mrna  (New England Biolabs)


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    Name:
    NEBNext Poly A mRNA Magnetic Isolation Module
    Description:
    NEBNext Poly A mRNA Magnetic Isolation Module 96 rxns
    Catalog Number:
    e7490l
    Price:
    242
    Size:
    96 rxns
    Category:
    mRNA Purification Kits
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    New England Biolabs mrna
    NEBNext Poly A mRNA Magnetic Isolation Module
    NEBNext Poly A mRNA Magnetic Isolation Module 96 rxns
    https://www.bioz.com/result/mrna/product/New England Biolabs
    Average 99 stars, based on 700 article reviews
    Price from $9.99 to $1999.99
    mrna - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Aging of Xenopus tropicalis Eggs Leads to Deadenylation of a Specific Set of Maternal mRNAs and Loss of Developmental Potential"

    Article Title: Aging of Xenopus tropicalis Eggs Leads to Deadenylation of a Specific Set of Maternal mRNAs and Loss of Developmental Potential

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013532

    Poly(A) tail reduction of specific maternal mRNA in aged eggs. Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT) 20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.
    Figure Legend Snippet: Poly(A) tail reduction of specific maternal mRNA in aged eggs. Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT) 20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.

    Techniques Used: Ligation, Polymerase Chain Reaction, Marker, Sequencing

    2) Product Images from "Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules"

    Article Title: Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.107862

    Generation of marmoset iPSCs with VEE-OKS-iM-iTomato. A) Structure of the reprogramming VEE-mRNA. B) Scheme of the reprogramming process. C) Image of cjFFs transfected with VEE-OKS-iM-iTomato at day 3 post-transfection. Transfected cells are recognizable by their red fluorescence. D) An intermediate primary colony at day 27 post-transfection with red VEE-OKS-iM-iTomato fluorescence. E) An intermediate primary colony at day 27 post-transfection with characteristic compact morphology and clearly defined borders (indicated with arrowheads). The cells beyond the upper and left border of the colony are non-reprogrammed cjFFs. F) An intermediate primary colony stained for AP. G) Marmoset iPSC colony after second round of reprogramming of the intermediate primary cells with IWR1, CHIR99021, CGP77675, LIF, and Forskolin. H) Morphology of marmoset iPSC colonies growing on Geltrex at P27. (All scale bars = 200 μm).
    Figure Legend Snippet: Generation of marmoset iPSCs with VEE-OKS-iM-iTomato. A) Structure of the reprogramming VEE-mRNA. B) Scheme of the reprogramming process. C) Image of cjFFs transfected with VEE-OKS-iM-iTomato at day 3 post-transfection. Transfected cells are recognizable by their red fluorescence. D) An intermediate primary colony at day 27 post-transfection with red VEE-OKS-iM-iTomato fluorescence. E) An intermediate primary colony at day 27 post-transfection with characteristic compact morphology and clearly defined borders (indicated with arrowheads). The cells beyond the upper and left border of the colony are non-reprogrammed cjFFs. F) An intermediate primary colony stained for AP. G) Marmoset iPSC colony after second round of reprogramming of the intermediate primary cells with IWR1, CHIR99021, CGP77675, LIF, and Forskolin. H) Morphology of marmoset iPSC colonies growing on Geltrex at P27. (All scale bars = 200 μm).

    Techniques Used: Transfection, Fluorescence, Staining

    3) Product Images from "In vitro Study of a Novel Stent Coating Using Modified CD39 Messenger RNA to Potentially Reduce Stent Angioplasty-Associated Complications"

    Article Title: In vitro Study of a Novel Stent Coating Using Modified CD39 Messenger RNA to Potentially Reduce Stent Angioplasty-Associated Complications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138375

    Vector map, generation and purification of CD39 mRNA. (A) Vector map of the pcDNA 3.3 plasmid containing the ENTDP-1/CD39 construct for mammalian expression. (B) Electrophoresis with a 1% agarose-TBE gel of the purified CD39 mRNA (1533 bp). eGFP mRNA (993 bp) was used as a loading control. 0.5–10 kB RNA ladder.
    Figure Legend Snippet: Vector map, generation and purification of CD39 mRNA. (A) Vector map of the pcDNA 3.3 plasmid containing the ENTDP-1/CD39 construct for mammalian expression. (B) Electrophoresis with a 1% agarose-TBE gel of the purified CD39 mRNA (1533 bp). eGFP mRNA (993 bp) was used as a loading control. 0.5–10 kB RNA ladder.

