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    Thermo Fisher superscript iii first strand synthesis supermix for qrt pcr
    Transcriptomic profiling reveals distinct clusters of genes regulated by IRF1, LXRβ and CEBPα. (a) Principal component analysis of Nanostring expression data (662 genes) demonstrating separation between each experimental condition. PC1 explained 34% while PC2 explained 14% of variance in the dataset. PC1 represents genes most strongly regulated by IRF1 while PC2 is representative of the LPS effect. (b) Heat map representing results from K-means clustering analysis of Nanostring gene expression data. Cluster 1 genes: upregulated by LPS and positively regulated by IRF1; Cluster 2 genes: negatively regulated by IRF1 under resting and activated conditions; Cluster 3 genes: down-regulated by LPS, positively regulated by IRF1; Cluster 4 genes: negatively regulated by all <t>three</t> TFs; Cluster 5 genes: down-regulated by IRF1 and LXRβ. (c) <t>qRT-PCR</t> validation of selected genes (Tyrobp, Spp1, Grn, C3ar1 and Lamp1) in IRF-1 siRNA treated microglia under resting and LPS-activated conditions (n=3 independent biological replicates, error bars represent standard error of mean, *P
    Superscript Iii First Strand Synthesis Supermix For Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii reverse transcriptase
    MEP-1, Mi-2, and Rpd3 involvement in piRNA-dependent repression. a Schematic representation of the krimper ( krimp ) piRNA-mediated repression assay after siRNA-mediated depletion of MEP-1, Mi-2, Rpd3, or Piwi in OSCs. Transfection of the apiRNA vector allows the production of antisense artificial piRNAs against krimp . Transfection of the GFPctrl vector is used as a piRNA-less negative control. b Fold change (relative to GFPctrl) in krimp <t>RNA</t> levels caused by apiRNA in OSCs previously transfected with the indicated siRNAs. RNA levels were quantified relative to RpL32 . Box plots display median (line), first and third quartiles (box), and highest/lowest value within 1.5× interquartile range (whiskers) for n = 6 values calculated over <t>three</t> independent samples. The siRNA effect was tested using the ANOVA test and differences using the pairwise t-test. P-values were calculated using sample data that displayed normal distribution (tested with the Shapiro–Wilkinson test). Variance homogeneity was tested with the Levene’s test and then the two-tailed Student’s t -test was used. *** P -value
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 102309 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii rnase h reverse transcriptase kit
    MEP-1, Mi-2, and Rpd3 involvement in piRNA-dependent repression. a Schematic representation of the krimper ( krimp ) piRNA-mediated repression assay after siRNA-mediated depletion of MEP-1, Mi-2, Rpd3, or Piwi in OSCs. Transfection of the apiRNA vector allows the production of antisense artificial piRNAs against krimp . Transfection of the GFPctrl vector is used as a piRNA-less negative control. b Fold change (relative to GFPctrl) in krimp <t>RNA</t> levels caused by apiRNA in OSCs previously transfected with the indicated siRNAs. RNA levels were quantified relative to RpL32 . Box plots display median (line), first and third quartiles (box), and highest/lowest value within 1.5× interquartile range (whiskers) for n = 6 values calculated over <t>three</t> independent samples. The siRNA effect was tested using the ANOVA test and differences using the pairwise t-test. P-values were calculated using sample data that displayed normal distribution (tested with the Shapiro–Wilkinson test). Variance homogeneity was tested with the Levene’s test and then the two-tailed Student’s t -test was used. *** P -value
    Superscript Iii Rnase H Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Transcriptomic profiling reveals distinct clusters of genes regulated by IRF1, LXRβ and CEBPα. (a) Principal component analysis of Nanostring expression data (662 genes) demonstrating separation between each experimental condition. PC1 explained 34% while PC2 explained 14% of variance in the dataset. PC1 represents genes most strongly regulated by IRF1 while PC2 is representative of the LPS effect. (b) Heat map representing results from K-means clustering analysis of Nanostring gene expression data. Cluster 1 genes: upregulated by LPS and positively regulated by IRF1; Cluster 2 genes: negatively regulated by IRF1 under resting and activated conditions; Cluster 3 genes: down-regulated by LPS, positively regulated by IRF1; Cluster 4 genes: negatively regulated by all three TFs; Cluster 5 genes: down-regulated by IRF1 and LXRβ. (c) qRT-PCR validation of selected genes (Tyrobp, Spp1, Grn, C3ar1 and Lamp1) in IRF-1 siRNA treated microglia under resting and LPS-activated conditions (n=3 independent biological replicates, error bars represent standard error of mean, *P

    Journal: Glia

    Article Title: Transcriptional regulation of homeostatic and disease-associated-microglial genes by IRF1, LXRβ and CEBPα

    doi: 10.1002/glia.23678

    Figure Lengend Snippet: Transcriptomic profiling reveals distinct clusters of genes regulated by IRF1, LXRβ and CEBPα. (a) Principal component analysis of Nanostring expression data (662 genes) demonstrating separation between each experimental condition. PC1 explained 34% while PC2 explained 14% of variance in the dataset. PC1 represents genes most strongly regulated by IRF1 while PC2 is representative of the LPS effect. (b) Heat map representing results from K-means clustering analysis of Nanostring gene expression data. Cluster 1 genes: upregulated by LPS and positively regulated by IRF1; Cluster 2 genes: negatively regulated by IRF1 under resting and activated conditions; Cluster 3 genes: down-regulated by LPS, positively regulated by IRF1; Cluster 4 genes: negatively regulated by all three TFs; Cluster 5 genes: down-regulated by IRF1 and LXRβ. (c) qRT-PCR validation of selected genes (Tyrobp, Spp1, Grn, C3ar1 and Lamp1) in IRF-1 siRNA treated microglia under resting and LPS-activated conditions (n=3 independent biological replicates, error bars represent standard error of mean, *P

    Article Snippet: Cells were cultured for 48h before to confirm the efficiency of siRNA-mediated gene silencing by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).

    Techniques: Expressing, Quantitative RT-PCR

    MEP-1, Mi-2, and Rpd3 involvement in piRNA-dependent repression. a Schematic representation of the krimper ( krimp ) piRNA-mediated repression assay after siRNA-mediated depletion of MEP-1, Mi-2, Rpd3, or Piwi in OSCs. Transfection of the apiRNA vector allows the production of antisense artificial piRNAs against krimp . Transfection of the GFPctrl vector is used as a piRNA-less negative control. b Fold change (relative to GFPctrl) in krimp RNA levels caused by apiRNA in OSCs previously transfected with the indicated siRNAs. RNA levels were quantified relative to RpL32 . Box plots display median (line), first and third quartiles (box), and highest/lowest value within 1.5× interquartile range (whiskers) for n = 6 values calculated over three independent samples. The siRNA effect was tested using the ANOVA test and differences using the pairwise t-test. P-values were calculated using sample data that displayed normal distribution (tested with the Shapiro–Wilkinson test). Variance homogeneity was tested with the Levene’s test and then the two-tailed Student’s t -test was used. *** P -value

    Journal: Nature Communications

    Article Title: The Mi-2 nucleosome remodeler and the Rpd3 histone deacetylase are involved in piRNA-guided heterochromatin formation

    doi: 10.1038/s41467-020-16635-5

    Figure Lengend Snippet: MEP-1, Mi-2, and Rpd3 involvement in piRNA-dependent repression. a Schematic representation of the krimper ( krimp ) piRNA-mediated repression assay after siRNA-mediated depletion of MEP-1, Mi-2, Rpd3, or Piwi in OSCs. Transfection of the apiRNA vector allows the production of antisense artificial piRNAs against krimp . Transfection of the GFPctrl vector is used as a piRNA-less negative control. b Fold change (relative to GFPctrl) in krimp RNA levels caused by apiRNA in OSCs previously transfected with the indicated siRNAs. RNA levels were quantified relative to RpL32 . Box plots display median (line), first and third quartiles (box), and highest/lowest value within 1.5× interquartile range (whiskers) for n = 6 values calculated over three independent samples. The siRNA effect was tested using the ANOVA test and differences using the pairwise t-test. P-values were calculated using sample data that displayed normal distribution (tested with the Shapiro–Wilkinson test). Variance homogeneity was tested with the Levene’s test and then the two-tailed Student’s t -test was used. *** P -value

