incubated with 1 μ M DZNep. After 18 h of treatment, cells were treated with 10 nM DHT and the cell lysates were harvested at the indicated time to perform RT-PCR with specific primers. Three independent experiments were conducted and statistically plotted. Columns, the average of three independent measurements; bars, mean±S.D. (** P

    Techniques Used: Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Incubation

    CEBPD activates the transcription of CASP8 but not CASP3 in PrCa cells. ( a ) CEBPD activates the transcription of CASP8 but not CASP3 . An RT-PCR assay was conducted using total RNAs harvested from transfectants with CEBPD or control expression vectors in PC-3 cells. Numbers below the images are the levels normalized to the GAPDH mRNA. ( b ) CEBPD induces the protein levels of procaspase 8 and procaspase 3 and their activations in LNCaP and PC-3 cells. Stable CEBPD-inducible cells were incubated with 100 μ M ZnSO 4 and total lysates were harvested at the indicated time for western blot analysis with the indicated antibodies. ( c ) CEBPD contributes to CASP8 reporter activity. The CASP8 reporters were independently co-transfected with CEBPD expression vectors in LNCaP or PC-3 cells. After 16 h of transfection, the cell lysates were harvested for a luciferase assay. The diagram represents the putative CEBPD-binding sites in the CASP8 promoter region. Columns, the average of three independent measurements in duplicate; bars, mean±S.D. (* P
    Figure Legend Snippet: CEBPD activates the transcription of CASP8 but not CASP3 in PrCa cells. ( a ) CEBPD activates the transcription of CASP8 but not CASP3 . An RT-PCR assay was conducted using total RNAs harvested from transfectants with CEBPD or control expression vectors in PC-3 cells. Numbers below the images are the levels normalized to the GAPDH mRNA. ( b ) CEBPD induces the protein levels of procaspase 8 and procaspase 3 and their activations in LNCaP and PC-3 cells. Stable CEBPD-inducible cells were incubated with 100 μ M ZnSO 4 and total lysates were harvested at the indicated time for western blot analysis with the indicated antibodies. ( c ) CEBPD contributes to CASP8 reporter activity. The CASP8 reporters were independently co-transfected with CEBPD expression vectors in LNCaP or PC-3 cells. After 16 h of transfection, the cell lysates were harvested for a luciferase assay. The diagram represents the putative CEBPD-binding sites in the CASP8 promoter region. Columns, the average of three independent measurements in duplicate; bars, mean±S.D. (* P

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

    CEBPD is an androgen-responsive gene. ( a ) The level of CEBPD is associated with the abundance of the AR. RT-PCR analysis and a western blot were conducted to detect the expression of CEBPD and AR, as indicated in LNCaP and PC-3 cells in DMEM supplemented with FBS (without charcoal treatment). ( b ) DHT stimulates the expression of CEBPD in LNCaP cells but not in PC-3 cells. RT-PCR analysis and western blot were conducted to detect the expression of CEBPD and AR at the indicated time. The LNCaP and PC-3 cells were cultured in DMEM supplemented with charcoal-treated FBS. ( c ) DHT activates the activity of a CEBPD reporter in LNCaP cells. LNCaP cells were transfected with the reporter pGL2-Basic vector carrying fragments of the CEBPD promoter (−1720 to +18 bp, −1000 to +18 bp or −360 to +18 bp). After 16 h of transfection, the transfectants were treated with 10 nM DHT for 6 h and the cell lysates were harvested for luciferase assay. Columns, the average of three independent experiments in duplicate measurements; bars, mean±S.D. (** P
    Figure Legend Snippet: CEBPD is an androgen-responsive gene. ( a ) The level of CEBPD is associated with the abundance of the AR. RT-PCR analysis and a western blot were conducted to detect the expression of CEBPD and AR, as indicated in LNCaP and PC-3 cells in DMEM supplemented with FBS (without charcoal treatment). ( b ) DHT stimulates the expression of CEBPD in LNCaP cells but not in PC-3 cells. RT-PCR analysis and western blot were conducted to detect the expression of CEBPD and AR at the indicated time. The LNCaP and PC-3 cells were cultured in DMEM supplemented with charcoal-treated FBS. ( c ) DHT activates the activity of a CEBPD reporter in LNCaP cells. LNCaP cells were transfected with the reporter pGL2-Basic vector carrying fragments of the CEBPD promoter (−1720 to +18 bp, −1000 to +18 bp or −360 to +18 bp). After 16 h of transfection, the transfectants were treated with 10 nM DHT for 6 h and the cell lysates were harvested for luciferase assay. Columns, the average of three independent experiments in duplicate measurements; bars, mean±S.D. (** P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Cell Culture, Activity Assay, Transfection, Plasmid Preparation, Luciferase

    13) Product Images from "An LRR/Malectin Receptor-Like Kinase Mediates Resistance to Non-adapted and Adapted Powdery Mildew Fungi in Barley and Wheat"

    Article Title: An LRR/Malectin Receptor-Like Kinase Mediates Resistance to Non-adapted and Adapted Powdery Mildew Fungi in Barley and Wheat

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.01836

    Effect of transiently expressing LEMK1 genes in barley and wheat on quantitative host resistance against adapted powdery mildew fungi. Barley and wheat orthologs of LEMK1 were transiently expressed in bombarded barley and wheat leaf segments, followed by inoculation with the adapted powdery mildew fungi Bgh and Bgt , respectively. In the case of HvLEMK1 , the gene was expressed under the control of its own promoter by bombarding an HvLEMK1 -containing BAC clone, and under the control of the CaMV 35S promoter as genomic BAC sub-clone (pIPKTA09_HvLEMK1_genomic) or full-length cDNA clone (pIPKTA09_HvLEMK1_cDNA). The corresponding wheat orthologs TaLEMK1_1 and TaLEMK1_2 were only expressed as full-length cDNA clones under the control of the CaMV 35S promoter (pIPKTA09_TaLEMK1_1_cDNA; pIPKTA09_TaLEMK1_2_cDNA). pGY1_TaPrx103 for over-expression of a pathogen-induced wheat class III peroxidase ( Schweizer, 2008 ) and BAC632F23 containing no annotated genes served as positive and negative controls, respectively. SI values were normalized to the empty over-expression vector pIPKTA09 and log(2) transformed. Mean values ± SEM of 5–10 independent experiments (pGY1_TaPrx103 in barley, 21 experiments) are shown. ∗ p
    Figure Legend Snippet: Effect of transiently expressing LEMK1 genes in barley and wheat on quantitative host resistance against adapted powdery mildew fungi. Barley and wheat orthologs of LEMK1 were transiently expressed in bombarded barley and wheat leaf segments, followed by inoculation with the adapted powdery mildew fungi Bgh and Bgt , respectively. In the case of HvLEMK1 , the gene was expressed under the control of its own promoter by bombarding an HvLEMK1 -containing BAC clone, and under the control of the CaMV 35S promoter as genomic BAC sub-clone (pIPKTA09_HvLEMK1_genomic) or full-length cDNA clone (pIPKTA09_HvLEMK1_cDNA). The corresponding wheat orthologs TaLEMK1_1 and TaLEMK1_2 were only expressed as full-length cDNA clones under the control of the CaMV 35S promoter (pIPKTA09_TaLEMK1_1_cDNA; pIPKTA09_TaLEMK1_2_cDNA). pGY1_TaPrx103 for over-expression of a pathogen-induced wheat class III peroxidase ( Schweizer, 2008 ) and BAC632F23 containing no annotated genes served as positive and negative controls, respectively. SI values were normalized to the empty over-expression vector pIPKTA09 and log(2) transformed. Mean values ± SEM of 5–10 independent experiments (pGY1_TaPrx103 in barley, 21 experiments) are shown. ∗ p

    Techniques Used: Expressing, BAC Assay, Clone Assay, Over Expression, Plasmid Preparation, Transformation Assay

    14) Product Images from "Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum)"

    Article Title: Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156319

    Gene expression and punicalagin accumulation in overexpression (A) and RNAi knockdown (B) hairy root lines of UGT84A23 or UGT84A24 . Changes in gene expression are represented on the primary y-axis as fold change relative to the vector transformed control, pK7WG2D-1 for overexpression lines and pHG8-1 for RNAi knockdown lines. Gene expression data presented are mean ± SD of three technical replicates for each line. Punicalagin accumulation (α and β isomers combined) is represented on the secondary y-axis as peak areas at 378 nm.
    Figure Legend Snippet: Gene expression and punicalagin accumulation in overexpression (A) and RNAi knockdown (B) hairy root lines of UGT84A23 or UGT84A24 . Changes in gene expression are represented on the primary y-axis as fold change relative to the vector transformed control, pK7WG2D-1 for overexpression lines and pHG8-1 for RNAi knockdown lines. Gene expression data presented are mean ± SD of three technical replicates for each line. Punicalagin accumulation (α and β isomers combined) is represented on the secondary y-axis as peak areas at 378 nm.

    Techniques Used: Expressing, Over Expression, Plasmid Preparation, Transformation Assay

    Reduced punicalagin accumulation and increased galloyl glucoside production in UGT84A23 and UGT84A24 double RNAi knockdown hairy root lines. (A) Gene expression and punicalagin accumulation in vector control and UGT84A23 and UGT84A24 double RNAi knockdown lines. Changes in gene expression are represented on the primary y-axis as fold change relative to the vector control pHG8-1. Gene expression data presented are mean ± SD of three technical replicates for each line. Punicalagin accumulation (α and β isomers combined) is represented on the secondary y-axis as peak areas at 378 nm. The RNAi constructs were derived from a chimera of the coding sequences (CDS) or the 3’ untranslated region (3’ UTR) of UGT84A23 and UGT84A24 . (B) Overlay of HPLC chromatograms from a representative vector transformed control (pHG8-1) and a representative double knockdown line of UGT84A23 and UGT84A24 (3’ UTR-2). Peaks that are only present in the double knockdown lines and those that show varied accumulation in vector control and double knockdown lines are indicated. (C) MS and MS/MS analyses of peaks indicated in (B). HPLC retention times and λ max of standards and the respective peaks in the 3’ UTR-2 hairy root line are also shown.
    Figure Legend Snippet: Reduced punicalagin accumulation and increased galloyl glucoside production in UGT84A23 and UGT84A24 double RNAi knockdown hairy root lines. (A) Gene expression and punicalagin accumulation in vector control and UGT84A23 and UGT84A24 double RNAi knockdown lines. Changes in gene expression are represented on the primary y-axis as fold change relative to the vector control pHG8-1. Gene expression data presented are mean ± SD of three technical replicates for each line. Punicalagin accumulation (α and β isomers combined) is represented on the secondary y-axis as peak areas at 378 nm. The RNAi constructs were derived from a chimera of the coding sequences (CDS) or the 3’ untranslated region (3’ UTR) of UGT84A23 and UGT84A24 . (B) Overlay of HPLC chromatograms from a representative vector transformed control (pHG8-1) and a representative double knockdown line of UGT84A23 and UGT84A24 (3’ UTR-2). Peaks that are only present in the double knockdown lines and those that show varied accumulation in vector control and double knockdown lines are indicated. (C) MS and MS/MS analyses of peaks indicated in (B). HPLC retention times and λ max of standards and the respective peaks in the 3’ UTR-2 hairy root line are also shown.

    Techniques Used: Expressing, Plasmid Preparation, Construct, Derivative Assay, High Performance Liquid Chromatography, Transformation Assay, Mass Spectrometry

    15) Product Images from "11β-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice"

    Article Title: 11β-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice

    Journal: Endocrinology

    doi: 10.1210/en.2016-1118

    HSD11B1 mRNA is up-regulated in activated human neutrophils. Human neutrophils were isolated from blood of three healthy individuals and treated for 4 hours with 100 ng/mL LPS, prior to RNA extraction and real-time measurement of HSD11B1 mRNA levels. Values are normalized to levels in untreated cells (black bars) and show fold induction of HSD11B1 mRNA after LPS (white bars) for single individuals.
    Figure Legend Snippet: HSD11B1 mRNA is up-regulated in activated human neutrophils. Human neutrophils were isolated from blood of three healthy individuals and treated for 4 hours with 100 ng/mL LPS, prior to RNA extraction and real-time measurement of HSD11B1 mRNA levels. Values are normalized to levels in untreated cells (black bars) and show fold induction of HSD11B1 mRNA after LPS (white bars) for single individuals.