    Techniques Used: Plasmid Preparation, Purification, Construct, Expressing, Electrophoresis

    4) Product Images from "Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/? gene expression via acetylation of nuclear factor of activated T cells c1"

    Article Title: Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/? gene expression via acetylation of nuclear factor of activated T cells c1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr162

    Ca 2+ -ionophore-induced MyHCI/β gene expression depends on phosphorylation of p300 and its acetyltransferase function. ( A ) Semiquantitative RT–PCR analysis of endogenous MyHCI/β and IId/x expression. Total RNA was isolated from C2C12 cells transfected with p300wt-, or p300DY-, or p300SA3-Myc, or empty Myc-tag expression vector. Cells were grown for 24 h in GM and then for 2 days in DM with or without Ca 2+ -ionophore. PCR products were separated on a 2% agarose gel and visualized with ethidium bromide staining. 18 s rRNA levels were used for normalization. The mRNA expression of transfected Myc-tagged p300, p300DY and p300SA3 was evaluated using a Myc-specific reverse and a p300 forward primer. ( B ) Immunofluorescence analysis of endogenous MyHCI/β expression in C2C12 myotubes. C2C12 cells were transiently transfected with p300wt-, or p300DY-, or p300SA3-Myc, or empty Myc-tag expression vector. Cells were grown for 24 h in GM and then for 4 days in DM with or without Ca 2+ -ionophore A23187 (0.1 µM). Cells were stained with an anti-MyHCI/β-antibody. MyHCI/β was visualized by a FITC-labeled secondary antibody. Fluorescence was detected by using an inverted fluorescence photomicroscope. MyHCI/β positive cells appear green. Nuclei were stained with DAPI. Scale bar, 100 µm. ( C ) Histogram illustrating the mean number of MyHCI/β positive cells per vision field ( n = 6) as examined by immunofluorescence analysis shown in ( B ). Western blot analysis using anti-c-Myc antibody illustrating p300 expression levels in cells transfected with Myc-tagged p300 expression vectors.
    Figure Legend Snippet: Ca 2+ -ionophore-induced MyHCI/β gene expression depends on phosphorylation of p300 and its acetyltransferase function. ( A ) Semiquantitative RT–PCR analysis of endogenous MyHCI/β and IId/x expression. Total RNA was isolated from C2C12 cells transfected with p300wt-, or p300DY-, or p300SA3-Myc, or empty Myc-tag expression vector. Cells were grown for 24 h in GM and then for 2 days in DM with or without Ca 2+ -ionophore. PCR products were separated on a 2% agarose gel and visualized with ethidium bromide staining. 18 s rRNA levels were used for normalization. The mRNA expression of transfected Myc-tagged p300, p300DY and p300SA3 was evaluated using a Myc-specific reverse and a p300 forward primer. ( B ) Immunofluorescence analysis of endogenous MyHCI/β expression in C2C12 myotubes. C2C12 cells were transiently transfected with p300wt-, or p300DY-, or p300SA3-Myc, or empty Myc-tag expression vector. Cells were grown for 24 h in GM and then for 4 days in DM with or without Ca 2+ -ionophore A23187 (0.1 µM). Cells were stained with an anti-MyHCI/β-antibody. MyHCI/β was visualized by a FITC-labeled secondary antibody. Fluorescence was detected by using an inverted fluorescence photomicroscope. MyHCI/β positive cells appear green. Nuclei were stained with DAPI. Scale bar, 100 µm. ( C ) Histogram illustrating the mean number of MyHCI/β positive cells per vision field ( n = 6) as examined by immunofluorescence analysis shown in ( B ). Western blot analysis using anti-c-Myc antibody illustrating p300 expression levels in cells transfected with Myc-tagged p300 expression vectors.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Immunofluorescence, Labeling, Fluorescence, Western Blot

    5) Product Images from "SMRT-Cappable-seq reveals complex operon variants in bacteria"