    Article Snippet: Five hundred nanograms of total RNA was reverse transcribed using random primers and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Transfection, Plasmid Preparation, Negative Control, Two Tailed Test

    The ovarian somatic depletion of MEP-1 or Mi-2 results in TE and TE reporter derepression in vivo. a X-Gal staining of egg chambers that contain the somatic TE reporter ( Gypsy-lacZ ) in which the indicated genes were knocked down by constitutive (left panel) or conditional (right panel) somatic RNAi. Conditional somatic knockdown was restricted to adult flies. The control was a shRNA against the white gene ( shwhite ). b , c Fold change (log2) in the steady-state RNA levels of the Gypsy-lacZ reporter (lacZ) and of three endogenous TEs in ovaries after constitutive ( tj-GAL4 > sh ) ( b ) or conditional ( t-jGAL4 ; tubGAL80 ts > sh ) ( c ) somatic knockdown, using the indicated RNAi. RNA levels were quantified relative to RpL32 levels and normalized to control knockdown ( shwhite ). Lines represent means ± SD for n = 3 biologically independent samples. Source data for b , c : Supplementary Data set 4 .

    Journal: Nature Communications

    Article Title: The Mi-2 nucleosome remodeler and the Rpd3 histone deacetylase are involved in piRNA-guided heterochromatin formation

    doi: 10.1038/s41467-020-16635-5

    Figure Lengend Snippet: The ovarian somatic depletion of MEP-1 or Mi-2 results in TE and TE reporter derepression in vivo. a X-Gal staining of egg chambers that contain the somatic TE reporter ( Gypsy-lacZ ) in which the indicated genes were knocked down by constitutive (left panel) or conditional (right panel) somatic RNAi. Conditional somatic knockdown was restricted to adult flies. The control was a shRNA against the white gene ( shwhite ). b , c Fold change (log2) in the steady-state RNA levels of the Gypsy-lacZ reporter (lacZ) and of three endogenous TEs in ovaries after constitutive ( tj-GAL4 > sh ) ( b ) or conditional ( t-jGAL4 ; tubGAL80 ts > sh ) ( c ) somatic knockdown, using the indicated RNAi. RNA levels were quantified relative to RpL32 levels and normalized to control knockdown ( shwhite ). Lines represent means ± SD for n = 3 biologically independent samples. Source data for b , c : Supplementary Data set 4 .

    Article Snippet: Five hundred nanograms of total RNA was reverse transcribed using random primers and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: In Vivo, Staining, shRNA

    Rpd3, Mi-2, and MEP-1 are implicated in TE epigenetic silencing in OSCs. a Scatterplots of RNA-seq reads in FPKM (fragments per kilobase of exon per million reads mapped) for 108 annotated D. melanogaster TEs in control knockdown (KD) ( siGFP ) vs. Piwi-KD ( sipiwi ) (left), Mi-2 KD ( siMi-2 ) (middle), and Rpd3-KD ( siRpd3 ) (right). TEs for which the expression level differed from control by more than two-fold in Piwi-KD (Piwi–piRNA-targeted TEs) are plotted in orange. Both x -axis and y -axis are a log10 scale. b A browser screenshot shows RNA-seq tracks upon GFP ( siGFP ), Piwi ( sipiwi ), Mi-2 ( siMi-2 ), and Rpd3 ( siRpd3 ) knockdown at the expanded ( ex ) locus. OSC-specific Gypsy insertion site is annotated; ex TSS is indicated with a dashed line. c RT-qPCR fold changes in steady-state RNA levels of three endogenous TEs ( Tabor , Gypsy , and mdg1 ) and of the expanded ( ex ) gene upon MEP-1 ( siMEP-1 ), Mi-2 ( siMi-2 ), Rpd3 ( siRpd3 ), or Piwi ( sipiwi ) knockdown using siRNAs. Dots show RNA values quantified relative to Rpl32 and normalized to control knockdown for n = 3 biologically independent samples, lines represent mean values ± SD (log2). d – f H3K9me3 ( d ), H3 ( e ), or H3K9ace ( f ) quantified by ChIP-qPCR at mdg1 and Gypsy TE genomic loci, as well as on the expanded ( ex ) and krimper ( krimp ) genes after knockdown using the indicated siRNA. Values relative to a positive control ( 1360-element for H3K9me3 or RpL32 for H3 and H3K9ace) were normalized to input (mean ± SD from n = 3 independent biological replicates). Source data for c – f : Supplementary Data set 3 .

    Journal: Nature Communications

    Article Title: The Mi-2 nucleosome remodeler and the Rpd3 histone deacetylase are involved in piRNA-guided heterochromatin formation

    doi: 10.1038/s41467-020-16635-5

    Figure Lengend Snippet: Rpd3, Mi-2, and MEP-1 are implicated in TE epigenetic silencing in OSCs. a Scatterplots of RNA-seq reads in FPKM (fragments per kilobase of exon per million reads mapped) for 108 annotated D. melanogaster TEs in control knockdown (KD) ( siGFP ) vs. Piwi-KD ( sipiwi ) (left), Mi-2 KD ( siMi-2 ) (middle), and Rpd3-KD ( siRpd3 ) (right). TEs for which the expression level differed from control by more than two-fold in Piwi-KD (Piwi–piRNA-targeted TEs) are plotted in orange. Both x -axis and y -axis are a log10 scale. b A browser screenshot shows RNA-seq tracks upon GFP ( siGFP ), Piwi ( sipiwi ), Mi-2 ( siMi-2 ), and Rpd3 ( siRpd3 ) knockdown at the expanded ( ex ) locus. OSC-specific Gypsy insertion site is annotated; ex TSS is indicated with a dashed line. c RT-qPCR fold changes in steady-state RNA levels of three endogenous TEs ( Tabor , Gypsy , and mdg1 ) and of the expanded ( ex ) gene upon MEP-1 ( siMEP-1 ), Mi-2 ( siMi-2 ), Rpd3 ( siRpd3 ), or Piwi ( sipiwi ) knockdown using siRNAs. Dots show RNA values quantified relative to Rpl32 and normalized to control knockdown for n = 3 biologically independent samples, lines represent mean values ± SD (log2). d – f H3K9me3 ( d ), H3 ( e ), or H3K9ace ( f ) quantified by ChIP-qPCR at mdg1 and Gypsy TE genomic loci, as well as on the expanded ( ex ) and krimper ( krimp ) genes after knockdown using the indicated siRNA. Values relative to a positive control ( 1360-element for H3K9me3 or RpL32 for H3 and H3K9ace) were normalized to input (mean ± SD from n = 3 independent biological replicates). Source data for c – f : Supplementary Data set 3 .

    Article Snippet: Five hundred nanograms of total RNA was reverse transcribed using random primers and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control

    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.

    Journal: Scientific Reports

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

    doi: 10.1038/srep02938

    Figure Lengend 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.

    Article Snippet: Briefly, 1 μg of RNA from distal colon tissue from AOM/DSS-treated and vehicle-treated control mice were reverse transcribed by using Superscript III (Invitrogen).

    Techniques: Injection, Mouse Assay, Staining

    Shu maps to the voltage-gated sodium channel gene paralytic. A , Mapping positions of the Shu mutation on the X chromosome. Red triangles and horizontal lines represent pairs of molecularly defined P-element insertions used to estimate the Shu mutation site; red X’s indicate the mutation sites deduced from the recombination rates between the corresponding P-element insertion and Shu . The estimated sites all reside within the para locus (CG9907). Boxes designate annotated genes near the para locus (based on FlyBase). B , DNA sequencing chromatogram identifying a G-to-A transition mutation (arrowheads) in the Shu genome at the position corresponding to the nucleotide 4249 in the para-RE cDNA (FlyBase). This mutation results in a methionine-to-isoleucine substitution at the amino acid position 1327. C , Schematic structural diagram of a Drosophila voltage-gated sodium channel. Arrow indicates the Shu mutation in the transmembrane segment S2 in homology domain III. The para GEFS+ mutation K1330T, which corresponds to a SCN1A mutation K1270T causing GEFS+ in humans ( Sun et al., 2012 ), lies three codons away from that of Shu . Also shown are the para DS mutation S1291R and the para bss1 mutation L1676F. D , Amino acid sequence alignment of Na v channels of different animal species. Note that the methionine residue, which is mutated to isoleucine in Shu , is present in all Na v channels.