    Techniques Used: Isolation, RNA Extraction

    16) Product Images from "Translocation of an antibody transgene requires AID and occurs by interchromosomal switching to all switch regions except the mu switch region"

    Article Title: Translocation of an antibody transgene requires AID and occurs by interchromosomal switching to all switch regions except the mu switch region

    Journal: European journal of immunology

    doi: 10.1002/eji.201041077

    AID is required for interchromosomal isotype switching. (A) Upper panels: Semi-quantitative PCR/Southern blot assays of the indicated mice immunized with Ars-KLH. Splenocytes were harvested for cDNA synthesis. The cDNAs were diluted as indicated and Cγ transcripts were amplified by PCR. PCR products were subjected to Southern blot analysis and hybridized to a transgene-specific probe (TND) to identify Cγ transcripts that are associated with VV29 VDJ segments (VV29-Cγ). As a loading control and for normalization, β-actin was amplified from cDNA that was diluted at 1:6400, 1:128000, 1:25600. Each set of three lanes represent data from a single mouse; sets 3 and 4 are from two different VV29:AID −/− mice. Lower panel: The band intensities of the Southern blots were quantified, normalized to β-actin (see materials and methods) and the data presented using a log 10 scale. Each bar represents data from a single mouse. Error bars represent standard deviation (SD) from three independent repetitions of the experiments using the same mice. (B) The translocation frequency was determined by stimulating B-cells isolated from the indicated mice with LPS and IL-4 for four days before RNA isolation and RT-PCR for Cγ transcripts. PCR products were subjected to Southern blot analysis and hybridized to a transgene-specific probe (TND) to identify VV29-Cγ transcripts. Amplification of β-actin serves as loading control. Each lane represents data from a single mouse.
    Figure Legend Snippet: AID is required for interchromosomal isotype switching. (A) Upper panels: Semi-quantitative PCR/Southern blot assays of the indicated mice immunized with Ars-KLH. Splenocytes were harvested for cDNA synthesis. The cDNAs were diluted as indicated and Cγ transcripts were amplified by PCR. PCR products were subjected to Southern blot analysis and hybridized to a transgene-specific probe (TND) to identify Cγ transcripts that are associated with VV29 VDJ segments (VV29-Cγ). As a loading control and for normalization, β-actin was amplified from cDNA that was diluted at 1:6400, 1:128000, 1:25600. Each set of three lanes represent data from a single mouse; sets 3 and 4 are from two different VV29:AID −/− mice. Lower panel: The band intensities of the Southern blots were quantified, normalized to β-actin (see materials and methods) and the data presented using a log 10 scale. Each bar represents data from a single mouse. Error bars represent standard deviation (SD) from three independent repetitions of the experiments using the same mice. (B) The translocation frequency was determined by stimulating B-cells isolated from the indicated mice with LPS and IL-4 for four days before RNA isolation and RT-PCR for Cγ transcripts. PCR products were subjected to Southern blot analysis and hybridized to a transgene-specific probe (TND) to identify VV29-Cγ transcripts. Amplification of β-actin serves as loading control. Each lane represents data from a single mouse.

    Techniques Used: Real-time Polymerase Chain Reaction, Southern Blot, Mouse Assay, Amplification, Polymerase Chain Reaction, Standard Deviation, Translocation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    17) Product Images from "BRCA1 Mutation Status and Follicular Fluid Exposure Alters NFκB Signaling and ISGylation in Human Fallopian Tube Epithelial Cells"

    Article Title: BRCA1 Mutation Status and Follicular Fluid Exposure Alters NFκB Signaling and ISGylation in Human Fallopian Tube Epithelial Cells

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.05.005

    Increased BRCA1 expression inhibits expression of NFκB target genes and alters the impact of FF exposure on expression of ISG15 and its interacting partners. (A) Left: Representative Western blot showing BRCA1 protein levels in OE-E6/E7 cells compared to ovarian cancer cell lines. UWB1.289 and UWB1.289+BRCA1 cells were used as a negative and positive control respectively. Right: Representative Western blot and comparison of BRCA1 levels in OE-Mock and OE-BRCA1 cells. Levels of HSP90 are shown as a loading control. Bars represent the mean ± SEM of three independent experiments relative to levels measured in OE-BRCA1 cells. * P =.0075, one-sample t test. (B-E) Comparison of TNFα (B), IL8 (C), ISG15 (D), and HRAS (E) transcript levels in OE-Mock and OE-BRCA1 cells as determined by RT-qPCR. Bars represent the mean ± SEM of three independent experiments relative to levels measured in OE-Mock cells. * P
    Figure Legend Snippet: Increased BRCA1 expression inhibits expression of NFκB target genes and alters the impact of FF exposure on expression of ISG15 and its interacting partners. (A) Left: Representative Western blot showing BRCA1 protein levels in OE-E6/E7 cells compared to ovarian cancer cell lines. UWB1.289 and UWB1.289+BRCA1 cells were used as a negative and positive control respectively. Right: Representative Western blot and comparison of BRCA1 levels in OE-Mock and OE-BRCA1 cells. Levels of HSP90 are shown as a loading control. Bars represent the mean ± SEM of three independent experiments relative to levels measured in OE-BRCA1 cells. * P =.0075, one-sample t test. (B-E) Comparison of TNFα (B), IL8 (C), ISG15 (D), and HRAS (E) transcript levels in OE-Mock and OE-BRCA1 cells as determined by RT-qPCR. Bars represent the mean ± SEM of three independent experiments relative to levels measured in OE-Mock cells. * P

    Techniques Used: Expressing, Western Blot, Positive Control, Quantitative RT-PCR

    18) Product Images from "Characterisation of CDKL5 Transcript Isoforms in Human and Mouse"

    Article Title: Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157758

    RNA-seq and 5’ and 3’-RACE mapping of mouse Cdkl5 transcripts. (A) Upper panels: RNA-seq data from brain (red) and testis (blue) datasets show reads mapping to the 5’ end of Cdkl5 (the y-axis indicates read count across the analysed region). Indicative numbers of RNA-seq reads spanning each exon junction are also shown, indicated by values and dotted lines joining exon boundaries. Middle panels: boxes representing each exon at the 5’ end of the gene are shown, aligned with those in the upper panels. Transcription Start Sites (TSSs) and splice events upstream of exon 2 are indicated. Coding regions are indicated by orange colouring and 5’-UTRs by black colouring. Lower panel: exonic sequences for each first exon are shown. TSSs, confirmed by sequencing of 5’-RACE products, are indicated by boxes; the major TSS is indicated by a solid box, minor TSSs are indicated by hatched boxes. (B) Upper panels: RNA-seq data from brain and testis datasets show reads mapping to the 3’ end of Cdkl5 ; exon boundary-spanning red counts are also shown, as in (A), above. Middle panels: the exon composition and splicing patterns at the 3’ end of each mouse isoform is shown, colouring as in A) above. Lower panel: sequences around each of the three polyadenylation signals and sites (pA) are shown; each was confirmed by sequencing of 3’-RACE mapping.
    Figure Legend Snippet: RNA-seq and 5’ and 3’-RACE mapping of mouse Cdkl5 transcripts. (A) Upper panels: RNA-seq data from brain (red) and testis (blue) datasets show reads mapping to the 5’ end of Cdkl5 (the y-axis indicates read count across the analysed region). Indicative numbers of RNA-seq reads spanning each exon junction are also shown, indicated by values and dotted lines joining exon boundaries. Middle panels: boxes representing each exon at the 5’ end of the gene are shown, aligned with those in the upper panels. Transcription Start Sites (TSSs) and splice events upstream of exon 2 are indicated. Coding regions are indicated by orange colouring and 5’-UTRs by black colouring. Lower panel: exonic sequences for each first exon are shown. TSSs, confirmed by sequencing of 5’-RACE products, are indicated by boxes; the major TSS is indicated by a solid box, minor TSSs are indicated by hatched boxes. (B) Upper panels: RNA-seq data from brain and testis datasets show reads mapping to the 3’ end of Cdkl5 ; exon boundary-spanning red counts are also shown, as in (A), above. Middle panels: the exon composition and splicing patterns at the 3’ end of each mouse isoform is shown, colouring as in A) above. Lower panel: sequences around each of the three polyadenylation signals and sites (pA) are shown; each was confirmed by sequencing of 3’-RACE mapping.

    Techniques Used: RNA Sequencing Assay, Sequencing

    Cdkl5 expression levels during development. (A-E) End-point and quantitative RT-PCR analysis of Cdkl5 transcript isoforms in total RNA isolated from mouse whole brains at different times of embryonic (E13-E20) and postnatal (P1–P45) development. Three animals were analysed at each time-point; one representative sample from each time-point is shown on the gel image. Expression levels in each assay are shown relative the P45 sample. (F) β-actin loading control. (G) Comparative expression levels of the mouse qRT-PCR assays using 2 -ΔCt analysis are shown, where all values are relative to the mCdkl5_1 assay of the P45 adult brain. All qRT-PCR assays were normalised to β-Actin endogenous controls and are shown as dot plots; bars indicate the standard error of the mean.
    Figure Legend Snippet: Cdkl5 expression levels during development. (A-E) End-point and quantitative RT-PCR analysis of Cdkl5 transcript isoforms in total RNA isolated from mouse whole brains at different times of embryonic (E13-E20) and postnatal (P1–P45) development. Three animals were analysed at each time-point; one representative sample from each time-point is shown on the gel image. Expression levels in each assay are shown relative the P45 sample. (F) β-actin loading control. (G) Comparative expression levels of the mouse qRT-PCR assays using 2 -ΔCt analysis are shown, where all values are relative to the mCdkl5_1 assay of the P45 adult brain. All qRT-PCR assays were normalised to β-Actin endogenous controls and are shown as dot plots; bars indicate the standard error of the mean.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation

    RNA-seq and 5’- and 3’-RACE mapping of human CDKL5 transcripts. (A) Upper panels: RNA-seq data from brain (red) and testis (blue) datasets show reads mapping to the 5’ end of CDKL5 (the y-axis indicates read count across the analysed region). Indicative numbers of RNA-seq reads spanning each exon junction are also shown, indicated by values and dotted lines joining exon boundaries. Middle panels: boxes representing each exon at the 5’ end of the gene are shown, aligned with those in the upper panels. Transcription Start Sites (TSSs) and splice even ts upstream of exon 2 are indicated. Coding regions are indicated by cyan colouring and 5’-UTRs by black colouring. Lower panel: exonic sequences for each first exon are shown. TSSs, confirmed by sequencing of 5’-RACE products, are indicated by boxes; the major TSS is indicated by a solid box, minor TSSs are indicated by hatched boxes. (B) Upper panels: RNA-seq data from brain and testis datasets show reads mapping to the 3’ end of CDKL5 ; exon boundary-spanning read counts are also shown, as in (A), above. Middle panels: the exon composition and splicing patterns at the 3’ end of each human isoform is indicated, colouring as in (A) above. Lower panel: sequences around each of the three polyadenylation signals and sites (pA) are shown; each was confirmed by 3’-RACE mapping.
    Figure Legend Snippet: RNA-seq and 5’- and 3’-RACE mapping of human CDKL5 transcripts. (A) Upper panels: RNA-seq data from brain (red) and testis (blue) datasets show reads mapping to the 5’ end of CDKL5 (the y-axis indicates read count across the analysed region). Indicative numbers of RNA-seq reads spanning each exon junction are also shown, indicated by values and dotted lines joining exon boundaries. Middle panels: boxes representing each exon at the 5’ end of the gene are shown, aligned with those in the upper panels. Transcription Start Sites (TSSs) and splice even ts upstream of exon 2 are indicated. Coding regions are indicated by cyan colouring and 5’-UTRs by black colouring. Lower panel: exonic sequences for each first exon are shown. TSSs, confirmed by sequencing of 5’-RACE products, are indicated by boxes; the major TSS is indicated by a solid box, minor TSSs are indicated by hatched boxes. (B) Upper panels: RNA-seq data from brain and testis datasets show reads mapping to the 3’ end of CDKL5 ; exon boundary-spanning read counts are also shown, as in (A), above. Middle panels: the exon composition and splicing patterns at the 3’ end of each human isoform is indicated, colouring as in (A) above. Lower panel: sequences around each of the three polyadenylation signals and sites (pA) are shown; each was confirmed by 3’-RACE mapping.