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria

    Journal: bioRxiv

    doi: 10.1101/262964

    Recovery of mRNA and rRNA by SMRT-Cappable-seq. The mRNA levels of protein coding genes (Gluc, hupB and rmf) and rRNA genes (rrlA and rrlH) were measured by qPCR using the cDNA obtained from E. coli grown in M9 medium. The recovery rates (in %, Y axis) was calculated as the amount of mRNA in the enriched fraction (after streptavidin) divided by the amount of mRNA in the control fraction (no streptavidin enrichment). Gluc is an in-vitro transcribed mRNA spiked in the E. coli total RNA as positive triphosphorylated control. HupB and rmf are endogenous protein coding genes representative of primary transcripts. rrlA and rrlH are rRNA representative of processed transcripts.
    Figure Legend Snippet: Recovery of mRNA and rRNA by SMRT-Cappable-seq. The mRNA levels of protein coding genes (Gluc, hupB and rmf) and rRNA genes (rrlA and rrlH) were measured by qPCR using the cDNA obtained from E. coli grown in M9 medium. The recovery rates (in %, Y axis) was calculated as the amount of mRNA in the enriched fraction (after streptavidin) divided by the amount of mRNA in the control fraction (no streptavidin enrichment). Gluc is an in-vitro transcribed mRNA spiked in the E. coli total RNA as positive triphosphorylated control. HupB and rmf are endogenous protein coding genes representative of primary transcripts. rrlA and rrlH are rRNA representative of processed transcripts.

    Techniques Used: Real-time Polymerase Chain Reaction, In Vitro

    6) Product Images from "A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2"

    Article Title: A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2

    Journal: Journal of Virology

    doi: 10.1128/JVI.02488-16

    Reovirus infection alters cellular mRNA splicing. (A) Venn diagram indicating differences in splicing based on MISO analysis of RNA-seq results. (B) Confirmation of differential expression of novel splicing variants using qRT-PCR. L929 cells were infected and stimulated with IFN-β using conditions identical to those used for RNA-seq. Transcripts with a specific exon skipped or spliced-in (as identified by RNA-seq) were quantitated by qRT-PCR for two representative genes. The results are expressed as a ratio of the two splicing events to capture the events comprising MISO analysis. The results of statistical analyses ( P
    Figure Legend Snippet: Reovirus infection alters cellular mRNA splicing. (A) Venn diagram indicating differences in splicing based on MISO analysis of RNA-seq results. (B) Confirmation of differential expression of novel splicing variants using qRT-PCR. L929 cells were infected and stimulated with IFN-β using conditions identical to those used for RNA-seq. Transcripts with a specific exon skipped or spliced-in (as identified by RNA-seq) were quantitated by qRT-PCR for two representative genes. The results are expressed as a ratio of the two splicing events to capture the events comprising MISO analysis. The results of statistical analyses ( P

    Techniques Used: Infection, RNA Sequencing Assay, Expressing, Quantitative RT-PCR

    7) Product Images from "Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD"

    Article Title: Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25743-8

    Down-regulated miRNA vs . up-regulated mRNAs pairs. ( A ) 459 miRNA - mRNA pair regulatory networks including 13 miRNAs and their targets. ( B ) A sub-network of 41 genes connected to more than 5 different miRNAs. ( C ) Venn diagram of miR-33-3p and miR-33-5p. ( D ) A sub-network of miR-33-3p and miR-33-5p and their target genes. The blue label represents the intersection target genes of the two miRNAs.
    Figure Legend Snippet: Down-regulated miRNA vs . up-regulated mRNAs pairs. ( A ) 459 miRNA - mRNA pair regulatory networks including 13 miRNAs and their targets. ( B ) A sub-network of 41 genes connected to more than 5 different miRNAs. ( C ) Venn diagram of miR-33-3p and miR-33-5p. ( D ) A sub-network of miR-33-3p and miR-33-5p and their target genes. The blue label represents the intersection target genes of the two miRNAs.

    Techniques Used:

    Up-regulated miRNA vs . down-regulated mRNA pairs. ( A ) 114 up-regulated miRNA vs . down-regulated mRNA pair regulatory networks with 6 miRNAs and their targets. Ellipse nodes represent miRNAs, and diamond nodes represent mRNAs; up-regulated and down-regulated miRNAs are shown in red and green, respectively. ( B ) A sub-network of Abcg8, Cyp1a1 and Tmem255a, each connected to 5 different miRNAs. ( C ) Venn diagram of miR-200b-3p, miR-200b-5p and miR-200c-3p. ( D ) A sub-network of miR-200b-3p, miR-200b-5p and miR-200c-3p and their target genes. The blue label represents the intersection target genes of the three miRNAs. ( E ) GO analysis of miR-200b-3p, miR-200b-5p and miR-200c-3p target genes; the x-axis represents gene counts, and the y-axis represents GO terms. ( F ) KEGG pathway analysis of miR-200b-3p, miR-200b-5p and miR-200c-3p target genes; the x-axis represents gene counts, and the y-axis represents KEGG pathway names.
    Figure Legend Snippet: Up-regulated miRNA vs . down-regulated mRNA pairs. ( A ) 114 up-regulated miRNA vs . down-regulated mRNA pair regulatory networks with 6 miRNAs and their targets. Ellipse nodes represent miRNAs, and diamond nodes represent mRNAs; up-regulated and down-regulated miRNAs are shown in red and green, respectively. ( B ) A sub-network of Abcg8, Cyp1a1 and Tmem255a, each connected to 5 different miRNAs. ( C ) Venn diagram of miR-200b-3p, miR-200b-5p and miR-200c-3p. ( D ) A sub-network of miR-200b-3p, miR-200b-5p and miR-200c-3p and their target genes. The blue label represents the intersection target genes of the three miRNAs. ( E ) GO analysis of miR-200b-3p, miR-200b-5p and miR-200c-3p target genes; the x-axis represents gene counts, and the y-axis represents GO terms. ( F ) KEGG pathway analysis of miR-200b-3p, miR-200b-5p and miR-200c-3p target genes; the x-axis represents gene counts, and the y-axis represents KEGG pathway names.

    Techniques Used:

    Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.
    Figure Legend Snippet: Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.

    Techniques Used: Expressing, RNA Expression

    8) Product Images from "Intradermal Delivery of Synthetic mRNA Using Hollow Microneedles for Efficient and Rapid Production of Exogenous Proteins in Skin"

    Article Title: Intradermal Delivery of Synthetic mRNA Using Hollow Microneedles for Efficient and Rapid Production of Exogenous Proteins in Skin

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.03.005

    Detection of Luciferase Activity after Transfection of HEK923 Cells with Synthetic hGLuc mRNA 3 × 10 5 HEK293 cells were incubated for 4 hr at 37°C with 0.2, 0.5, or 1.5 μg mRNA complexed with Lipofectamine 2000. Then transfection complexes were discarded, and fresh medium was added to the cells. After 24 hr, the luciferase activity (RLUs) was detected in the supernatants and cell lysates. Cells treated with medium or medium and Lipofectamine 2000 (L2000) served as negative controls. Results are shown as mean ± SEM (n = 3). Statistical differences were determined using two-way ANOVA followed by Bonferroni’s multiple comparisons test (**p
    Figure Legend Snippet: Detection of Luciferase Activity after Transfection of HEK923 Cells with Synthetic hGLuc mRNA 3 × 10 5 HEK293 cells were incubated for 4 hr at 37°C with 0.2, 0.5, or 1.5 μg mRNA complexed with Lipofectamine 2000. Then transfection complexes were discarded, and fresh medium was added to the cells. After 24 hr, the luciferase activity (RLUs) was detected in the supernatants and cell lysates. Cells treated with medium or medium and Lipofectamine 2000 (L2000) served as negative controls. Results are shown as mean ± SEM (n = 3). Statistical differences were determined using two-way ANOVA followed by Bonferroni’s multiple comparisons test (**p