    Journal: eNeuro

    Article Title: Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic

    doi: 10.1523/ENEURO.0221-16.2016

    Figure Lengend Snippet: Shu maps to the voltage-gated sodium channel gene paralytic. A , Mapping positions of the Shu mutation on the X chromosome. Red triangles and horizontal lines represent pairs of molecularly defined P-element insertions used to estimate the Shu mutation site; red X’s indicate the mutation sites deduced from the recombination rates between the corresponding P-element insertion and Shu . The estimated sites all reside within the para locus (CG9907). Boxes designate annotated genes near the para locus (based on FlyBase). B , DNA sequencing chromatogram identifying a G-to-A transition mutation (arrowheads) in the Shu genome at the position corresponding to the nucleotide 4249 in the para-RE cDNA (FlyBase). This mutation results in a methionine-to-isoleucine substitution at the amino acid position 1327. C , Schematic structural diagram of a Drosophila voltage-gated sodium channel. Arrow indicates the Shu mutation in the transmembrane segment S2 in homology domain III. The para GEFS+ mutation K1330T, which corresponds to a SCN1A mutation K1270T causing GEFS+ in humans ( Sun et al., 2012 ), lies three codons away from that of Shu . Also shown are the para DS mutation S1291R and the para bss1 mutation L1676F. D , Amino acid sequence alignment of Na v channels of different animal species. Note that the methionine residue, which is mutated to isoleucine in Shu , is present in all Na v channels.

    Article Snippet: Reverse-transcription PCR analysis For semiquantitative and real-time reverse-transcription (RT)-PCR, RNA was extracted using the methods described above, and single-strand cDNA libraries were synthesized with DNase I-treated RNA using Superscript III reverse transcriptase kit (Invitrogen) or, in the real-time experiments, iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Mutagenesis, DNA Sequencing, Sequencing

    Nova2 expression levels and AS of its target are regulated in ECs. ( a ) RT–qPCR analysis of Nova2 and Nova1 mRNA expression levels in mouse ECs grown as confluent or sparse (left), in E9.5 and E15.5 mouse whole brain (centre), and RT–qPCR analysis of Muscleblind family members ( Mbnl1 , Mbnl2 and Mbnl3 ) in mouse ECs grown at different densities (right). ( b ) Immunoblotting analysis of Nova2 levels in mouse confluent and sparse ECs (left panel; Tubulin as the loading control) and in confluent ECs and the mouse brain cortex (right panel; GAPDH as loading control). ( c ) Nova2 mRNA and protein expression levels in HUVECs grown at different densities. ( d ) RT–qPCR analysis of Nova2 during endothelial differentiation of mouse ES cells at the indicated times. ( e ) Immunoblotting analysis of Nova2 in mouse EC lines derived from whole embryo, fetal heart and adult lung; Actin as the loading control. In all histograms, error bars indicate ±s.d. calculated from three independent experiments ( n =3). ( f ) RT–PCR analysis of AS of a known Nova2 target ( Ankyrin3 / Ank3 ) in mouse ECs (confluent and sparse; left), during endothelial differentiation of mouse ES cells (centre) and in mouse ECs of different origins (right). The schematic representation of the mouse gene structure (AS exon in grey; constitutive exons in black), the YCAY cluster predicted to function as Nova2 silencer (blue dot) and the Nova2-regulated exon-skipping event (blue bars) are also shown. NISS, Nova intronic splicing silencer. The percentage of exon inclusion is shown. Asterisk indicates a nonspecific PCR product.

    Journal: Nature Communications

    Article Title: The alternative splicing factor Nova2 regulates vascular development and lumen formation

    doi: 10.1038/ncomms9479

    Figure Lengend Snippet: Nova2 expression levels and AS of its target are regulated in ECs. ( a ) RT–qPCR analysis of Nova2 and Nova1 mRNA expression levels in mouse ECs grown as confluent or sparse (left), in E9.5 and E15.5 mouse whole brain (centre), and RT–qPCR analysis of Muscleblind family members ( Mbnl1 , Mbnl2 and Mbnl3 ) in mouse ECs grown at different densities (right). ( b ) Immunoblotting analysis of Nova2 levels in mouse confluent and sparse ECs (left panel; Tubulin as the loading control) and in confluent ECs and the mouse brain cortex (right panel; GAPDH as loading control). ( c ) Nova2 mRNA and protein expression levels in HUVECs grown at different densities. ( d ) RT–qPCR analysis of Nova2 during endothelial differentiation of mouse ES cells at the indicated times. ( e ) Immunoblotting analysis of Nova2 in mouse EC lines derived from whole embryo, fetal heart and adult lung; Actin as the loading control. In all histograms, error bars indicate ±s.d. calculated from three independent experiments ( n =3). ( f ) RT–PCR analysis of AS of a known Nova2 target ( Ankyrin3 / Ank3 ) in mouse ECs (confluent and sparse; left), during endothelial differentiation of mouse ES cells (centre) and in mouse ECs of different origins (right). The schematic representation of the mouse gene structure (AS exon in grey; constitutive exons in black), the YCAY cluster predicted to function as Nova2 silencer (blue dot) and the Nova2-regulated exon-skipping event (blue bars) are also shown. NISS, Nova intronic splicing silencer. The percentage of exon inclusion is shown. Asterisk indicates a nonspecific PCR product.

    Article Snippet: Total RNA was extracted from 24-hpf pooled embryos with TRIzol reagent (Invitrogen), purified with the RNeasy Mini Kit (QIAGEN) and retro-transcribed with d(T)18 oligo or gene-specific primer and Superscript III RT (Invitrogen) and then analysed in PCR for AS modification of nova2 targets.

    Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    ) with modifications, using Invitrogen Superscript III RNase H- reverse transcriptase and 32 P-labeled primers for 40 min at 52°C. Lanes U to A, sequencing ladders generated by PCR with pRZ6-2 template and 32 P-labeled primers in the presence of ddATP, ddCTP, ddGTP, and ddUTP by utilizing the Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH). All samples were run on an 8% polyacrylamide-8 M urea sequencing gel. The nucleotide sequence of the (+)-PSTVd is given on the left, and the arrows point to the bands corresponding to reverse transcription termination at A99.

    Journal: Journal of Virology

    Article Title: Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo ▿

    doi: 10.1128/JVI.01781-06

    Figure Lengend Snippet: ) with modifications, using Invitrogen Superscript III RNase H- reverse transcriptase and 32 P-labeled primers for 40 min at 52°C. Lanes U to A, sequencing ladders generated by PCR with pRZ6-2 template and 32 P-labeled primers in the presence of ddATP, ddCTP, ddGTP, and ddUTP by utilizing the Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH). All samples were run on an 8% polyacrylamide-8 M urea sequencing gel. The nucleotide sequence of the (+)-PSTVd is given on the left, and the arrows point to the bands corresponding to reverse transcription termination at A99.

    Article Snippet: The reverse transcription was carried out following the protocols described by Baumstark et al. ( ) with modifications, using primer R1 (5′-AAACCCTGTTTCGGCGGGAATTAC-3′, complementary to position 154-179 on the PSTVd genome) (Fig. ) and the SuperScript III RNase H- reverse transcriptase (Invitrogen, Carlsbad, CA) at 52°C for 40 min. As shown in Fig. , reverse transcription terminated predominantly at A99 when the cross-linked PSTVd RNA was used as the template (lane cI), compared to the situation when the non-cross-linked circular PSTVd RNA was used as the template (lane c).