    Techniques Used: RNA Sequencing Assay, Sequencing

    19) Product Images from "Self-renewal of CD133hi cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer"

    Article Title: Self-renewal of CD133hi cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer

    Journal: Nature Communications

    doi: 10.1038/ncomms10442

    IL6R blockade increases ER and decreases Stat3/Notch3/CD133 expression restoring HT sensitivity. ( a ) Metastatic tumour cells, isolated from Fulv-resistant metastases (MCF7:FulvR, Supplementary Table 2 ), were cultured in the presence of vehicle (Placebo) or Fulv (10 μM), and growth was determined after 10 days using CalceinAM fluorescent probe: partial (sensitive, HTS) or complete hormone-resistant features (HTR) were generated. Data are reported as mean (fluorescence)±s.e.m. of the last time point of the growth curve (three biological replicates with three technical replicates each). ( b ) Fold increase in the expression of CD133, Stat3, IL6 and Notch3 mRNA using qPCR from HTR and HTS cells treated with Fulv (see a , HTS transcript as reference). ( c ) Representative images of Notch3 expression by IHC of lung metastases from placebo versus TamR mice (scale bar, 20 μm). ( d ) Proliferation (CalceinAM fluorescence) in Stat3 or Notch3 stably silenced HTR cells (shS3, shN3) derived from FulvR-Met and propagated in vitro ( a ) in the presence/absence of Fulv (10 μM). Data are reported as the mean (fluorescence)±s.e.m. of each time point of the growth curve (three biological replicates with three technical replicates each). ( e ) Number-No- of II-MS ( > 100 μm) from FulvR-metastatic cells (MetMS) expressing stable shS3 and shN3 cultured in the presence/absence of Fulv (10 μM) and/or tamoxifen (1 μM, 14 days). ( f ) qPCR of CD133, Notch3 and ER mRNA (fold increase Fulv versus untreated cells) in shS3-FulvR-metastatic cells cultured in the presence/absence of Fulv in vitro (10 μM, 7 days). ( g ) Number of II-MS (diameter > 100 μm) from FACS-isolated (10 3 cells per well) CD44 hi and CD133 hi cells from TamR-Met and FulvR-Met ( Supplementary Table 2 ) treated with Tam (1 μM) or Fulv (10 μM) and tocilizumab (50 μg ml −1 ), either alone or in combination (7 days). ( h ) Western blot analysis and IHC images for ER expression from HTR cancer cell lines (ZR751, BT474) and xenografts (MCF7) following treatment with Fulv (10 μM, 14 days) in presence/absence of tocilizumab (50 μg ml −1 , treatment for 14 days) and after shS3 (scale bar, 50 μm). Data are reported as mean±s.d. of three independent experiments ( n =3; b , e , f , g ). P values (* P
    Figure Legend Snippet: IL6R blockade increases ER and decreases Stat3/Notch3/CD133 expression restoring HT sensitivity. ( a ) Metastatic tumour cells, isolated from Fulv-resistant metastases (MCF7:FulvR, Supplementary Table 2 ), were cultured in the presence of vehicle (Placebo) or Fulv (10 μM), and growth was determined after 10 days using CalceinAM fluorescent probe: partial (sensitive, HTS) or complete hormone-resistant features (HTR) were generated. Data are reported as mean (fluorescence)±s.e.m. of the last time point of the growth curve (three biological replicates with three technical replicates each). ( b ) Fold increase in the expression of CD133, Stat3, IL6 and Notch3 mRNA using qPCR from HTR and HTS cells treated with Fulv (see a , HTS transcript as reference). ( c ) Representative images of Notch3 expression by IHC of lung metastases from placebo versus TamR mice (scale bar, 20 μm). ( d ) Proliferation (CalceinAM fluorescence) in Stat3 or Notch3 stably silenced HTR cells (shS3, shN3) derived from FulvR-Met and propagated in vitro ( a ) in the presence/absence of Fulv (10 μM). Data are reported as the mean (fluorescence)±s.e.m. of each time point of the growth curve (three biological replicates with three technical replicates each). ( e ) Number-No- of II-MS ( > 100 μm) from FulvR-metastatic cells (MetMS) expressing stable shS3 and shN3 cultured in the presence/absence of Fulv (10 μM) and/or tamoxifen (1 μM, 14 days). ( f ) qPCR of CD133, Notch3 and ER mRNA (fold increase Fulv versus untreated cells) in shS3-FulvR-metastatic cells cultured in the presence/absence of Fulv in vitro (10 μM, 7 days). ( g ) Number of II-MS (diameter > 100 μm) from FACS-isolated (10 3 cells per well) CD44 hi and CD133 hi cells from TamR-Met and FulvR-Met ( Supplementary Table 2 ) treated with Tam (1 μM) or Fulv (10 μM) and tocilizumab (50 μg ml −1 ), either alone or in combination (7 days). ( h ) Western blot analysis and IHC images for ER expression from HTR cancer cell lines (ZR751, BT474) and xenografts (MCF7) following treatment with Fulv (10 μM, 14 days) in presence/absence of tocilizumab (50 μg ml −1 , treatment for 14 days) and after shS3 (scale bar, 50 μm). Data are reported as mean±s.d. of three independent experiments ( n =3; b , e , f , g ). P values (* P

    Techniques Used: Expressing, Isolation, Cell Culture, Generated, Fluorescence, Real-time Polymerase Chain Reaction, Immunohistochemistry, Mouse Assay, Stable Transfection, Derivative Assay, In Vitro, Mass Spectrometry, FACS, Western Blot

    20) Product Images from "Creation of the two isoforms of rodent NKG2D was driven by a B1 retrotransposon insertion"

    Article Title: Creation of the two isoforms of rodent NKG2D was driven by a B1 retrotransposon insertion

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp174

    Detection of polymerase II promoter in SINE elements. ( A ) RLM-5′RACE of mouse Nkg2d . Sequence shown corresponds to chr6:129 572 548–29 572 772 of the mouse genome from UCSC Genome Browser (mm9 assembly). Underlined sequence represents the mB1 element inserted in front of exon 1b. Single stars mark transcription start sites found in C57BL/6 mouse spleen. Stacked double stars mark transcription start sites found in LNK mouse cell line. ATG1 start codon for mNKG2D-L is in brackets. Splice donor site of exon 1b is denoted with an arrow. ( B ) Detection of Nkg2d splice forms in LNK, RNK16, EL4 and NIH 3T3 cell lines with RT–PCR. The mouse exon–intron structure of Nkg2d is shown with exons labelled in roman numerals. Primers used are shown as arrowheads. ( C ) 5′RACE of rat Nkg2d . Sequence shown corresponds to chr4:166 923 873–166 924 142 of the rat genome from UCSC Genome Browser (rn4 assembly). Single stars mark transcription start sites found in Sprague–Dawley rat spleen. Underlined sequence represents the rB1 element. ATG1 start codon for rNKG2D-L is in brackets. Splice donor site of exon 1b is denoted with an arrow. ( D ) Luciferase reporter assay of SINE elements. Fragments including the rodent SINEs and 391-bp upstream of the mouse SINE were cloned into pGL4B and assayed using a dual-luciferase system. Promoter activity was normalized to co-transfected Renilla luciferase activity and calculated as fold above pGL4B. Data indicate mean (±SD) of three independent experiments.
    Figure Legend Snippet: Detection of polymerase II promoter in SINE elements. ( A ) RLM-5′RACE of mouse Nkg2d . Sequence shown corresponds to chr6:129 572 548–29 572 772 of the mouse genome from UCSC Genome Browser (mm9 assembly). Underlined sequence represents the mB1 element inserted in front of exon 1b. Single stars mark transcription start sites found in C57BL/6 mouse spleen. Stacked double stars mark transcription start sites found in LNK mouse cell line. ATG1 start codon for mNKG2D-L is in brackets. Splice donor site of exon 1b is denoted with an arrow. ( B ) Detection of Nkg2d splice forms in LNK, RNK16, EL4 and NIH 3T3 cell lines with RT–PCR. The mouse exon–intron structure of Nkg2d is shown with exons labelled in roman numerals. Primers used are shown as arrowheads. ( C ) 5′RACE of rat Nkg2d . Sequence shown corresponds to chr4:166 923 873–166 924 142 of the rat genome from UCSC Genome Browser (rn4 assembly). Single stars mark transcription start sites found in Sprague–Dawley rat spleen. Underlined sequence represents the rB1 element. ATG1 start codon for rNKG2D-L is in brackets. Splice donor site of exon 1b is denoted with an arrow. ( D ) Luciferase reporter assay of SINE elements. Fragments including the rodent SINEs and 391-bp upstream of the mouse SINE were cloned into pGL4B and assayed using a dual-luciferase system. Promoter activity was normalized to co-transfected Renilla luciferase activity and calculated as fold above pGL4B. Data indicate mean (±SD) of three independent experiments.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Reporter Assay, Clone Assay, Activity Assay, Transfection

    Transient knockdown reveals requirement of GABP for full expression of Nkg2d-S but not Nkg2d-L . LNK cells were treated with GABPα or negative control Accell siRNA. Transcript levels of Gabpα , Nkg2d-S and Nkg2d-L were assayed by real-time RT–PCR and normalized to the levels of Gapdh . mRNA levels from untreated cells (incubated with carrier media but no siRNA) were set to one. Results are the mean (±SD) of three independent experiments. * P = 0.03 (Student's t -test).
    Figure Legend Snippet: Transient knockdown reveals requirement of GABP for full expression of Nkg2d-S but not Nkg2d-L . LNK cells were treated with GABPα or negative control Accell siRNA. Transcript levels of Gabpα , Nkg2d-S and Nkg2d-L were assayed by real-time RT–PCR and normalized to the levels of Gapdh . mRNA levels from untreated cells (incubated with carrier media but no siRNA) were set to one. Results are the mean (±SD) of three independent experiments. * P = 0.03 (Student's t -test).

    Techniques Used: Expressing, Negative Control, Quantitative RT-PCR, Incubation

    21) Product Images from "HAX-1: A Novel P-Body Protein"

    Article Title: HAX-1: A Novel P-Body Protein

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2014.2657

    HAX-1 silencing affects vimentin expression levels. (A) Relative vimentin mRNA levels obtained by quantitative PCR for untreated and arsenite-treated cells with silenced HAX-1 (miR-HAX-1) versus silencing control (miR-NEG). Values represent mean±SEM of three biological repeats. Statistical significance assessed by t -test, p -values: 0.015, 0.014 for the control and arsenite-treated cells, respectively. *Indicates statistical significance. (B) Western blot showing vimentin expression levels in the HAX-1 knockdown (miR-HAX-1) and control (miR-NEG) cells. The percentage of expression was calculated using ImageJ, relative to GAPDH content.
    Figure Legend Snippet: HAX-1 silencing affects vimentin expression levels. (A) Relative vimentin mRNA levels obtained by quantitative PCR for untreated and arsenite-treated cells with silenced HAX-1 (miR-HAX-1) versus silencing control (miR-NEG). Values represent mean±SEM of three biological repeats. Statistical significance assessed by t -test, p -values: 0.015, 0.014 for the control and arsenite-treated cells, respectively. *Indicates statistical significance. (B) Western blot showing vimentin expression levels in the HAX-1 knockdown (miR-HAX-1) and control (miR-NEG) cells. The percentage of expression was calculated using ImageJ, relative to GAPDH content.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    22) Product Images from "Neonatal Infection with Species C Adenoviruses Confirmed in Viable Cord Blood Lymphocytes"

    Article Title: Neonatal Infection with Species C Adenoviruses Confirmed in Viable Cord Blood Lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119256

    Detection of the ETV6-RUNX1 fusion transcript by reverse transcription followed by PCR. 200 ng of RNA purified from umbilical vein cord blood lymphocytes was reverse transcribed using random hexamers as primers. (A) The integrity of the RNA in the numbered cord blood samples was confirmed by amplification of a 384 bp sequence spanning the junction of exons 3 and 4 of the β-2-microglobulin gene. Positive controls included RNA from the BJAB and UoCB4 cell lines (+RT). Negative controls included salmon sperm DNA (SS DNA) and the exclusion of reverse transcriptase (NRT). Lanes containing the 100 bp DNA ladder reference are indicated by M. (B) A 200 bp amplicon obtained from a nested PCR assay indicates the presence of the ETV6-RUNX1 transcript. Representative results show the products of three technical replicates from four numbered cord blood samples, salmon sperm DNA as a negative control, and a 10 -2 dilution of the ETV6-RUNX1 -positive UoCB4 cells among BJAB cells as a positive control. Samples in which at least two of three technical replicates were detected from cDNA generated independently in two laboratories and in which no product was observed when reverse transcriptase was omitted were considered to contain the ETV6-RUNX1 fusion transcript. (C) The ETV6-RUNX1 amplicon generated from cord blood sample 392 was subjected to sequencing with the primers used to generate the product. The sequencing electropherogram from one of two reactions demonstrates the structure of the expected fusion transcript.
    Figure Legend Snippet: Detection of the ETV6-RUNX1 fusion transcript by reverse transcription followed by PCR. 200 ng of RNA purified from umbilical vein cord blood lymphocytes was reverse transcribed using random hexamers as primers. (A) The integrity of the RNA in the numbered cord blood samples was confirmed by amplification of a 384 bp sequence spanning the junction of exons 3 and 4 of the β-2-microglobulin gene. Positive controls included RNA from the BJAB and UoCB4 cell lines (+RT). Negative controls included salmon sperm DNA (SS DNA) and the exclusion of reverse transcriptase (NRT). Lanes containing the 100 bp DNA ladder reference are indicated by M. (B) A 200 bp amplicon obtained from a nested PCR assay indicates the presence of the ETV6-RUNX1 transcript. Representative results show the products of three technical replicates from four numbered cord blood samples, salmon sperm DNA as a negative control, and a 10 -2 dilution of the ETV6-RUNX1 -positive UoCB4 cells among BJAB cells as a positive control. Samples in which at least two of three technical replicates were detected from cDNA generated independently in two laboratories and in which no product was observed when reverse transcriptase was omitted were considered to contain the ETV6-RUNX1 fusion transcript. (C) The ETV6-RUNX1 amplicon generated from cord blood sample 392 was subjected to sequencing with the primers used to generate the product. The sequencing electropherogram from one of two reactions demonstrates the structure of the expected fusion transcript.