    Techniques: Labeling, Sequencing, Generated, Polymerase Chain Reaction

    GSK-3 β differentially affects the binding of NF- κ B p65 and p50 to the BCL2L1 and BIRC3 promoters. ( a , b ) qRT-PCR analysis of MEF and Panc04.03 cells following TNF α -stimulation. Note that Bcl-xL and cIAP2 transcripts, but not IKB α , were reduced in Gsk-3 β- null MEFs compared with WT control ( a ). Similarly, Bcl-xL and cIAP2, but not IKB α mRNA expression was reduced in TNF α -treated Panc04.03 cells transduced with lentiviral GSK-3 β shRNA ( b ). ( c – e ). ChIP analysis of Panc04.03 cells treated with GSK-3i (0.5 μ M LY2064827) or diluent (0.1% DMSO) for 16 h. The relative amount of DNA precipitated with the specified antibodies was accessed by qPCR using three sets of primers targeting promoter regions containing the depicted NF- κ B sites. The qPCR was performed in triplicate; and the result is normalized to diluent control

    Journal: Cell Death & Disease

    Article Title: Differential activity of GSK-3 isoforms regulates NF-κB and TRAIL- or TNFα induced apoptosis in pancreatic cancer cells

    doi: 10.1038/cddis.2014.102

    Figure Lengend Snippet: GSK-3 β differentially affects the binding of NF- κ B p65 and p50 to the BCL2L1 and BIRC3 promoters. ( a , b ) qRT-PCR analysis of MEF and Panc04.03 cells following TNF α -stimulation. Note that Bcl-xL and cIAP2 transcripts, but not IKB α , were reduced in Gsk-3 β- null MEFs compared with WT control ( a ). Similarly, Bcl-xL and cIAP2, but not IKB α mRNA expression was reduced in TNF α -treated Panc04.03 cells transduced with lentiviral GSK-3 β shRNA ( b ). ( c – e ). ChIP analysis of Panc04.03 cells treated with GSK-3i (0.5 μ M LY2064827) or diluent (0.1% DMSO) for 16 h. The relative amount of DNA precipitated with the specified antibodies was accessed by qPCR using three sets of primers targeting promoter regions containing the depicted NF- κ B sites. The qPCR was performed in triplicate; and the result is normalized to diluent control

    Article Snippet: RNA extraction and qRT-PCR RNA extraction using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), generation of cDNA with Superscript III reverse transcription Kit (Invitrogen) and PCR using the comparative CT method with the SYBR Green PCR Master Mix (Applied Biosystems, Grand Island, NY, USA) and the ABI Prism 7900TM Sequence Detection System have previously been described.

    Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Transduction, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    CKII Phosphorylation of Spt6 Regulates Chromatin Integrity during Transcription (A) Schematic of the SRG1 and SER3 loci showing their expression patterns in WT and mutant strains, as indicated by red (WT) and green (mutant) arrows. (B) Representative RNA-seq tracks showing an increase in the expression level of the SER3 gene in WT, spt6 S8 → A8 , spt6 S8 → E8 , and spt6–1004 allele. (C) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6–1004 mutant strain. (D) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. (E) ChIP analysis of histone H3 levels across SRG1 and SER3 was performed with WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. Amplicons are indicated below the schematic diagram of the genes. Quantitative real-time PCR and ChIP data are represented as means ± SDs of three independent biological experiments. Asterisks indicate significance values (**p

    Journal: Cell reports

    Article Title: Casein Kinase II Phosphorylation of Spt6 Enforces Transcriptional Fidelity by Maintaining Spn1-Spt6 Interaction

    doi: 10.1016/j.celrep.2018.11.089

    Figure Lengend Snippet: CKII Phosphorylation of Spt6 Regulates Chromatin Integrity during Transcription (A) Schematic of the SRG1 and SER3 loci showing their expression patterns in WT and mutant strains, as indicated by red (WT) and green (mutant) arrows. (B) Representative RNA-seq tracks showing an increase in the expression level of the SER3 gene in WT, spt6 S8 → A8 , spt6 S8 → E8 , and spt6–1004 allele. (C) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6–1004 mutant strain. (D) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. (E) ChIP analysis of histone H3 levels across SRG1 and SER3 was performed with WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. Amplicons are indicated below the schematic diagram of the genes. Quantitative real-time PCR and ChIP data are represented as means ± SDs of three independent biological experiments. Asterisks indicate significance values (**p

    Article Snippet: The isolated RNA was treated with 10 U of RNase-free DNase (Promega) for 30 minutes, followed by RNA cleanup (QIAGEN RNeasy Mini Kit, 74106). cDNA was synthesized from one mg of total RNA using random hexamer primers and Superscript Reverse Transcriptase III (Thermo-Fisher Scientific, 108–80044).

    Techniques: Expressing, Mutagenesis, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    The roles of STAT3 and NF-κB in the induction of miR-21 by IFN DU145 cells were (A) transfected with DN-F705 STAT3 (F705) or (B) transduced with lentivirus encoding shRNA for STAT3, and treated with human IFNα (1000 IU/ml for 6 h). (C,D) MEFs from wild type mice or mice in which the p65 subunit of NF-κB was knocked out (p65 −/− ) were treated with murine IFN (1000 IU/ml) or LPS (40 ng/ml) as indicated for 6 h. (C) miR-21, or (D) miR-296 or miR-351 expression was determined by qPCR and expressed relative to U6 RNA levels. Data represent the mean +/− SD of at least three experiments performed in duplicate.

    Journal: Cancer research

    Article Title: Interferon induces miR-21 through a STAT3-dependent pathway as a suppressive negative feedback on interferon-induced apoptosis

    doi: 10.1158/0008-5472.CAN-10-2579

    Figure Lengend Snippet: The roles of STAT3 and NF-κB in the induction of miR-21 by IFN DU145 cells were (A) transfected with DN-F705 STAT3 (F705) or (B) transduced with lentivirus encoding shRNA for STAT3, and treated with human IFNα (1000 IU/ml for 6 h). (C,D) MEFs from wild type mice or mice in which the p65 subunit of NF-κB was knocked out (p65 −/− ) were treated with murine IFN (1000 IU/ml) or LPS (40 ng/ml) as indicated for 6 h. (C) miR-21, or (D) miR-296 or miR-351 expression was determined by qPCR and expressed relative to U6 RNA levels. Data represent the mean +/− SD of at least three experiments performed in duplicate.

    Article Snippet: PolyA-tailed RNA (6 μl) was reverse-transcribed into first-strand cDNA using Superscript III transcriptase (Invitrogen) with the oligo-dT adapter primer 5′GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′.

    Techniques: Transfection, Transduction, shRNA, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    IFN induces miR-21 in a variety of cell types (A) Human U87 glioma, DU145 and PC3 prostate cancer cells, and skin fibroblasts, and B16 mouse melanoma were treated with IFN (1000 IU/ml, for 6 h) prior to RNA extraction. DU145 cells were treated with IFN (1000 IU/ml) for the indicated times, or with the indicated concentrations of IFN for 6 h prior to RNA extraction and miR-21 (B) or ISG15 (C) expression was determined by qPCR. Expression was normalized to expression of U6 RNA for miR-21 and B-actin for ISG15, respectively. Data represent the mean +/− SD of at least three experiments performed in duplicate.

    Journal: Cancer research

    Article Title: Interferon induces miR-21 through a STAT3-dependent pathway as a suppressive negative feedback on interferon-induced apoptosis

    doi: 10.1158/0008-5472.CAN-10-2579

    Figure Lengend Snippet: IFN induces miR-21 in a variety of cell types (A) Human U87 glioma, DU145 and PC3 prostate cancer cells, and skin fibroblasts, and B16 mouse melanoma were treated with IFN (1000 IU/ml, for 6 h) prior to RNA extraction. DU145 cells were treated with IFN (1000 IU/ml) for the indicated times, or with the indicated concentrations of IFN for 6 h prior to RNA extraction and miR-21 (B) or ISG15 (C) expression was determined by qPCR. Expression was normalized to expression of U6 RNA for miR-21 and B-actin for ISG15, respectively. Data represent the mean +/− SD of at least three experiments performed in duplicate.

    Article Snippet: PolyA-tailed RNA (6 μl) was reverse-transcribed into first-strand cDNA using Superscript III transcriptase (Invitrogen) with the oligo-dT adapter primer 5′GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′.

    Techniques: RNA Extraction, Expressing, Real-time Polymerase Chain Reaction

    The HERV-K LTR does not possess a functional initiator element, but silencing of Sp1 and Sp3 reduces pGL3_pck30 promoter activity. (A) HERV-K transcription initiation is not mediated by an initiator element (Inr). The putative Inr wild-type sequence, TCAGTAA, was changed into a heptamer of adenines in the LTR reporter pGL3_pck30_Inr construct. Luciferase activities of pGL3_pck30 (wild type) and the pGL3_pck30_Inr mutant were determined in GH and Mel-C9 cells at 24 h posttransfection. The promoter activity of LTRpck30 was set to 100% for each cell line. The results are means ± SD for six individual experiments. (B) Immunoblot analysis of endogenous Sp1 and Sp3 protein levels in HeLa, HCl Mel 19, GH, and Mel-C9 cell lines. Fifty micrograms of cell lysate was used. Actin served as a loading control. (C) GH and Mel-C9 cells were cotransfected with pGL3_pck30 DNA and 150 ng specific siRNA or with 150 ng unrelated siRNA. % RLU are given as means ± SD for three individual transfections. The mock level was set to 100%. HERV-K LTR activity was significantly reduced by Sp1 as well as Sp3 ablation. (D) Sp1 or Sp3 knockdown reduced HERV-K promoter activity. Mel-C9 and GH cells were transfected with 150 or 300 ng of either Sp1- or Sp3-specific siRNA. Immunoblot analysis using 30 μg of protein revealed that the amounts of Sp1 and Sp3 proteins were significantly reduced by 150 ng of the respective siRNA at 48 h posttransfection. Actin was used to control for equal loading of cell lysates.

    Journal: Journal of Virology

    Article Title: Expression of the Human Endogenous Retrovirus (HERV) Group HML-2/HERV-K Does Not Depend on Canonical Promoter Elements but Is Regulated by Transcription Factors Sp1 and Sp3 ▿

    doi: 10.1128/JVI.02539-10

    Figure Lengend Snippet: The HERV-K LTR does not possess a functional initiator element, but silencing of Sp1 and Sp3 reduces pGL3_pck30 promoter activity. (A) HERV-K transcription initiation is not mediated by an initiator element (Inr). The putative Inr wild-type sequence, TCAGTAA, was changed into a heptamer of adenines in the LTR reporter pGL3_pck30_Inr construct. Luciferase activities of pGL3_pck30 (wild type) and the pGL3_pck30_Inr mutant were determined in GH and Mel-C9 cells at 24 h posttransfection. The promoter activity of LTRpck30 was set to 100% for each cell line. The results are means ± SD for six individual experiments. (B) Immunoblot analysis of endogenous Sp1 and Sp3 protein levels in HeLa, HCl Mel 19, GH, and Mel-C9 cell lines. Fifty micrograms of cell lysate was used. Actin served as a loading control. (C) GH and Mel-C9 cells were cotransfected with pGL3_pck30 DNA and 150 ng specific siRNA or with 150 ng unrelated siRNA. % RLU are given as means ± SD for three individual transfections. The mock level was set to 100%. HERV-K LTR activity was significantly reduced by Sp1 as well as Sp3 ablation. (D) Sp1 or Sp3 knockdown reduced HERV-K promoter activity. Mel-C9 and GH cells were transfected with 150 or 300 ng of either Sp1- or Sp3-specific siRNA. Immunoblot analysis using 30 μg of protein revealed that the amounts of Sp1 and Sp3 proteins were significantly reduced by 150 ng of the respective siRNA at 48 h posttransfection. Actin was used to control for equal loading of cell lysates.

    Article Snippet: The TSS and termination sites of HERV-K were identified using a GeneRacer kit with SuperScript III reverse transcriptase (RT) (Invitrogen) according to the manufacturer's instructions.

    Techniques: Functional Assay, Activity Assay, Sequencing, Construct, Luciferase, Mutagenesis, Transfection

    HERV-K LTR reporter plasmids analyzed with a dual-luciferase reporter system. GH (germ cell tumor) (A), Mel-C9 (melanoma) (B), and HCl Mel 19 (melanoma) (C) cells were transfected with pGL3_pck30, pGL3_LTR21, pGL3_basic (promoterless), or pGL3_control (SV40 promoter) DNA. (D) GH cells were also transfected with pGL3_pck30 DNA treated with the CpG dinucleotide-methylating enzyme SssI (pGL3_pck30meth). % RLU are shown as means ± standard deviations (SD) for six (A to C) or three (D) individual transfections. LTR21 was inactive in all cell lines (A to C, 4th bars), and LTRpck30 was active in GH and Mel-C9 cells (A and B, 3rd bars) but was silenced upon methylation of CpG (D, 2nd bar).

    Journal: Journal of Virology

    Article Title: Expression of the Human Endogenous Retrovirus (HERV) Group HML-2/HERV-K Does Not Depend on Canonical Promoter Elements but Is Regulated by Transcription Factors Sp1 and Sp3 ▿

    doi: 10.1128/JVI.02539-10

    Figure Lengend Snippet: HERV-K LTR reporter plasmids analyzed with a dual-luciferase reporter system. GH (germ cell tumor) (A), Mel-C9 (melanoma) (B), and HCl Mel 19 (melanoma) (C) cells were transfected with pGL3_pck30, pGL3_LTR21, pGL3_basic (promoterless), or pGL3_control (SV40 promoter) DNA. (D) GH cells were also transfected with pGL3_pck30 DNA treated with the CpG dinucleotide-methylating enzyme SssI (pGL3_pck30meth). % RLU are shown as means ± standard deviations (SD) for six (A to C) or three (D) individual transfections. LTR21 was inactive in all cell lines (A to C, 4th bars), and LTRpck30 was active in GH and Mel-C9 cells (A and B, 3rd bars) but was silenced upon methylation of CpG (D, 2nd bar).

    Article Snippet: The TSS and termination sites of HERV-K were identified using a GeneRacer kit with SuperScript III reverse transcriptase (RT) (Invitrogen) according to the manufacturer's instructions.

    Techniques: Luciferase, Transfection, Methylation

    Three GC boxes mediate transcriptional activity of the HERV-K LTR.

    Journal: Journal of Virology

    Article Title: Expression of the Human Endogenous Retrovirus (HERV) Group HML-2/HERV-K Does Not Depend on Canonical Promoter Elements but Is Regulated by Transcription Factors Sp1 and Sp3 ▿

    doi: 10.1128/JVI.02539-10

    Figure Lengend Snippet: Three GC boxes mediate transcriptional activity of the HERV-K LTR.

    Article Snippet: The TSS and termination sites of HERV-K were identified using a GeneRacer kit with SuperScript III reverse transcriptase (RT) (Invitrogen) according to the manufacturer's instructions.

    Techniques: Activity Assay

    RNA editing of EEF-derived miRNA. (A) Graphic illustration of A-to-I editing, which is catalyzed by ADAR enzymes. miRNA editing can occur in seed regions (as shown here) or outside of the seed regions. This graph was created with BioRender. (B) Boxplots showing the editing levels (median with interquartile range), in control and KA samples at the two different time points for six edited sites detected in EEF miRNAs. (C) The table shows evidence supporting the edited sites and (D) sequence motifs of the edited sites. This shows the nucleotide preferences of all sequences surrounding the six edited sites [three-bases long, with the edited nucleotide Adenosine (A) is centered]. All the six edited sites have preferable sequence motifs for ADAR enzymes, which is (1) over-represented uridine (U) but depleted guanosine (G) at the nucleotides before the edited sites and (2) over-represented guanosine at the nucleotides after the edited sites. Confidence of edited site ( Li et al., 2018 ) reported in brain ( Alon et al., 2012 ) and reported in exosomes ( Nigita et al., 2018 ).