    Techniques Used: Polymerase Chain Reaction, Purification, Amplification, Sequencing, Nested PCR, Negative Control, Positive Control, Generated

    23) Product Images from "5′-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5′-halves"

    Article Title: 5′-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5′-halves

    Journal: RNA

    doi: 10.1261/rna.058024.116

    TaqMan qRT-PCR method to analyze the 5′-terminal nucleotide of mature tRNA HisGUG . ( A ) Schematic representation of the TaqMan qRT-PCR analysis used to quantify each 5′-terminal variant of mature tRNA HisGUG . ( B ) Sequences and/or positions of the mature tRNA HisGUG and the following TaqMan qPCR components: adapter, AS-disrupter, primers, and TaqMan probe. ( C ) Under the indicated conditions, this method was applied to synthetic mature tRNA HisGUG containing G −1 . The reaction containing only T4 RNA ligase ( far left ) was set to one, and fold changes relative to this reference are shown; bars indicate SD from three independent experiments. Amplified cDNA bands observed in native PAGE after 40 cycles of PCR are also shown. ( D ) Synthetic mature tRNAs HisGUG starting from G 1 , G −1 , and U −1 ), and the relative abundances of detected tRNAs containing each 5′-terminal nucleotide are shown. Bars indicate SD from three independent experiments. N.D. indicates that the reaction did not amplify detectable cDNA signals.
    Figure Legend Snippet: TaqMan qRT-PCR method to analyze the 5′-terminal nucleotide of mature tRNA HisGUG . ( A ) Schematic representation of the TaqMan qRT-PCR analysis used to quantify each 5′-terminal variant of mature tRNA HisGUG . ( B ) Sequences and/or positions of the mature tRNA HisGUG and the following TaqMan qPCR components: adapter, AS-disrupter, primers, and TaqMan probe. ( C ) Under the indicated conditions, this method was applied to synthetic mature tRNA HisGUG containing G −1 . The reaction containing only T4 RNA ligase ( far left ) was set to one, and fold changes relative to this reference are shown; bars indicate SD from three independent experiments. Amplified cDNA bands observed in native PAGE after 40 cycles of PCR are also shown. ( D ) Synthetic mature tRNAs HisGUG starting from G 1 , G −1 , and U −1 ), and the relative abundances of detected tRNAs containing each 5′-terminal nucleotide are shown. Bars indicate SD from three independent experiments. N.D. indicates that the reaction did not amplify detectable cDNA signals.

    Techniques Used: Quantitative RT-PCR, Variant Assay, Real-time Polymerase Chain Reaction, Amplification, Clear Native PAGE, Polymerase Chain Reaction

    Variations in 5′-terminal nucleotide from mature tRNA HisGUG expressed in BT-474 cells. ( A ) Of note, 70- to 90-nt mature tRNA fractions of BT-474 cells were subjected to TaqMan qRT-PCR quantification of each 5′-terminal variant of mature tRNA HisGUG ), and the relative abundances of mature tRNAs containing each 5′-terminal nucleotide are shown. Bars indicate SD from three independent experiments. N.D. indicates that the reaction did not amplify detectable cDNA signals. ( B ) A primer extension assay to analyze the 5′-terminal positions of mature tRNA HisGUG was performed using the 70- to 90-nt mature tRNA fraction from BT-474 cells.
    Figure Legend Snippet: Variations in 5′-terminal nucleotide from mature tRNA HisGUG expressed in BT-474 cells. ( A ) Of note, 70- to 90-nt mature tRNA fractions of BT-474 cells were subjected to TaqMan qRT-PCR quantification of each 5′-terminal variant of mature tRNA HisGUG ), and the relative abundances of mature tRNAs containing each 5′-terminal nucleotide are shown. Bars indicate SD from three independent experiments. N.D. indicates that the reaction did not amplify detectable cDNA signals. ( B ) A primer extension assay to analyze the 5′-terminal positions of mature tRNA HisGUG was performed using the 70- to 90-nt mature tRNA fraction from BT-474 cells.

    Techniques Used: Quantitative RT-PCR, Variant Assay, Primer Extension Assay

    24) Product Images from "ARID1a-DNA Interactions Are Required for Promoter Occupancy by SWI/SNF"

    Article Title: ARID1a-DNA Interactions Are Required for Promoter Occupancy by SWI/SNF

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01008-12

    The V1068G mutation does not affect the stability or the catalytic activity of BAF-A complexes. (A) Quantitative fluorescent Western blots (WB) of Arid1a +/+ and Arid1a V1068G/V1068G E9.5 whole-embryo lysates probed with SWI/SNF subunit-specific antibodies. Graph represents the average ± SEM of the normalized band intensity from three age- or littermate-matched whole-embryo lysates. Band intensity measurements are plotted as a ratio to β-actin signals, and wild-type (wt) measurements were set at 1. mut, mutant. (B to E) Coimmunoprecipitation of SWI/SNF complex subunits or cullin-2 from wild-type or Arid1a V1068G / V1068G MEFs. Shown are Western blot panels containing input protein, mock-precipitated (protein A/G-agarose bead only) protein, or protein precipitated with antibodies specific for ARID1a, BRG1, or BRM. Western blots were probed with antibodies specific for ARID1a, BRG1, BRM, INI1/SNF5, BAF60a, and cullin-2. (F) Mononucleosome disruption by wild-type and mutant ARID1a-containing complexes increases PstI restriction site exposure. Fixed amounts of anti-ARID1a immunopurified fractions from either wild-type or Arid1a V1068G / V1068G MEFs were incubated with limiting amounts of gel-purified, radiolabeled mononucleosomes over a 40-min time course in the presence of ATP. Reaction mixtures containing wild-type or mutant anti-ARID1a fractions were quenched at 10-min intervals. Reaction mixtures containing ATPγS or mock-purified (protein A/G-agarose bead only) fractions and incubated over the entire time course served as controls. Naked DNA (lane 1) or mononucleosomes (lane 2) incubated without immunopurified material are shown. Uncut fractions were plotted as a ratio of the total DNA. Error bars in panel F represent the standard deviations, and significant differences were calculated using a two-tailed Student t test (*, P
    Figure Legend Snippet: The V1068G mutation does not affect the stability or the catalytic activity of BAF-A complexes. (A) Quantitative fluorescent Western blots (WB) of Arid1a +/+ and Arid1a V1068G/V1068G E9.5 whole-embryo lysates probed with SWI/SNF subunit-specific antibodies. Graph represents the average ± SEM of the normalized band intensity from three age- or littermate-matched whole-embryo lysates. Band intensity measurements are plotted as a ratio to β-actin signals, and wild-type (wt) measurements were set at 1. mut, mutant. (B to E) Coimmunoprecipitation of SWI/SNF complex subunits or cullin-2 from wild-type or Arid1a V1068G / V1068G MEFs. Shown are Western blot panels containing input protein, mock-precipitated (protein A/G-agarose bead only) protein, or protein precipitated with antibodies specific for ARID1a, BRG1, or BRM. Western blots were probed with antibodies specific for ARID1a, BRG1, BRM, INI1/SNF5, BAF60a, and cullin-2. (F) Mononucleosome disruption by wild-type and mutant ARID1a-containing complexes increases PstI restriction site exposure. Fixed amounts of anti-ARID1a immunopurified fractions from either wild-type or Arid1a V1068G / V1068G MEFs were incubated with limiting amounts of gel-purified, radiolabeled mononucleosomes over a 40-min time course in the presence of ATP. Reaction mixtures containing wild-type or mutant anti-ARID1a fractions were quenched at 10-min intervals. Reaction mixtures containing ATPγS or mock-purified (protein A/G-agarose bead only) fractions and incubated over the entire time course served as controls. Naked DNA (lane 1) or mononucleosomes (lane 2) incubated without immunopurified material are shown. Uncut fractions were plotted as a ratio of the total DNA. Error bars in panel F represent the standard deviations, and significant differences were calculated using a two-tailed Student t test (*, P

    Techniques Used: Mutagenesis, Activity Assay, Western Blot, Incubation, Purification, Two Tailed Test

    25) Product Images from "Sporadic colon cancer murine models demonstrate the value of autoantibody detection for preclinical cancer diagnosis"

    Article Title: Sporadic colon cancer murine models demonstrate the value of autoantibody detection for preclinical cancer diagnosis

    Journal: Scientific Reports

    doi: 10.1038/srep02938

    DSS-only and AOM-only models of sporadic colon carcinogenesis. (a) Top, AOM (10 mg/kg) was weekly injected during six weeks. Bottom, fresh 2.5% DSS was given in drinking water during five-day cycles (gray areas) followed by regular drinking water for 16 days. Blood samples were collected at the end of the protocols and also for AOM-treated mice at day 63. (b) Representative images of distal colon macroscopic appearance of DSS-treated (day 63) and AOM-treated mice at day 63 and 250. Tumors are highlighted with black arrows. (c) Representative haematoxylin/eosin staining sections of distal colon morphology of DSS- and AOM-treated mice and control mice. No significant changes were observed for AOM-treated mouse at day 63. DSS-treated mice showed presence of adenomas and different grade of dysplasia (DSS 1, DSS 2 and DSS 3) at day 63, whereas DSS 4 animal showed low grade nonmalignant dysplastic lesions and DSS 5 showed no changes respect to normal mucosa. (d) At day 250, three AOM-treated mice showed infiltrating but non-invasive tumors (AOM 2, AOM 3 and AOM 5), whereas the remaining animals (AOM 1 and AOM 4) showed small flat non-polipoid adenomas with dysplasia. (c, d) Black arrows highlight the observed lesions. Images are shown at 200x magnification.
    Figure Legend Snippet: DSS-only and AOM-only models of sporadic colon carcinogenesis. (a) Top, AOM (10 mg/kg) was weekly injected during six weeks. Bottom, fresh 2.5% DSS was given in drinking water during five-day cycles (gray areas) followed by regular drinking water for 16 days. Blood samples were collected at the end of the protocols and also for AOM-treated mice at day 63. (b) Representative images of distal colon macroscopic appearance of DSS-treated (day 63) and AOM-treated mice at day 63 and 250. Tumors are highlighted with black arrows. (c) Representative haematoxylin/eosin staining sections of distal colon morphology of DSS- and AOM-treated mice and control mice. No significant changes were observed for AOM-treated mouse at day 63. DSS-treated mice showed presence of adenomas and different grade of dysplasia (DSS 1, DSS 2 and DSS 3) at day 63, whereas DSS 4 animal showed low grade nonmalignant dysplastic lesions and DSS 5 showed no changes respect to normal mucosa. (d) At day 250, three AOM-treated mice showed infiltrating but non-invasive tumors (AOM 2, AOM 3 and AOM 5), whereas the remaining animals (AOM 1 and AOM 4) showed small flat non-polipoid adenomas with dysplasia. (c, d) Black arrows highlight the observed lesions. Images are shown at 200x magnification.

    Techniques Used: Injection, Mouse Assay, Staining

    26) Product Images from "LincRNA landscape in human lymphocytes highlights regulation of T cell differentiation by linc-MAF-4"

    Article Title: LincRNA landscape in human lymphocytes highlights regulation of T cell differentiation by linc-MAF-4

    Journal: Nature immunology

    doi: 10.1038/ni.3093

    Identification of lincRNAs expressed in human lymphocyte subsets RNA-seq data generated from 63 lymphocyte samples were processed according to two different strategies: quantification of lincRNAs already annotated in public resources and de novo Genome Based Transcripts Reconstruction for the quantification of new lincRNAs expressed in human lymphocytes. Three methods for the identification of new transcripts were adopted: Reference Annotation Based assembly by Cufflinks with two different aligners (TopHat and STAR) and an approach that integrates Trinity and PASA software. Only transcripts reconstructed by at least two assemblers were considered. Novel transcripts were filtered with a computational analysis pipeline to select for lincRNAs. The number of lincRNA genes and transcripts identified in lymphocytes subsets is indicated.
    Figure Legend Snippet: Identification of lincRNAs expressed in human lymphocyte subsets RNA-seq data generated from 63 lymphocyte samples were processed according to two different strategies: quantification of lincRNAs already annotated in public resources and de novo Genome Based Transcripts Reconstruction for the quantification of new lincRNAs expressed in human lymphocytes. Three methods for the identification of new transcripts were adopted: Reference Annotation Based assembly by Cufflinks with two different aligners (TopHat and STAR) and an approach that integrates Trinity and PASA software. Only transcripts reconstructed by at least two assemblers were considered. Novel transcripts were filtered with a computational analysis pipeline to select for lincRNAs. The number of lincRNA genes and transcripts identified in lymphocytes subsets is indicated.

    Techniques Used: RNA Sequencing Assay, Generated, Software

    27) Product Images from "The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus"

    Article Title: The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138789

    CBLC ubiquitin ligase activity is required for Golgi but not SRC levels regulation. (A) Requirement of CBLC RING domain. HeLa wild type (WT) or stably expressing siRNA-resistant-CBLC (CBLC-GFP) or siRNA-resistant-CBLC RING mutant (C351A-GFP) cells were transfected with deconvoluted CBLC-7 siRNA. Golgi stained with Giantin. Top right: Schematic diagram, exogenous CBLC contains three-point mutations at CBLC-7 siRNA-binding site. Bottom right: Fragmentation Index measured in quadruplicates on at least 400 cells per condition. Error bars show SD statistical significance (p) measured by unpaired Student’s t-test. (**) represents p
    Figure Legend Snippet: CBLC ubiquitin ligase activity is required for Golgi but not SRC levels regulation. (A) Requirement of CBLC RING domain. HeLa wild type (WT) or stably expressing siRNA-resistant-CBLC (CBLC-GFP) or siRNA-resistant-CBLC RING mutant (C351A-GFP) cells were transfected with deconvoluted CBLC-7 siRNA. Golgi stained with Giantin. Top right: Schematic diagram, exogenous CBLC contains three-point mutations at CBLC-7 siRNA-binding site. Bottom right: Fragmentation Index measured in quadruplicates on at least 400 cells per condition. Error bars show SD statistical significance (p) measured by unpaired Student’s t-test. (**) represents p

    Techniques Used: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Transfection, Staining, Binding Assay

    28) Product Images from "Association Analysis of a Microsatellite Repeat in the TRIB1 Gene With Prostate Cancer Risk, Aggressiveness and Survival"

    Article Title: Association Analysis of a Microsatellite Repeat in the TRIB1 Gene With Prostate Cancer Risk, Aggressiveness and Survival

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2018.00428

    Predicted secondary structures of the two TRIB1 mRNA transcripts. Showing two examples of the differences observed in the predicted TRIB1 mRNA structure of the TRIB1 transcript variant one with the three (A) and four (B) TTTTG- TRIB1 repeats alleles; and of the TRIB1 transcript variant two with the three (C) and four (D) TTTTG- TRIB1 repeats alleles. The dotted squares highlight the secondary structures differences in the final structure. Data obtained from mfold web server v.3 ( http://www.bioinfo.rpi.edu/applications/mfold .).
    Figure Legend Snippet: Predicted secondary structures of the two TRIB1 mRNA transcripts. Showing two examples of the differences observed in the predicted TRIB1 mRNA structure of the TRIB1 transcript variant one with the three (A) and four (B) TTTTG- TRIB1 repeats alleles; and of the TRIB1 transcript variant two with the three (C) and four (D) TTTTG- TRIB1 repeats alleles. The dotted squares highlight the secondary structures differences in the final structure. Data obtained from mfold web server v.3 ( http://www.bioinfo.rpi.edu/applications/mfold .).

    Techniques Used: Variant Assay

    29) Product Images from "BST2/Tetherin Inhibits Dengue Virus Release from Human Hepatoma Cells"

    Article Title: BST2/Tetherin Inhibits Dengue Virus Release from Human Hepatoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051033

    BST2 inhibits DENV spread via cell-to-cell transmission. The cells were infected with DENV at a MOI of 0.01 or 10 for 1 h and culture media were replaced with media containing 0.5% methocellulose to prevent cell-free virus infection and cultured for 2 days. (A) Representative DENV-infected cell foci from cultures of the three cell lines. The infected cell foci and cell viability were revealed by In-Cell Western assay by using of antibody against DENV E protein and Sapphire 700 staining, respectively. The indicated gray values of the dots were quantified by using of an Odyssey Infrared Imaging System (LI-COR Biotechnology). (B) The average infectious foci number per well in 24-well plate and the average DENV-infected cell number per focus from 100 foci were plotted. (C) The intracellular DENV RNA was determined for the cells infected with DENV at MOI of 10 by qRT-PCR assay. The values were presented as percentage of values from the Huh7-BST2 and Huh7-BST2CV5 cells compared with that from parent Huh7 cells. The experiment was performed in 3 replicates to generate statistically sufficient data. p values were calculated using Student's t test.
    Figure Legend Snippet: BST2 inhibits DENV spread via cell-to-cell transmission. The cells were infected with DENV at a MOI of 0.01 or 10 for 1 h and culture media were replaced with media containing 0.5% methocellulose to prevent cell-free virus infection and cultured for 2 days. (A) Representative DENV-infected cell foci from cultures of the three cell lines. The infected cell foci and cell viability were revealed by In-Cell Western assay by using of antibody against DENV E protein and Sapphire 700 staining, respectively. The indicated gray values of the dots were quantified by using of an Odyssey Infrared Imaging System (LI-COR Biotechnology). (B) The average infectious foci number per well in 24-well plate and the average DENV-infected cell number per focus from 100 foci were plotted. (C) The intracellular DENV RNA was determined for the cells infected with DENV at MOI of 10 by qRT-PCR assay. The values were presented as percentage of values from the Huh7-BST2 and Huh7-BST2CV5 cells compared with that from parent Huh7 cells. The experiment was performed in 3 replicates to generate statistically sufficient data. p values were calculated using Student's t test.

    Techniques Used: Transmission Assay, Infection, Cell Culture, In-Cell ELISA, Staining, Imaging, Quantitative RT-PCR

    30) Product Images from "Reprogramming the antigen specificity of B cells using genome-editing technologies"

    Article Title: Reprogramming the antigen specificity of B cells using genome-editing technologies

    Journal: eLife

    doi: 10.7554/eLife.42995

    V3-74 Universal B cell engineering strategy. The annotated donor DNA highlights 5’ and 3’ CRISPR-Cas9 target sequences (PAM sites are mutated in the donor plasmid). When this donor was co-nucleofected into Ramos B cells with the 5’ and 3’ cas9 plasmids, roughly 0.35% of cells could bind C108 SOSIP-APC three days later by FACS (top left). C108 enriched cells were further cultured. gDNA and mRNA from these cells was purified. RT-PCR with PG9 specific primers showed a band made up of PG9-IgM from engineered cells but not non-engineered controls. Out/out PCR on gDNA template across V374 to the J6 intron could amplify bands of the expected size from engineered cells only using three different primer sets (top right). Sanger sequencing of all bands show successful engineering and expression of PG9-IgM in this cell line.
    Figure Legend Snippet: V3-74 Universal B cell engineering strategy. The annotated donor DNA highlights 5’ and 3’ CRISPR-Cas9 target sequences (PAM sites are mutated in the donor plasmid). When this donor was co-nucleofected into Ramos B cells with the 5’ and 3’ cas9 plasmids, roughly 0.35% of cells could bind C108 SOSIP-APC three days later by FACS (top left). C108 enriched cells were further cultured. gDNA and mRNA from these cells was purified. RT-PCR with PG9 specific primers showed a band made up of PG9-IgM from engineered cells but not non-engineered controls. Out/out PCR on gDNA template across V374 to the J6 intron could amplify bands of the expected size from engineered cells only using three different primer sets (top right). Sanger sequencing of all bands show successful engineering and expression of PG9-IgM in this cell line.

    Techniques Used: CRISPR, Plasmid Preparation, FACS, Cell Culture, Purification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Expressing

    31) Product Images from "Identification of Fhit as a Post-transcriptional Effector of Thymidine Kinase 1 Expression"

    Article Title: Identification of Fhit as a Post-transcriptional Effector of Thymidine Kinase 1 Expression

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagrm.2017.01.005

    Fhit increases polysome-bound TK1 mRNA A. Cytoplasmic extracts from H1299 E1 and D1 induced cells were separated on a 10-50% sucrose gradient and fractions were monitored continuously at 254 nm. A representative gradient (of 3 independent gradients) is shown for each cell line and a representative Western blot of Fhit expression is shown in the inset. B. Even-numbered fractions were spiked with 1 ng of luciferase RNA as a control for sample recovery, and recovered RNA was analyzed by RT-qPCR for TK1 and luciferase. TK1 was normalized to luciferase and plotted as the percent of mRNA in each gradient fraction relative to input. Results shown are the means ± SEM of three independent experiments each performed in triplicate.
    Figure Legend Snippet: Fhit increases polysome-bound TK1 mRNA A. Cytoplasmic extracts from H1299 E1 and D1 induced cells were separated on a 10-50% sucrose gradient and fractions were monitored continuously at 254 nm. A representative gradient (of 3 independent gradients) is shown for each cell line and a representative Western blot of Fhit expression is shown in the inset. B. Even-numbered fractions were spiked with 1 ng of luciferase RNA as a control for sample recovery, and recovered RNA was analyzed by RT-qPCR for TK1 and luciferase. TK1 was normalized to luciferase and plotted as the percent of mRNA in each gradient fraction relative to input. Results shown are the means ± SEM of three independent experiments each performed in triplicate.

    Techniques Used: Western Blot, Expressing, Luciferase, Quantitative RT-PCR

    32) Product Images from "The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl− co-transporters"

    Article Title: The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl− co-transporters

    Journal: Biochemical Journal

    doi: 10.1042/BJ20131478

    STOCK1S-50699 inhibits phosphorylation of endogenous KCC isoforms at Site-2 and Site-4 in ES and HEK-293 cells ES ( A ) or HEK-293 ( B ) cells were exposed for 30 min to either basic control isotonic conditions or hypotonic low Cl − conditions (to activate the SPAK/OSR1 pathway), and then treated under the same conditions with the indicated doses of the SPAK/OSR1 CCT domain inhibitor STOCK1S-50699 [ 30 ] for an additional 30 min. The lysates were subjected to immunoprecipitation (IP) with the indicated KCC3A Site-1, Site-2 and Site-4 phospho-antibodies that recognize all KCC isoforms (see Figure 3 ). The immunoprecipitates were then immunoblotted (IB) employing the indicated specific KCC isoform antibodies. Cells lysates were also subjected to immunoblot analysis with the indicated total and phospho-specific antibodies. Similar results were obtained in three separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: STOCK1S-50699 inhibits phosphorylation of endogenous KCC isoforms at Site-2 and Site-4 in ES and HEK-293 cells ES ( A ) or HEK-293 ( B ) cells were exposed for 30 min to either basic control isotonic conditions or hypotonic low Cl − conditions (to activate the SPAK/OSR1 pathway), and then treated under the same conditions with the indicated doses of the SPAK/OSR1 CCT domain inhibitor STOCK1S-50699 [ 30 ] for an additional 30 min. The lysates were subjected to immunoprecipitation (IP) with the indicated KCC3A Site-1, Site-2 and Site-4 phospho-antibodies that recognize all KCC isoforms (see Figure 3 ). The immunoprecipitates were then immunoblotted (IB) employing the indicated specific KCC isoform antibodies. Cells lysates were also subjected to immunoblot analysis with the indicated total and phospho-specific antibodies. Similar results were obtained in three separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Immunoprecipitation

    WNK–SPAK/OSR1 regulation and phosphorylation of endogenous KCCs Previously described matched wild-type and double OSR1 185A/185A /SPAK 243A/243A knockin ES cells [ 29 ], in which SPAK/OSR1 are inactive as the T-loop phosphorylation site that is phosphorylated by WNK isoforms is ablated, were incubated with either basic control isotonic medium (open bars) or hypotonic high K + medium (closed bars) for 30 min in the presence of 1 mM ouabain (an inhibitor of the plasma membrane Na + /K + -ATPase sodium pump) and 0.1 mM bumetanide (inhibitor of NKCC1). 86 Rb + uptake proceeded for 10 min and was then quantified by scintillation counting. 86 Rb + uptake c.p.m. values were normalized per mg of protein for each condition and plotted for both isotonic and hypotonic conditions. The lysates were also subjected to immunoprecipitation (IP) with the indicated KCC3A Site-1 and Site-2 phospho-antibodies that recognize all KCC isoforms (see Figure 3 ). The immunoprecipitates were then immunoblotted employing the indicated specific KCC isoform antibodies. Molecular masses are indicated in kDa on the left-hand side of the Western blots (lower panel). Similar results were obtained for three separate experiments.
    Figure Legend Snippet: WNK–SPAK/OSR1 regulation and phosphorylation of endogenous KCCs Previously described matched wild-type and double OSR1 185A/185A /SPAK 243A/243A knockin ES cells [ 29 ], in which SPAK/OSR1 are inactive as the T-loop phosphorylation site that is phosphorylated by WNK isoforms is ablated, were incubated with either basic control isotonic medium (open bars) or hypotonic high K + medium (closed bars) for 30 min in the presence of 1 mM ouabain (an inhibitor of the plasma membrane Na + /K + -ATPase sodium pump) and 0.1 mM bumetanide (inhibitor of NKCC1). 86 Rb + uptake proceeded for 10 min and was then quantified by scintillation counting. 86 Rb + uptake c.p.m. values were normalized per mg of protein for each condition and plotted for both isotonic and hypotonic conditions. The lysates were also subjected to immunoprecipitation (IP) with the indicated KCC3A Site-1 and Site-2 phospho-antibodies that recognize all KCC isoforms (see Figure 3 ). The immunoprecipitates were then immunoblotted employing the indicated specific KCC isoform antibodies. Molecular masses are indicated in kDa on the left-hand side of the Western blots (lower panel). Similar results were obtained for three separate experiments.