    Journal: Frontiers in Neuroscience

    Article Title: Altered Biogenesis and MicroRNA Content of Hippocampal Exosomes Following Experimental Status Epilepticus

    doi: 10.3389/fnins.2019.01404

    Figure Lengend Snippet: RNA editing of EEF-derived miRNA. (A) Graphic illustration of A-to-I editing, which is catalyzed by ADAR enzymes. miRNA editing can occur in seed regions (as shown here) or outside of the seed regions. This graph was created with BioRender. (B) Boxplots showing the editing levels (median with interquartile range), in control and KA samples at the two different time points for six edited sites detected in EEF miRNAs. (C) The table shows evidence supporting the edited sites and (D) sequence motifs of the edited sites. This shows the nucleotide preferences of all sequences surrounding the six edited sites [three-bases long, with the edited nucleotide Adenosine (A) is centered]. All the six edited sites have preferable sequence motifs for ADAR enzymes, which is (1) over-represented uridine (U) but depleted guanosine (G) at the nucleotides before the edited sites and (2) over-represented guanosine at the nucleotides after the edited sites. Confidence of edited site ( Li et al., 2018 ) reported in brain ( Alon et al., 2012 ) and reported in exosomes ( Nigita et al., 2018 ).

    Article Snippet: For analysis of transcripts, complementary DNA (cDNA) was produced from 1 μg of the total RNA by reverse transcription using Superscript III Reverse Transcriptase enzyme (Invitrogen).

    Techniques: Derivative Assay, Sequencing

    Induction of Id1-3 ( A ), endogenous expression of p21 and p53 ( B ), and expression of p53 ( C ), p21 ( D ), p27 ( E ), p107 ( F ) and p130 ( G ) upon recombinant BMP4 administration to cell cultures 24hrs after administration of 40ng/ml recombinant BMP4. Messenger RNA expression levels of Id1, Id2 and Id3 are significantly up-regulated in WERI-Rb-1 ( A ) upon a BMP4 administration, whereas no changes in p53, p21, p27, p107 and p130 expression level were detectable ( C-G ). Endogeneous levels of p21 and p53 are, however, elevated in WERI-Rb1 cells ( B ). Data were gained from real-time PCR analyses of three independent experiments. Cells that were only treated with 0.1% BSA in 4mM HCl, the solvent for recombinant human BMP4, were taken as a control (ctr.) group for A and C-G , healthy human retina was used as a reference in B and the expression level was set as 1, respectively.

    Journal: International Journal of Biological Sciences

    Article Title: Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells

    doi:

    Figure Lengend Snippet: Induction of Id1-3 ( A ), endogenous expression of p21 and p53 ( B ), and expression of p53 ( C ), p21 ( D ), p27 ( E ), p107 ( F ) and p130 ( G ) upon recombinant BMP4 administration to cell cultures 24hrs after administration of 40ng/ml recombinant BMP4. Messenger RNA expression levels of Id1, Id2 and Id3 are significantly up-regulated in WERI-Rb-1 ( A ) upon a BMP4 administration, whereas no changes in p53, p21, p27, p107 and p130 expression level were detectable ( C-G ). Endogeneous levels of p21 and p53 are, however, elevated in WERI-Rb1 cells ( B ). Data were gained from real-time PCR analyses of three independent experiments. Cells that were only treated with 0.1% BSA in 4mM HCl, the solvent for recombinant human BMP4, were taken as a control (ctr.) group for A and C-G , healthy human retina was used as a reference in B and the expression level was set as 1, respectively.

    Article Snippet: Equal amounts of RNA were subjected to an RT-reaction using oligo(dt)20 primers (Invitrogen), employing the SuperScript III reverse transcriptase system (Invitrogen) and following the manufacturer`s instructions.

    Techniques: Expressing, Recombinant, RNA Expression, Real-time Polymerase Chain Reaction

    Growth kinetic of recombinant MVs carrying mutations in the SAM binding site. Vero cells were infected with each recombinant MV at an MOI of 0.01; supernatant and cells were harvested after three freeze-thaw cycles, and virus titer was determined by plaque assay in Vero cells. Parental rMV-Hu191 reached a peak titer after 72h post-infection (hpi) whereas peak titer for the mutants ranged from 84 to 96 hpi. Virus titer for the mutants detected at different time points were different from the parental rMV- Hu191. * =p

    Journal: Virology

    Article Title: Enhancement of safety and immunogenicity of the Chinese Hu191 measles virus vaccine by alteration of the S-adenosylmethionine (SAM) binding site in the large polymerase protein

    doi: 10.1016/j.virol.2018.02.022

    Figure Lengend Snippet: Growth kinetic of recombinant MVs carrying mutations in the SAM binding site. Vero cells were infected with each recombinant MV at an MOI of 0.01; supernatant and cells were harvested after three freeze-thaw cycles, and virus titer was determined by plaque assay in Vero cells. Parental rMV-Hu191 reached a peak titer after 72h post-infection (hpi) whereas peak titer for the mutants ranged from 84 to 96 hpi. Virus titer for the mutants detected at different time points were different from the parental rMV- Hu191. * =p

    Article Snippet: Viral RNA was extracted from 200 μl of MV-Hu191 using an RNeasy mini-kit (Qiagen), and reverse-transcribed using Super Script® III reverse transcriptase (Invitrogen) and Random Primer Mix (NEB).

    Techniques: Recombinant, Binding Assay, Infection, Plaque Assay

    Coronin 1A was dispensable for osteoclastogenesis. ( a – c ) BMMs were transduced with CSII-CMV-MCS-IRES2-Venus (empty vector) lentivirus or with lentivirus expressing coronin 1A, and then were cultured with M-CSF (10 ng/mL) and RANKL (50 ng/mL) to differentiate into osteoclasts. ( a , b ) Cultured cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). The numbers of TRAP-positive multinucleated cells (MNCs) ( > 3 nuclei) were counted. Data are means ± SD of three independent experiments. Scale bars, 200 μm. (Student’s t-test), NS: not-statistically significant. ( c ) Expression of osteoclast marker genes, Nfatc1, Dcstamp, Atp6v0d2, Acp5 and Ctsk during osteoclastogenesis. Total RNA was extracted from the cultured cells at the indicated time points and subjected to real-time PCR. (Student’s t-test), NS: not-statistically significant. ( d – f ) BMMs were transfected with luciferase- or coronin 1A-specific siRNA, and then were incubated with M-CSF and RANKL. ( d , e ), Osteoclast differentiation of the control and coronin 1A knockdown cells. The numbers of TRAP-positive MNCs ( > 3 nuclei) were counted. Data are means ± SD of three independent experiments. Scale bars, 200 μm. (Student’s t-test), NS: not-statistically significant. ( f ) Expression of osteoclast marker genes during osteoclast differentiation. Data are representative of three experiments. (Student’s t-test), NS: not-statistically significant.

    Journal: Scientific Reports

    Article Title: Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K

    doi: 10.1038/srep41710

    Figure Lengend Snippet: Coronin 1A was dispensable for osteoclastogenesis. ( a – c ) BMMs were transduced with CSII-CMV-MCS-IRES2-Venus (empty vector) lentivirus or with lentivirus expressing coronin 1A, and then were cultured with M-CSF (10 ng/mL) and RANKL (50 ng/mL) to differentiate into osteoclasts. ( a , b ) Cultured cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). The numbers of TRAP-positive multinucleated cells (MNCs) ( > 3 nuclei) were counted. Data are means ± SD of three independent experiments. Scale bars, 200 μm. (Student’s t-test), NS: not-statistically significant. ( c ) Expression of osteoclast marker genes, Nfatc1, Dcstamp, Atp6v0d2, Acp5 and Ctsk during osteoclastogenesis. Total RNA was extracted from the cultured cells at the indicated time points and subjected to real-time PCR. (Student’s t-test), NS: not-statistically significant. ( d – f ) BMMs were transfected with luciferase- or coronin 1A-specific siRNA, and then were incubated with M-CSF and RANKL. ( d , e ), Osteoclast differentiation of the control and coronin 1A knockdown cells. The numbers of TRAP-positive MNCs ( > 3 nuclei) were counted. Data are means ± SD of three independent experiments. Scale bars, 200 μm. (Student’s t-test), NS: not-statistically significant. ( f ) Expression of osteoclast marker genes during osteoclast differentiation. Data are representative of three experiments. (Student’s t-test), NS: not-statistically significant.

    Article Snippet: First-strand cDNA was then produced from total RNA using Reverse Transcriptase SuperScript III (Invitrogen).

    Techniques: Transduction, Plasmid Preparation, Expressing, Cell Culture, Staining, Marker, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Incubation

    Modulation of genes related to cancer specific pathway in oralspheres. (a) Gene expression profiling of cancer pathway finder specific array was performed using RNA from parental and oralspheres derived from OSC19 cells. OSC19 spheres displayed an increased expression of genes related to cell migration and metastasis (e.g., ANG1, ITGB3, MMP9 and c-Fos). (b) qRT-PCR analyses of MMP9 in control and oralspheres from OSC19 and Cal27. Expression of MMP9 is normalized to GAPDH. The results are presented as means of three different experiments with standard errors (**, p

    Journal: Scientific Reports

    Article Title: Identification of molecular signature of head and neck cancer stem-like cells

    doi: 10.1038/srep07819

    Figure Lengend Snippet: Modulation of genes related to cancer specific pathway in oralspheres. (a) Gene expression profiling of cancer pathway finder specific array was performed using RNA from parental and oralspheres derived from OSC19 cells. OSC19 spheres displayed an increased expression of genes related to cell migration and metastasis (e.g., ANG1, ITGB3, MMP9 and c-Fos). (b) qRT-PCR analyses of MMP9 in control and oralspheres from OSC19 and Cal27. Expression of MMP9 is normalized to GAPDH. The results are presented as means of three different experiments with standard errors (**, p

    Article Snippet: 1 μg of RNA was reverse-transcribed using superscript III reverse transcription (Invitrogen, Life technologies).

    Techniques: Expressing, Derivative Assay, Migration, Quantitative RT-PCR

    B cell development in Igh ε/ε and Igh γ1/γ1 mice is impaired. ( A ) Schematic representations of the Igh ε ( Top ) and Igh γ1 ( Bottom ) alleles. ( B ) FACS plots of live CD19 + and B220 + bone marrow cells ( Top plots) as well as live B220 + CD19 + and BCR − ( Bottom plots). Mature recirculating B cells (B220 hi BCR + ), immature B cells (B220 int BCR + ), and pro–B cell (B220 lo BCR − CD43 + ) frequencies are indicated ( n = 6). ( C ) FACS plots of splenic lymphocytes showing CD19 expression versus IgM, IgE, or IgG1 from the indicated mice ( n = 6). ( D and E ) Dot graph showing summary statistics of percentages ( D ), and total number ( E ), of splenic B cells from the indicated mice. Each dot represents one mouse ( n = 4–9). ( F ) Semiquantitative PCR analyses of J H -proximal V H 7183 and J H -distal V H J558 family rearrangements in sorted bone marrow pro-B cells from indicated mice. Dlg5 was amplified as a loading control. Threefold serial dilutions are shown. Results are typical of three experiments. Bands corresponding to rearrangements to various J H segments are indicated. ( G ) FACS plots showing intracellular Igμ, Igε, and Igγ1 heavy chain in pro-B cells (CD19 + B220 lo Igκ − CD43 + ) from bone marrows of the indicated mice. Results are typical of at least four experiments. ( H ) Percentage of intracellular Igμ, Igε, and Igγ1 heavy chain in BM pro-B cells. Each dot represents one mouse ( n = 5). ( I ) Semiquantitative PCR analyses of V κ Igl chain rearrangements in magnetically separated bone marrow B220 + cells from indicated mice. Intronic Ig κ was amplified as a loading control. Threefold serial dilutions are shown. Bands corresponding to rearrangements to various J κ segments are indicated on the Left . Results are typical of four experiments. ( J ) Quantitative PCR analyses of V κ to J κ1 Igl chain rearrangement relative to β -actin DNA in purified B220 + BM cells from the indicated mice. Expression is shown as fold change relative to wild-type levels. ** P

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

    Article Title: IgH isotype-specific B cell receptor expression influences B cell fate

    doi: 10.1073/pnas.1704962114

    Figure Lengend Snippet: B cell development in Igh ε/ε and Igh γ1/γ1 mice is impaired. ( A ) Schematic representations of the Igh ε ( Top ) and Igh γ1 ( Bottom ) alleles. ( B ) FACS plots of live CD19 + and B220 + bone marrow cells ( Top plots) as well as live B220 + CD19 + and BCR − ( Bottom plots). Mature recirculating B cells (B220 hi BCR + ), immature B cells (B220 int BCR + ), and pro–B cell (B220 lo BCR − CD43 + ) frequencies are indicated ( n = 6). ( C ) FACS plots of splenic lymphocytes showing CD19 expression versus IgM, IgE, or IgG1 from the indicated mice ( n = 6). ( D and E ) Dot graph showing summary statistics of percentages ( D ), and total number ( E ), of splenic B cells from the indicated mice. Each dot represents one mouse ( n = 4–9). ( F ) Semiquantitative PCR analyses of J H -proximal V H 7183 and J H -distal V H J558 family rearrangements in sorted bone marrow pro-B cells from indicated mice. Dlg5 was amplified as a loading control. Threefold serial dilutions are shown. Results are typical of three experiments. Bands corresponding to rearrangements to various J H segments are indicated. ( G ) FACS plots showing intracellular Igμ, Igε, and Igγ1 heavy chain in pro-B cells (CD19 + B220 lo Igκ − CD43 + ) from bone marrows of the indicated mice. Results are typical of at least four experiments. ( H ) Percentage of intracellular Igμ, Igε, and Igγ1 heavy chain in BM pro-B cells. Each dot represents one mouse ( n = 5). ( I ) Semiquantitative PCR analyses of V κ Igl chain rearrangements in magnetically separated bone marrow B220 + cells from indicated mice. Intronic Ig κ was amplified as a loading control. Threefold serial dilutions are shown. Bands corresponding to rearrangements to various J κ segments are indicated on the Left . Results are typical of four experiments. ( J ) Quantitative PCR analyses of V κ to J κ1 Igl chain rearrangement relative to β -actin DNA in purified B220 + BM cells from the indicated mice. Expression is shown as fold change relative to wild-type levels. ** P

    Article Snippet: Cell mRNA was isolated using Dynabeads mRNA DIRECT Micro Purification Kit (ambion/Life technologies), followed by DNA elimination with gDNA wipeout (Qiagen). cDNA was obtained using SuperScript III reverse transcription system (Invitrogen) using anchored Oligo(dT)20 primers (Invitrogen).

    Techniques: Mouse Assay, FACS, Expressing, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Purification

    qRT-PCR validation of microarray results for a selected number of genes. cDNA was generated from total RNA independently prepared from small intestine epithelial cells isolated from three 3 month old Muc2 −/− mice and 3 age, and gender

    Journal: Cancer research

    Article Title: Interaction of Muc2 and Apc on Wnt signaling and in intestinal tumorigenesis: potential role of chronic inflammation

    doi: 10.1158/0008-5472.CAN-08-0598

    Figure Lengend Snippet: qRT-PCR validation of microarray results for a selected number of genes. cDNA was generated from total RNA independently prepared from small intestine epithelial cells isolated from three 3 month old Muc2 −/− mice and 3 age, and gender

    Article Snippet: RNA was reverse transcribed into cDNA (SuperScriptIII reverse transcriptase, Invitrogen).