    Techniques Used: Knock-In, Incubation, Immunoprecipitation, Western Blot

    33) Product Images from "Mutations across Murine Hepatitis Virus nsp4 Alter Virus Fitness and Membrane Modifications"

    Article Title: Mutations across Murine Hepatitis Virus nsp4 Alter Virus Fitness and Membrane Modifications

    Journal: Journal of Virology

    doi: 10.1128/JVI.02776-14

    Competition assay of nsp4 mutants. DBT cells were infected with the indicated pairs of viruses at a total MOI of 0.1 PFU/cell at a ratio of 1:1. When cells were at least 50% involved in cytopathic effect, supernatants were collected, and cell monolayers were harvested in TRIzol. Supernatants were used for subsequent passages, for a total of three passages. Total RNA was extracted, and nsp4 amplicons were generated by RT-PCR and sequenced. For residues of interest, the area under the peak was calculated using MacVector, version 13. Then, the percentage of nucleotides of virus A to virus B was calculated and plotted on the graph. Error bars represent standard errors of the means ( n ≥ 2).
    Figure Legend Snippet: Competition assay of nsp4 mutants. DBT cells were infected with the indicated pairs of viruses at a total MOI of 0.1 PFU/cell at a ratio of 1:1. When cells were at least 50% involved in cytopathic effect, supernatants were collected, and cell monolayers were harvested in TRIzol. Supernatants were used for subsequent passages, for a total of three passages. Total RNA was extracted, and nsp4 amplicons were generated by RT-PCR and sequenced. For residues of interest, the area under the peak was calculated using MacVector, version 13. Then, the percentage of nucleotides of virus A to virus B was calculated and plotted on the graph. Error bars represent standard errors of the means ( n ≥ 2).

    Techniques Used: Competitive Binding Assay, Infection, Generated, Reverse Transcription Polymerase Chain Reaction

    34) Product Images from "Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis"

    Article Title: Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

    Journal: International Journal for Parasitology: Drugs and Drug Resistance

    doi: 10.1016/j.ijpddr.2014.09.004

    Kinetic study of the influence of Akt inhibitors on schistosomula viability. S. mansoni schistosomula were incubated with different concentrations (50 nM–10 μM) of Akt inhibitor IV (A4), Akt inhibitor X (A7) and PDK1/Akt/Flt Dual Pathway inhibitor (A8). Percentages of alive schistosomula were determined under microscope at 24 h, 48 h and 72 h. Results are expressed as the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Kinetic study of the influence of Akt inhibitors on schistosomula viability. S. mansoni schistosomula were incubated with different concentrations (50 nM–10 μM) of Akt inhibitor IV (A4), Akt inhibitor X (A7) and PDK1/Akt/Flt Dual Pathway inhibitor (A8). Percentages of alive schistosomula were determined under microscope at 24 h, 48 h and 72 h. Results are expressed as the mean ± SEM of three independent experiments.

    Techniques Used: Incubation, Microscopy

    35) Product Images from "Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA"

    Article Title: Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12358

    HigB expression affects tmRNA in M. tuberculosis ΔTAC. RNA was extracted from mid-exponential cultures treated with ATc for 2 h, 6 h or 24 h and cDNA was prepared for qRT-PCR analysis.A–C. Northern blots probing for tmRNA (A and B) and 5S (C). Transcript sizes are relative to the position of RNA marker and tmRNA cleavage products are indicated with an arrow.D. Quantitative RT-PCR of transcripts of interest. All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between vector control and HigB overexpression strain is marked by an asterisk (*) for P
    Figure Legend Snippet: HigB expression affects tmRNA in M. tuberculosis ΔTAC. RNA was extracted from mid-exponential cultures treated with ATc for 2 h, 6 h or 24 h and cDNA was prepared for qRT-PCR analysis.A–C. Northern blots probing for tmRNA (A and B) and 5S (C). Transcript sizes are relative to the position of RNA marker and tmRNA cleavage products are indicated with an arrow.D. Quantitative RT-PCR of transcripts of interest. All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between vector control and HigB overexpression strain is marked by an asterisk (*) for P

    Techniques Used: Expressing, Quantitative RT-PCR, Northern Blot, Marker, Standard Deviation, Plasmid Preparation, Over Expression

    36) Product Images from "SAG/RBX2 E3 Ubiquitin Ligase Differentially Regulates Inflammatory Responses of Myeloid Cell Subsets"

    Article Title: SAG/RBX2 E3 Ubiquitin Ligase Differentially Regulates Inflammatory Responses of Myeloid Cell Subsets

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02882

    Increased TNFα and decreased induction of genes responsive to LPS in Sag-deficient neutrophils and bone marrow. (A,B) Splenocytes from LysM-Cre − / Sag fl / fl (WT) and LysM-Cre + / Sag fl / fl (KO) mice ( n = 5 for each genotype) were stimulated with LPS (100 ng/ml) and TNFα production by CD11b + Gr-1 + neutrophils was analyzed by flow cytometry (A) 6 h and (B) 18 h following stimulation. (C) Protein analysis by western blot of p65 isoform of NF-κB protein using nuclear [N] and cytosolic [C] fractions of neutrophils isolated from blood of WT and KO mice stimulated concurrently with LPS (100 ng/ml) for 1 h. Densitometric analysis of p65 NF-κB presence in the nuclear fraction and cytosolic fraction is shown underneath the blot. Parp and Procaspase-3 serve as biomarkers of nuclear and cytoplasmic fractions, respectively. (D,E) Affymetrix microarray analysis upon stimulation with LPS for 6 h: Bone marrow cells were isolated from two individual WT and KO mice with i.p. injection of PBS or LPS (25 mg/kg body weight) for 6 h, followed by RNA extraction and Affymetrix microarray analysis. (D) The expression of genes was compared in WT or KO bone marrow in response to LPS stimulation. (E) GO enrichment analysis was performed in a total of 1,141 genes whose expression was specifically altered in KO bone marrow in response to LPS stimulation. The expression heatmap of genes annotated with term “response to lipopolysaccharide” was generated. (F) Validation of microarray results by qRT-PCR: Bone marrow was isolated from three individual WT and KO mice i.p. injected with PBS or LPS (25 mg/kg body weight) for 6 h, followed by RNA extraction and qRT-PCR analysis. * p
    Figure Legend Snippet: Increased TNFα and decreased induction of genes responsive to LPS in Sag-deficient neutrophils and bone marrow. (A,B) Splenocytes from LysM-Cre − / Sag fl / fl (WT) and LysM-Cre + / Sag fl / fl (KO) mice ( n = 5 for each genotype) were stimulated with LPS (100 ng/ml) and TNFα production by CD11b + Gr-1 + neutrophils was analyzed by flow cytometry (A) 6 h and (B) 18 h following stimulation. (C) Protein analysis by western blot of p65 isoform of NF-κB protein using nuclear [N] and cytosolic [C] fractions of neutrophils isolated from blood of WT and KO mice stimulated concurrently with LPS (100 ng/ml) for 1 h. Densitometric analysis of p65 NF-κB presence in the nuclear fraction and cytosolic fraction is shown underneath the blot. Parp and Procaspase-3 serve as biomarkers of nuclear and cytoplasmic fractions, respectively. (D,E) Affymetrix microarray analysis upon stimulation with LPS for 6 h: Bone marrow cells were isolated from two individual WT and KO mice with i.p. injection of PBS or LPS (25 mg/kg body weight) for 6 h, followed by RNA extraction and Affymetrix microarray analysis. (D) The expression of genes was compared in WT or KO bone marrow in response to LPS stimulation. (E) GO enrichment analysis was performed in a total of 1,141 genes whose expression was specifically altered in KO bone marrow in response to LPS stimulation. The expression heatmap of genes annotated with term “response to lipopolysaccharide” was generated. (F) Validation of microarray results by qRT-PCR: Bone marrow was isolated from three individual WT and KO mice i.p. injected with PBS or LPS (25 mg/kg body weight) for 6 h, followed by RNA extraction and qRT-PCR analysis. * p

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Western Blot, Isolation, Microarray, Injection, RNA Extraction, Expressing, Generated, Quantitative RT-PCR

    37) Product Images from "Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins"

    Article Title: Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw441

    TuMV-GA6 and its utilization in slippage analysis. ( A ) Schematic of the TuMV genome showing the additional inserted 2nd-GA6 slip site. Coloured boxes indicate the polyprotein cleavage products. GFP followed by an NIa protease cleavage site is inserted between P1 and HC-Pro in the infectious clone. A small insert containing an additional GAAAAAA sequence with flanking nucleotides (2nd-GA6) was inserted between GFP and the NIa protease cleavage site as shown below the genome diagram (TuMV WT sequence shown). Regions corresponding to restriction nuclease SmaI and NheI recognition sites enabling manipulation of the 2nd-GA6 insert are indicated in lowercase. The position of the conserved GAAAAAA sequence at the 5′ end of the pipo ORF (WT-GA6) is indicated with an arrow. ( B ) Insertions and deletions at the WT-GA6 site. RNA was extracted at 11 d p.i. from systemically infected leaves of plants inoculated with various TuMV constructs (differences at the 2nd-GA6 site, not at WT-GA6) and subjected to targeted high-throughput sequencing. The occurrence of single or double nucleotide deletions or insertions within the slip site sequence (AAAAAA) at the WT-GA6 site was determined from 95 different biological samples ( n = 95). Frequencies show the mean percentage of modified reads per library. Error bars indicate standard deviations. ( C ) Comparative analysis of deletions and insertions at the 2nd-GA6 and WT-GA6 sites. Plants were inoculated with viruses containing different 2nd-GA6 sites (TuMV WT, TuMV WT-L and PISPO WT) but WT TuMV sequence at the WT-GA6 site. The 2nd-GA6 sequence for each construct is shown below the graph. Systemically infected leaves were analysed as described in (A). For each construct three biological samples were tested ( n = 3). The mean frequencies of deletions or insertions at the 2nd-GA6 site of each construct are shown in the left panel and the corresponding WT-GA6 data are shown in the right panel. Error bars indicate standard deviations; black dots mark individual samples for the single-nucleotide insertion data.
    Figure Legend Snippet: TuMV-GA6 and its utilization in slippage analysis. ( A ) Schematic of the TuMV genome showing the additional inserted 2nd-GA6 slip site. Coloured boxes indicate the polyprotein cleavage products. GFP followed by an NIa protease cleavage site is inserted between P1 and HC-Pro in the infectious clone. A small insert containing an additional GAAAAAA sequence with flanking nucleotides (2nd-GA6) was inserted between GFP and the NIa protease cleavage site as shown below the genome diagram (TuMV WT sequence shown). Regions corresponding to restriction nuclease SmaI and NheI recognition sites enabling manipulation of the 2nd-GA6 insert are indicated in lowercase. The position of the conserved GAAAAAA sequence at the 5′ end of the pipo ORF (WT-GA6) is indicated with an arrow. ( B ) Insertions and deletions at the WT-GA6 site. RNA was extracted at 11 d p.i. from systemically infected leaves of plants inoculated with various TuMV constructs (differences at the 2nd-GA6 site, not at WT-GA6) and subjected to targeted high-throughput sequencing. The occurrence of single or double nucleotide deletions or insertions within the slip site sequence (AAAAAA) at the WT-GA6 site was determined from 95 different biological samples ( n = 95). Frequencies show the mean percentage of modified reads per library. Error bars indicate standard deviations. ( C ) Comparative analysis of deletions and insertions at the 2nd-GA6 and WT-GA6 sites. Plants were inoculated with viruses containing different 2nd-GA6 sites (TuMV WT, TuMV WT-L and PISPO WT) but WT TuMV sequence at the WT-GA6 site. The 2nd-GA6 sequence for each construct is shown below the graph. Systemically infected leaves were analysed as described in (A). For each construct three biological samples were tested ( n = 3). The mean frequencies of deletions or insertions at the 2nd-GA6 site of each construct are shown in the left panel and the corresponding WT-GA6 data are shown in the right panel. Error bars indicate standard deviations; black dots mark individual samples for the single-nucleotide insertion data.