    Techniques: Quantitative RT-PCR, Microarray, Generated, Isolation, Mouse Assay

    Heterogeneous expression pattern of YFP + and YFP - signals within the aneuploid population was not due to the karyotypic variations. ( A ) A schematic representation of the genetic manipulations used to generate isogenic diploid and triploid strains from the parental WT haploid strain, which has nuclear localization sequence-tagged YFP inserted into the HML locus. ( B ) FACS sorting of YFP - vs YFP + cells from the strain with 2x Chr III and 1x Chr X gain. Red and yellow line outlines the population of YFP - and YFP + cells sorted for the qPCR analysis in C and D, respectively. ( C–D ) qPCR karyotyping of YFP - ( C ) and YFP + ( D ) cell population sorted from the strain with 2x Chr III and 1x Chr X gain. Chromosome copy numbers of sixteen yeast chromosomes are plotted as mean and SD of three technical replicates. ( E–G ) Confocal images of YFP fluorescence, taken at the indicated time points during time-lapse imaging, show transitions between repression and derepression of the HML locus in proliferating lineages of aneuploid yeast cells with the following karyotypes: ( E ) Gain of III, III, X; ( F ) Loss of I, V, VII, VIII, XI (basal ploidy, 2N); and ( G ) Gain of I, X, XII, XIII. Arrows point to mother cells that switched from the silenced to the desilenced state. The circle outlines the boundary of the cell. Scale bar, 4 µm.

    Journal: eLife

    Article Title: Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast

    doi: 10.7554/eLife.27991

    Figure Lengend Snippet: Heterogeneous expression pattern of YFP + and YFP - signals within the aneuploid population was not due to the karyotypic variations. ( A ) A schematic representation of the genetic manipulations used to generate isogenic diploid and triploid strains from the parental WT haploid strain, which has nuclear localization sequence-tagged YFP inserted into the HML locus. ( B ) FACS sorting of YFP - vs YFP + cells from the strain with 2x Chr III and 1x Chr X gain. Red and yellow line outlines the population of YFP - and YFP + cells sorted for the qPCR analysis in C and D, respectively. ( C–D ) qPCR karyotyping of YFP - ( C ) and YFP + ( D ) cell population sorted from the strain with 2x Chr III and 1x Chr X gain. Chromosome copy numbers of sixteen yeast chromosomes are plotted as mean and SD of three technical replicates. ( E–G ) Confocal images of YFP fluorescence, taken at the indicated time points during time-lapse imaging, show transitions between repression and derepression of the HML locus in proliferating lineages of aneuploid yeast cells with the following karyotypes: ( E ) Gain of III, III, X; ( F ) Loss of I, V, VII, VIII, XI (basal ploidy, 2N); and ( G ) Gain of I, X, XII, XIII. Arrows point to mother cells that switched from the silenced to the desilenced state. The circle outlines the boundary of the cell. Scale bar, 4 µm.

    Article Snippet: Quantitative reverse transcriptase-PCR (qPCR) analysis RNA was extracted as described above and cDNA was prepared from 2 µg of the resulting total RNA using the Super-Script III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using SYBR Green real-time PCR master mix (Quanta Biosciences, Beverly, MA) and analyzed by standard procedures ( ).

    Techniques: Expressing, Sequencing, FACS, Real-time Polymerase Chain Reaction, Fluorescence, Imaging

    HM desilencing in disome X cells correlates with increased H4K16 acetylation and reduced Sir2 enrichment across HM loci. ( A–B ) Bottom: The plots show levels of H4K16 acetylation across the HML ( A ) and HMR ( B ) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-H4K16ac chromatin immunoprecipitation (ChIP) followed by quantitative RT-PCR (qPCR) analysis. Top: Schematics of the HM loci indicate the genomic positioning of primer sets A to F used for qPCR. Plots show the mean and SD from three biological replicates. *p

    Journal: eLife

    Article Title: Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast

    doi: 10.7554/eLife.27991

    Figure Lengend Snippet: HM desilencing in disome X cells correlates with increased H4K16 acetylation and reduced Sir2 enrichment across HM loci. ( A–B ) Bottom: The plots show levels of H4K16 acetylation across the HML ( A ) and HMR ( B ) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-H4K16ac chromatin immunoprecipitation (ChIP) followed by quantitative RT-PCR (qPCR) analysis. Top: Schematics of the HM loci indicate the genomic positioning of primer sets A to F used for qPCR. Plots show the mean and SD from three biological replicates. *p

    Article Snippet: Quantitative reverse transcriptase-PCR (qPCR) analysis RNA was extracted as described above and cDNA was prepared from 2 µg of the resulting total RNA using the Super-Script III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using SYBR Green real-time PCR master mix (Quanta Biosciences, Beverly, MA) and analyzed by standard procedures ( ).

    Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Expression of genes related to reprogramming in porcine blastocysts cultured for 7 days. mRNA was extracted from blastocysts cultured in the absence or presence of 500 ng/mL ascorbic acid. Gene expression was analysed by real-time reverse transcription-polymerase chain reaction. Black bar: control group, white bar: ascorbic acid group. Values are the mean±standard error of the mean of three independent experiments. VC, ascorbic acid, Asterisks (*) indicate p

    Journal: Asian-Australasian Journal of Animal Sciences

    Article Title: Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa)

    doi: 10.5713/ajas.16.0818

    Figure Lengend Snippet: Expression of genes related to reprogramming in porcine blastocysts cultured for 7 days. mRNA was extracted from blastocysts cultured in the absence or presence of 500 ng/mL ascorbic acid. Gene expression was analysed by real-time reverse transcription-polymerase chain reaction. Black bar: control group, white bar: ascorbic acid group. Values are the mean±standard error of the mean of three independent experiments. VC, ascorbic acid, Asterisks (*) indicate p

    Article Snippet: First-strand cDNA was synthesized by reverse transcription (RT) of mRNA using an Oligo(dT)12–18 primer and SuperScript TM III Reverse Transcriptase (Invitrogen Co., Grand Island, NY, USA).

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    Efficiency of TET3 knockdown in porcine oocytes. Porcine oocytes were injected into siRNA for eGFP as control or three siRNA for TET3. After injection, TET3 mRNA levels were detected and normalized to control. * p

    Journal: Asian-Australasian Journal of Animal Sciences

    Article Title: Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa)

    doi: 10.5713/ajas.16.0818

    Figure Lengend Snippet: Efficiency of TET3 knockdown in porcine oocytes. Porcine oocytes were injected into siRNA for eGFP as control or three siRNA for TET3. After injection, TET3 mRNA levels were detected and normalized to control. * p

    Article Snippet: First-strand cDNA was synthesized by reverse transcription (RT) of mRNA using an Oligo(dT)12–18 primer and SuperScript TM III Reverse Transcriptase (Invitrogen Co., Grand Island, NY, USA).

    Techniques: Injection

    BMMSCs express Tet1, Tet2, and Tet3. a , b Both human (h) and mouse (m) BMMSCs expressed Tet1, Tet2, and Tet3, as assessed by western blotting ( a ) and qPCR ( b ). c Immunocytofluorescent staining showed that CD146-positive BMMSCs expressed Tet1, Tet2, and Tet3. Scale bar, 50 μm. Results are from three independent experiments. *** p

    Journal: Nature Communications

    Article Title: Tet1 and Tet2 maintain mesenchymal stem cell homeostasis via demethylation of the P2rX7 promoter

    doi: 10.1038/s41467-018-04464-6

    Figure Lengend Snippet: BMMSCs express Tet1, Tet2, and Tet3. a , b Both human (h) and mouse (m) BMMSCs expressed Tet1, Tet2, and Tet3, as assessed by western blotting ( a ) and qPCR ( b ). c Immunocytofluorescent staining showed that CD146-positive BMMSCs expressed Tet1, Tet2, and Tet3. Scale bar, 50 μm. Results are from three independent experiments. *** p

    Article Snippet: For qPCR of mRNA, we used SuperScript III Reverse Transcriptase (RT) kit (Invitrogen) to prepare the complementary DNA (cDNA). qPCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and gene-specific primers.

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Staining