    Techniques Used: Sequencing, Infection, Construct, Next-Generation Sequencing, Modification

    Substitution profiles at WT and mutated slip site sequences. The name and number of samples analysed is presented at the top of each subfigure. The mean frequency (blue line) of detected substitutions at each nucleotide position is shown in the upper graph of each subfigure. Error bars indicate standard deviations; for samples with two or three replicates, blue dots denote individual datapoints. The average rates of single-nucleotide insertions (ins) and deletions (del) occurring at the slip site are shown at right. The lower graph of each subfigure shows the nucleotide distribution of detected substitutions at each position. Mutations to other than the original sequence are indicated as follows: U/T—red, A—green, G—grey, C—blue. For clarity, the average total mutation rate for each position (same value as the blue line in the upper plot) is shown on top of each bar. ( A ) Mutations occurring at the WT-GA6 site across all tested constructs; n = 95 (at position +8 [asterisk], n = 94 due to the removal of a single deviantly high [by ∼2 orders of magnitude] data point). Note the different y-axis scale in this subfigure. ( B ) Mutations at the 2nd-GA6 site of construct TuMV WT. ( C ) Mutations detected using TuMV DNA (plasmid) as source, a control for mutations potentially introduced during library amplification and sequencing. ( D ) Mutations detected from in planta expressed TuMV RNA, a control for mutations potentially introduced during reverse transcription, library amplification and sequencing. ( E ) Mutations at the 2nd-GA6 site of construct TuMV RC.
    Figure Legend Snippet: Substitution profiles at WT and mutated slip site sequences. The name and number of samples analysed is presented at the top of each subfigure. The mean frequency (blue line) of detected substitutions at each nucleotide position is shown in the upper graph of each subfigure. Error bars indicate standard deviations; for samples with two or three replicates, blue dots denote individual datapoints. The average rates of single-nucleotide insertions (ins) and deletions (del) occurring at the slip site are shown at right. The lower graph of each subfigure shows the nucleotide distribution of detected substitutions at each position. Mutations to other than the original sequence are indicated as follows: U/T—red, A—green, G—grey, C—blue. For clarity, the average total mutation rate for each position (same value as the blue line in the upper plot) is shown on top of each bar. ( A ) Mutations occurring at the WT-GA6 site across all tested constructs; n = 95 (at position +8 [asterisk], n = 94 due to the removal of a single deviantly high [by ∼2 orders of magnitude] data point). Note the different y-axis scale in this subfigure. ( B ) Mutations at the 2nd-GA6 site of construct TuMV WT. ( C ) Mutations detected using TuMV DNA (plasmid) as source, a control for mutations potentially introduced during library amplification and sequencing. ( D ) Mutations detected from in planta expressed TuMV RNA, a control for mutations potentially introduced during reverse transcription, library amplification and sequencing. ( E ) Mutations at the 2nd-GA6 site of construct TuMV RC.

    Techniques Used: Sequencing, Mutagenesis, Construct, Plasmid Preparation, Amplification

    Detection of slippage events at mutated slip site sequences. ( A and B ) Plants were inoculated with various 2nd-GA6 constructs and systemically infected leaves harvested at 11 d p.i. Total RNA was extracted and subjected to targeted high-throughput sequencing. For each mutant three biological samples were used ( n = 3). The sequences at the 2nd-GA6 site are shown below each graph, with mutated nucleotides indicated in red. The mean frequencies of deletions or insertions at the 2nd-GA6 site of each construct are shown in the left panel and the corresponding WT-GA6 data are shown in the right panel. Error bars indicate standard deviations; black dots mark individual samples for the single-nucleotide insertion data. Reference values for TuMV WT, PISPO WT and SPS WT 2nd-GA6 (0.92, 2.65 and 2.43%) are indicated with horizontal blue dotted lines.
    Figure Legend Snippet: Detection of slippage events at mutated slip site sequences. ( A and B ) Plants were inoculated with various 2nd-GA6 constructs and systemically infected leaves harvested at 11 d p.i. Total RNA was extracted and subjected to targeted high-throughput sequencing. For each mutant three biological samples were used ( n = 3). The sequences at the 2nd-GA6 site are shown below each graph, with mutated nucleotides indicated in red. The mean frequencies of deletions or insertions at the 2nd-GA6 site of each construct are shown in the left panel and the corresponding WT-GA6 data are shown in the right panel. Error bars indicate standard deviations; black dots mark individual samples for the single-nucleotide insertion data. Reference values for TuMV WT, PISPO WT and SPS WT 2nd-GA6 (0.92, 2.65 and 2.43%) are indicated with horizontal blue dotted lines.

    Techniques Used: Construct, Infection, Next-Generation Sequencing, Mutagenesis

    Detection of slippage events at mutated slip site sequences. ( A–F ) Plants were inoculated with various 2nd-GA6 constructs and systemically infected leaves harvested at 11 d p.i. Total RNA was extracted and subjected to targeted high-throughput sequencing. For most mutants three biological samples were used ( n = 3), for TuMV +A, TuMV +AA, TuMV dnGtoC and TuMV dnGtoU only two biological samples were used ( n = 2). The sequences at the 2nd-GA6 site are shown below each graph, with mutated nucleotides indicated in red. The mean frequencies of deletions or insertions at the 2nd-GA6 site of each construct are shown in the left panel and the corresponding WT-GA6 data are shown in the right panel. Error bars indicate standard deviations; black dots mark individual samples for the single-nucleotide insertion data. Reference values for TuMV WT and PISPO WT 2nd-GA6 (0.92 and 2.65%) are indicated with horizontal blue dotted lines.
    Figure Legend Snippet: Detection of slippage events at mutated slip site sequences. ( A–F ) Plants were inoculated with various 2nd-GA6 constructs and systemically infected leaves harvested at 11 d p.i. Total RNA was extracted and subjected to targeted high-throughput sequencing. For most mutants three biological samples were used ( n = 3), for TuMV +A, TuMV +AA, TuMV dnGtoC and TuMV dnGtoU only two biological samples were used ( n = 2). The sequences at the 2nd-GA6 site are shown below each graph, with mutated nucleotides indicated in red. The mean frequencies of deletions or insertions at the 2nd-GA6 site of each construct are shown in the left panel and the corresponding WT-GA6 data are shown in the right panel. Error bars indicate standard deviations; black dots mark individual samples for the single-nucleotide insertion data. Reference values for TuMV WT and PISPO WT 2nd-GA6 (0.92 and 2.65%) are indicated with horizontal blue dotted lines.

    Techniques Used: Construct, Infection, Next-Generation Sequencing

    38) Product Images from "New Genomic Structure for Prostate Cancer Specific Gene PCA3 within BMCC1: Implications for Prostate Cancer Detection and Progression"

    Article Title: New Genomic Structure for Prostate Cancer Specific Gene PCA3 within BMCC1: Implications for Prostate Cancer Detection and Progression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004995

    Complexity of PCA3 transcripts. (A) Partial PCA3 gene structure as originally reported by Bussemakers et al. [15] with 4 exons (open boxes ∼ not to scale) with alternate splicing of exon 2 and three alternate transcription termination sites in exon 4. 5′ RACE experiments (Supplementary Fig. S1 ) identified 4 novel PCA3 transcription initiation sites (isoforms 1–4 marked by vertical arrows pointing down with nucleotide sequence below located 1150 bp, 699 bp, 640 bp and 136 bp upstream of the original initiation site (renamed here isoform 5). 3′ RACE identified four novel polyadenylation sites (7 in total*) located in exon 4. The size of exon 1 is expanded from the original 120 bp to 1270 bp. Isoform 4 ( PCA3-4 ) is the most highly expressed of the four novel isoforms. Four overlapping ORFs initiate from a single ‘ATG’ start site (vertical arrow pointing up) within PCA3-4 and terminate within one of the alternatively spliced exons (2a or 2b or 2c) or within exon 3. (B) RT-PCR amplification of BPH, PCa and PCa metastasis samples using a forward primer from within the novel PCA3-4 transcription start site and a reverse primer from novel exon 2a. (C) Complete structure of the PCA3 gene. Shading identifies the newly identified regions of the PCA3 gene which has 6 exons with alternate splicing of exon 2a (93 bp) and exon 2b (93 bp) and exon 2c (original exon 2, 165 bp). and (D) RT-PCR amplification of PCA3 using the same forward primer and a reverse primer from novel exon 2b and (E) RT-PCR amplification of PCA3 using a forward primer from PCA3-5F (within the original transcription start site) and a reverse primer spanning exons 1 and 3 [15] .
    Figure Legend Snippet: Complexity of PCA3 transcripts. (A) Partial PCA3 gene structure as originally reported by Bussemakers et al. [15] with 4 exons (open boxes ∼ not to scale) with alternate splicing of exon 2 and three alternate transcription termination sites in exon 4. 5′ RACE experiments (Supplementary Fig. S1 ) identified 4 novel PCA3 transcription initiation sites (isoforms 1–4 marked by vertical arrows pointing down with nucleotide sequence below located 1150 bp, 699 bp, 640 bp and 136 bp upstream of the original initiation site (renamed here isoform 5). 3′ RACE identified four novel polyadenylation sites (7 in total*) located in exon 4. The size of exon 1 is expanded from the original 120 bp to 1270 bp. Isoform 4 ( PCA3-4 ) is the most highly expressed of the four novel isoforms. Four overlapping ORFs initiate from a single ‘ATG’ start site (vertical arrow pointing up) within PCA3-4 and terminate within one of the alternatively spliced exons (2a or 2b or 2c) or within exon 3. (B) RT-PCR amplification of BPH, PCa and PCa metastasis samples using a forward primer from within the novel PCA3-4 transcription start site and a reverse primer from novel exon 2a. (C) Complete structure of the PCA3 gene. Shading identifies the newly identified regions of the PCA3 gene which has 6 exons with alternate splicing of exon 2a (93 bp) and exon 2b (93 bp) and exon 2c (original exon 2, 165 bp). and (D) RT-PCR amplification of PCA3 using the same forward primer and a reverse primer from novel exon 2b and (E) RT-PCR amplification of PCA3 using a forward primer from PCA3-5F (within the original transcription start site) and a reverse primer spanning exons 1 and 3 [15] .

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

    39) Product Images from "New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood"

    Article Title: New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113052

    Generation of a new temperature-sensitive Sendai virus vector, TS12KOS. ( a ) Comparison of schematic structures among the newly constructed Sendai virus (SeV) vector, TS12KOS, and previous vectors. The TS12KOS vector contains three point mutations in the RNA polymerase–related gene (P) and carries the coding sequences of KLF4 (K), OCT3/4 (O), and SOX2 (S) in the KOS direction. In comparison, the HNL/TS15 c-Myc vector carries two additional mutations, L1361C and L1558I, in the large polymerase (L) gene and an exogenous c- MYC cDNA sequence inserted between the hemagglutinin-neuraminidase (HN) and L genes, and the conventional vectors individually carry three reprogramming factors as indicated. ( b ) iPS cell generation from human skin-derived fibroblasts. The efficiency of iPS cell generation was significantly higher using the TS12KOS vector than with the conventional vectors at all multiplicities of infection (MOI) tested. iPSC colonies were identified on day 28 of induction by the appearance of alkaline phosphatase-positive (AP + ) colonies with embryonic stem (ES) cell-like colony morphology. N1, N2, and N3 represent individual healthy volunteers. Experiments were conducted in triplicate (mean ± SD). * P
    Figure Legend Snippet: Generation of a new temperature-sensitive Sendai virus vector, TS12KOS. ( a ) Comparison of schematic structures among the newly constructed Sendai virus (SeV) vector, TS12KOS, and previous vectors. The TS12KOS vector contains three point mutations in the RNA polymerase–related gene (P) and carries the coding sequences of KLF4 (K), OCT3/4 (O), and SOX2 (S) in the KOS direction. In comparison, the HNL/TS15 c-Myc vector carries two additional mutations, L1361C and L1558I, in the large polymerase (L) gene and an exogenous c- MYC cDNA sequence inserted between the hemagglutinin-neuraminidase (HN) and L genes, and the conventional vectors individually carry three reprogramming factors as indicated. ( b ) iPS cell generation from human skin-derived fibroblasts. The efficiency of iPS cell generation was significantly higher using the TS12KOS vector than with the conventional vectors at all multiplicities of infection (MOI) tested. iPSC colonies were identified on day 28 of induction by the appearance of alkaline phosphatase-positive (AP + ) colonies with embryonic stem (ES) cell-like colony morphology. N1, N2, and N3 represent individual healthy volunteers. Experiments were conducted in triplicate (mean ± SD). * P

    Techniques Used: Plasmid Preparation, Construct, Sequencing, Derivative Assay, Infection

    40) Product Images from "Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast"

    Article Title: Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast

    Journal: Nature Communications

    doi: 10.1038/ncomms6576

    Two distinct lncRNAs are transcribed from a bidirectional promoter upstream of tgp1 + . ( a ) Previously published strand-specific RNA-Seq analysis (Rhind et al ., 31 ) upstream of SPBC1271.09/ tgp1 + , represented as reads per kilobase per million (RPKM). Location of qPCR primer pairs and probes for Northern analysis are shown below. ( b ) Rbp1 ChIP–qPCR experiments performed in wild-type cells. ( c , e , g ) Northern analysis of nc-1343 , nc-tgp1 and tgp1 + transcript levels in wild-type, rrp6Δ , mmi1Δ and 1343Δ , respectively. ( d , f , h ) RT–qPCR experiments measured nc-1343 , nc-tgp1 and tgp1 + transcript levels in wild-type, rrp6Δ , mmi1Δ and 1343Δ , respectively. Error bars represent s.e.m. resulting from at least three independent replicates.
    Figure Legend Snippet: Two distinct lncRNAs are transcribed from a bidirectional promoter upstream of tgp1 + . ( a ) Previously published strand-specific RNA-Seq analysis (Rhind et al ., 31 ) upstream of SPBC1271.09/ tgp1 + , represented as reads per kilobase per million (RPKM). Location of qPCR primer pairs and probes for Northern analysis are shown below. ( b ) Rbp1 ChIP–qPCR experiments performed in wild-type cells. ( c , e , g ) Northern analysis of nc-1343 , nc-tgp1 and tgp1 + transcript levels in wild-type, rrp6Δ , mmi1Δ and 1343Δ , respectively. ( d , f , h ) RT–qPCR experiments measured nc-1343 , nc-tgp1 and tgp1 + transcript levels in wild-type, rrp6Δ , mmi1Δ and 1343Δ , respectively. Error bars represent s.e.m. resulting from at least three independent replicates.

    Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Northern Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR

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    Real-time Polymerase Chain Reaction:

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    Amplification:

    Article Title: Deficiency in the nuclear long noncoding RNA Charme causes myogenic defects and heart remodeling in mice
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    Quantitation Assay:

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    Quantitative RT-PCR:

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    Purification:

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    SYBR Green Assay:

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    Polymerase Chain Reaction:

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    Thermo Fisher superscript iii rt
    EML1 and EML3 associate with similar regions of CaLCuV DNA A. (A) Linear representation of the circular dsDNA A genome component (2,583 bp), showing viral open reading frames (shaded boxes), the intergenic region (IR) containing divergent promoters and transcription start sites (right-angle arrows), viral transcripts (arrows), and the amplicons (Amp 1, 2, and 3) tested in ChIP experiments. (B) ChIP-qPCR was performed with nuclear extracts from silique and floral tissues of CaLCuV-infected GFP-EML1 transgenic plants and Ler-0 control plants, using GFP antibody and primers amplifying the indicated amplicons. An amplicon within the FLC promoter served as a negative control. (C) ChIP-qPCR was performed with nuclear extracts from N. benthamiana leaf tissue coinfiltrated with constructs to express CaLCuV DNA A, CaLCuV DNA B, and <t>FLAG-EML3</t> or FLAG-GFP (control), using FLAG antibody and primers amplifying the indicated amplicons. Bars indicate standard errors for data compiled from at least <t>three</t> biological replicates, each with at least two technical replicates.
    Superscript Iii Rt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii
    Validation of antisense transcripts. a Nanostring nCounter assays: controls. <t>RNA</t> was isolated from the <t>three</t> indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top
    Superscript Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including ( A ) mTOR complexes, ( B ) mTORC1-positive regulation, and ( C ) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total <t>RNA</t> samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of <t>three</t> independent experiments; * P
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    EML1 and EML3 associate with similar regions of CaLCuV DNA A. (A) Linear representation of the circular dsDNA A genome component (2,583 bp), showing viral open reading frames (shaded boxes), the intergenic region (IR) containing divergent promoters and transcription start sites (right-angle arrows), viral transcripts (arrows), and the amplicons (Amp 1, 2, and 3) tested in ChIP experiments. (B) ChIP-qPCR was performed with nuclear extracts from silique and floral tissues of CaLCuV-infected GFP-EML1 transgenic plants and Ler-0 control plants, using GFP antibody and primers amplifying the indicated amplicons. An amplicon within the FLC promoter served as a negative control. (C) ChIP-qPCR was performed with nuclear extracts from N. benthamiana leaf tissue coinfiltrated with constructs to express CaLCuV DNA A, CaLCuV DNA B, and FLAG-EML3 or FLAG-GFP (control), using FLAG antibody and primers amplifying the indicated amplicons. Bars indicate standard errors for data compiled from at least three biological replicates, each with at least two technical replicates.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: EML1 and EML3 associate with similar regions of CaLCuV DNA A. (A) Linear representation of the circular dsDNA A genome component (2,583 bp), showing viral open reading frames (shaded boxes), the intergenic region (IR) containing divergent promoters and transcription start sites (right-angle arrows), viral transcripts (arrows), and the amplicons (Amp 1, 2, and 3) tested in ChIP experiments. (B) ChIP-qPCR was performed with nuclear extracts from silique and floral tissues of CaLCuV-infected GFP-EML1 transgenic plants and Ler-0 control plants, using GFP antibody and primers amplifying the indicated amplicons. An amplicon within the FLC promoter served as a negative control. (C) ChIP-qPCR was performed with nuclear extracts from N. benthamiana leaf tissue coinfiltrated with constructs to express CaLCuV DNA A, CaLCuV DNA B, and FLAG-EML3 or FLAG-GFP (control), using FLAG antibody and primers amplifying the indicated amplicons. Bars indicate standard errors for data compiled from at least three biological replicates, each with at least two technical replicates.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Infection, Transgenic Assay, Amplification, Negative Control, Construct

    Mutant eml1 plants exhibit hypersusceptibility and eml3 plants show increased tolerance to CaLCuV infection. (A) Illustration of transposon/T-DNA insertion sites (triangles) and EMS mutations (asterisks) within EML1 and EML3 genes. Arrows indicate the positions of forward (F) and reverse (R) primers used to amplify transcript cDNAs. UTR, untranslated region. (B) Relative EML1 and EML3 mRNA levels, measured by RT-qPCR, in extracts from silique tissue of eml mutants and cognate wild-type plants using the 2 −ΔΔC T method. Values were normalized to values for PP2A (At1g13320). (C) Representative photographs of mock-inoculated and CaLCuV-infected wild-type Ler-0 plants and eml1 plants (top) and wild-type Col-0 and eml3 plants (bottom). (D) Bolt heights were measured from CaLCuV-infected plants (Col-0, n = 96; eml3 , n = 46; Ler-0, n = 45; eml1 , n = 67), mock-inoculated plants (Col-0, n = 9; eml3 , n = 9; Ler-0, n = 11; eml1 , n = 19), and TCV-infected plants (Col-0, n = 70; eml3 , n = 62; Ler-0, n = 80; eml1 , n = 51). The average bolt height per plant was calculated for each treatment, and distributions are depicted in box-and-whisker plots. (E) Representative photographs and bolt measurements of CaLCuV-infected, transgenic GFP-EML1 plants ( n = 68) and eml1 plants ( n = 34). Bolt heights were measured and plotted as described above for panel D. (F) Levels of CaLCuV DNA A and DNA B in silique and floral tissues of eml1 and eml3 plants, collected at 10, 12, and 14 days postinoculation (dpi), were quantified by qPCR, normalized to 18S ribosomal DNA levels, and compared to viral DNA levels in cognate wild-type plants. Data presented in panels B and D to F were compiled from a minimum of three biological replicates. Significance values were determined using Student's two-tailed t test. Bars in panels B and F indicate standard errors.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: Mutant eml1 plants exhibit hypersusceptibility and eml3 plants show increased tolerance to CaLCuV infection. (A) Illustration of transposon/T-DNA insertion sites (triangles) and EMS mutations (asterisks) within EML1 and EML3 genes. Arrows indicate the positions of forward (F) and reverse (R) primers used to amplify transcript cDNAs. UTR, untranslated region. (B) Relative EML1 and EML3 mRNA levels, measured by RT-qPCR, in extracts from silique tissue of eml mutants and cognate wild-type plants using the 2 −ΔΔC T method. Values were normalized to values for PP2A (At1g13320). (C) Representative photographs of mock-inoculated and CaLCuV-infected wild-type Ler-0 plants and eml1 plants (top) and wild-type Col-0 and eml3 plants (bottom). (D) Bolt heights were measured from CaLCuV-infected plants (Col-0, n = 96; eml3 , n = 46; Ler-0, n = 45; eml1 , n = 67), mock-inoculated plants (Col-0, n = 9; eml3 , n = 9; Ler-0, n = 11; eml1 , n = 19), and TCV-infected plants (Col-0, n = 70; eml3 , n = 62; Ler-0, n = 80; eml1 , n = 51). The average bolt height per plant was calculated for each treatment, and distributions are depicted in box-and-whisker plots. (E) Representative photographs and bolt measurements of CaLCuV-infected, transgenic GFP-EML1 plants ( n = 68) and eml1 plants ( n = 34). Bolt heights were measured and plotted as described above for panel D. (F) Levels of CaLCuV DNA A and DNA B in silique and floral tissues of eml1 and eml3 plants, collected at 10, 12, and 14 days postinoculation (dpi), were quantified by qPCR, normalized to 18S ribosomal DNA levels, and compared to viral DNA levels in cognate wild-type plants. Data presented in panels B and D to F were compiled from a minimum of three biological replicates. Significance values were determined using Student's two-tailed t test. Bars in panels B and F indicate standard errors.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Mutagenesis, Infection, Quantitative RT-PCR, Whisker Assay, Transgenic Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

    Viral gene expression is upregulated in eml1 plants. (A) EML1 and EML3 transcript levels were measured by RT-qPCR in extracts from silique tissue of mock-inoculated and CaLCuV-infected plants. Transcripts were quantified by the 2 −ΔΔC T method. (B and C) Viral transcripts from the CaLCuV A genome ( AL1 and CP ) and B genome ( BL1 and BR1 ) were measured by RT-qPCR and compared in silique and floral tissues of Ler-0 and eml1 mutant plants (B) or Col-0 and eml3 plants (C). (D) The ratio of viral dsDNA to ssDNA was measured by DNA blotting (left). Locations of dsDNA and ssDNA, as well as background signals (asterisks), are shown. The percentage of each form relative to the total (dsDNA plus ssDNA) is shown to the right. Bars indicate standard errors for a minimum of three biological replicates, each with at least two technical replicates. Significance values were determined using Student's two-tailed t test.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: Viral gene expression is upregulated in eml1 plants. (A) EML1 and EML3 transcript levels were measured by RT-qPCR in extracts from silique tissue of mock-inoculated and CaLCuV-infected plants. Transcripts were quantified by the 2 −ΔΔC T method. (B and C) Viral transcripts from the CaLCuV A genome ( AL1 and CP ) and B genome ( BL1 and BR1 ) were measured by RT-qPCR and compared in silique and floral tissues of Ler-0 and eml1 mutant plants (B) or Col-0 and eml3 plants (C). (D) The ratio of viral dsDNA to ssDNA was measured by DNA blotting (left). Locations of dsDNA and ssDNA, as well as background signals (asterisks), are shown. The percentage of each form relative to the total (dsDNA plus ssDNA) is shown to the right. Bars indicate standard errors for a minimum of three biological replicates, each with at least two technical replicates. Significance values were determined using Student's two-tailed t test.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Expressing, Quantitative RT-PCR, Infection, Mutagenesis, Two Tailed Test

    Agenet domains have an incomplete aromatic cage. Multiple-sequence alignments comparing the Agenet domains of EML1-4, three Arabidopsis Tudor/PWWP/MBT superfamily domain proteins (AT1G80810, AT4G31880, and AT5G10950) sharing a high percentage of amino acid sequence similarity with EML Agenet domains, and the Tudor domains of the structural animal homologs PHF1 and PHF19 were constructed using ClustalW2. Highlighted residues in PHF1 and PHF19 are characterized, conserved aromatic residues necessary for the formation of the aromatic cage for H3K36me3 binding. Asterisks mark identical residues. Dots mark similar residues with various degrees of conservation.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: Agenet domains have an incomplete aromatic cage. Multiple-sequence alignments comparing the Agenet domains of EML1-4, three Arabidopsis Tudor/PWWP/MBT superfamily domain proteins (AT1G80810, AT4G31880, and AT5G10950) sharing a high percentage of amino acid sequence similarity with EML Agenet domains, and the Tudor domains of the structural animal homologs PHF1 and PHF19 were constructed using ClustalW2. Highlighted residues in PHF1 and PHF19 are characterized, conserved aromatic residues necessary for the formation of the aromatic cage for H3K36me3 binding. Asterisks mark identical residues. Dots mark similar residues with various degrees of conservation.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Sequencing, Construct, Binding Assay

    Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top

    Journal: Genome Biology

    Article Title: Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection

    doi: 10.1186/s13059-017-1329-5

    Figure Lengend Snippet: Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top

    Article Snippet: Per reaction, 1 μg total RNA was used and transcribed using SuperScript III with random hexamer primers (both Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Isolation, Infection, Transformation Assay, Expressing

    DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

    Journal: Emerging Microbes & Infections

    Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

    doi: 10.1080/22221751.2020.1713705

    Figure Lengend Snippet: DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

    Article Snippet: In brief, viral RNA was isolated from cell culture supernatant using the NucleoSpin RNA Virus kit (Macherey-Nagel), reverse-transcribed into cDNA using the Superscript III one step RT–PCR system (ThermoFisher Scientific) and analyzed on a LightCycler 480 qPCR cycler platform (Roche) with primers and conditions as specified for the upE assay [ ].

    Techniques: Binding Assay, Infection, Expressing, Mutagenesis, Negative Control, Incubation, Polymerase Chain Reaction

    Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including ( A ) mTOR complexes, ( B ) mTORC1-positive regulation, and ( C ) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total RNA samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of three independent experiments; * P

    Journal: Drug Design, Development and Therapy

    Article Title: Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells

    doi: 10.2147/DDDT.S95900

    Figure Lengend Snippet: Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including ( A ) mTOR complexes, ( B ) mTORC1-positive regulation, and ( C ) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total RNA samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of three independent experiments; * P

    Article Snippet: Two micrograms of total RNA was subjected to reverse transcription using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). qPCR was performed as previously described.

    Techniques: Inhibition, Real-time Polymerase Chain Reaction