    Techniques Used: Luciferase, Activity Assay, Transfection, Incubation

    Delivery of Synthetic hGLuc mRNA into Porcine Skin Using Hollow Microneedles and Analysis of Protein Expression (1) Porcine skin detached from the outer side of pigs’ ears was trimmed and punched into 1-mm-thick pieces with 1.5-cm diameter and disinfected. The structure of the skin is shown schematically. S.c., stratum corneum ; E, epidermis; D, dermis. (2) Lipoplexes were generated by incubation of 1.5 μg hGLuc mRNA with 1.5 μl Lipofectamine 2000 in a total volume of 35 μL OptiMEM I reduced serum-free medium for 20 min at RT. The mixture was injected into the skin using MicronJet600 microneedles. (3) After washing with DPBS, the skin was transferred into a ThinCert insert, which served as a permeable barrier between the skin and the surrounding medium. (4) The skin was incubated air-exposed in 1.5 mL human endothelial cell culture medium in one well of a 12-well plate from 24 to 72 hr at 37°C and 5% CO 2 . The microinjected lipoplexes can enter the cells via endocytosis. After the release of mRNA into the cytosol, mRNA is translated by ribosomes into protein. (5) After the appropriate incubation time, the produced hGLuc protein is detected by luciferase assay in the surrounding medium as well as in the skin.
    Figure Legend Snippet: Delivery of Synthetic hGLuc mRNA into Porcine Skin Using Hollow Microneedles and Analysis of Protein Expression (1) Porcine skin detached from the outer side of pigs’ ears was trimmed and punched into 1-mm-thick pieces with 1.5-cm diameter and disinfected. The structure of the skin is shown schematically. S.c., stratum corneum ; E, epidermis; D, dermis. (2) Lipoplexes were generated by incubation of 1.5 μg hGLuc mRNA with 1.5 μl Lipofectamine 2000 in a total volume of 35 μL OptiMEM I reduced serum-free medium for 20 min at RT. The mixture was injected into the skin using MicronJet600 microneedles. (3) After washing with DPBS, the skin was transferred into a ThinCert insert, which served as a permeable barrier between the skin and the surrounding medium. (4) The skin was incubated air-exposed in 1.5 mL human endothelial cell culture medium in one well of a 12-well plate from 24 to 72 hr at 37°C and 5% CO 2 . The microinjected lipoplexes can enter the cells via endocytosis. After the release of mRNA into the cytosol, mRNA is translated by ribosomes into protein. (5) After the appropriate incubation time, the produced hGLuc protein is detected by luciferase assay in the surrounding medium as well as in the skin.

    Techniques Used: Expressing, Generated, Incubation, Injection, Cell Culture, Produced, Luciferase

    9) Product Images from "A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions"

    Article Title: A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.09.025

    Schematic Representation of the CimiR System for Expressing the Circularized AntamiRNAs or miRNA Antagonists (A and B) Overall structure (A) and the essential elements (B) of the CimiR system. Briefly, the consensus sequences of the RNA-splicing branch site (B) and polypyrimidine track (ppy) were constructed at the 5′ end of the antamiRNA locus, while the donor site sequence (D) and a 20-mer random sequence (R) were engineered at the 3′ end of AntamiR, which were flanked by the 100-bp inverted repeat sequence derived from mouse Rosa26 genomic sequence. (C) Formation of a circularized antamiR (or CimiR). Upon transcription driven by the hEFH promoter, the antamiR-containing pre-mRNA is processed through cis backsplicing mechanism to yield a circularized antamiR (CimiR) and a splicing by-product. The expression system was constructed on the basis of retroviral vector pSEBR, which expresses blasticidin selection marker and RFP-tracking marker. The same CimiR expression cassette was constructed in an adenoviral shuttle vector pAdTrace-CimiR as well.
    Figure Legend Snippet: Schematic Representation of the CimiR System for Expressing the Circularized AntamiRNAs or miRNA Antagonists (A and B) Overall structure (A) and the essential elements (B) of the CimiR system. Briefly, the consensus sequences of the RNA-splicing branch site (B) and polypyrimidine track (ppy) were constructed at the 5′ end of the antamiRNA locus, while the donor site sequence (D) and a 20-mer random sequence (R) were engineered at the 3′ end of AntamiR, which were flanked by the 100-bp inverted repeat sequence derived from mouse Rosa26 genomic sequence. (C) Formation of a circularized antamiR (or CimiR). Upon transcription driven by the hEFH promoter, the antamiR-containing pre-mRNA is processed through cis backsplicing mechanism to yield a circularized antamiR (CimiR) and a splicing by-product. The expression system was constructed on the basis of retroviral vector pSEBR, which expresses blasticidin selection marker and RFP-tracking marker. The same CimiR expression cassette was constructed in an adenoviral shuttle vector pAdTrace-CimiR as well.

    Techniques Used: Expressing, Construct, Sequencing, Derivative Assay, Plasmid Preparation, Selection, Marker

    10) Product Images from "Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis"

    Article Title: Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis

    Journal: Cell Division

    doi: 10.1186/s13008-018-0042-4

    Spindle assembly and chromosome alignment demand hypoacetylation of tubulin K40 during meiosis. a Typical images of MI control oocytes, Kif18a siRNA oocytes and Kif18a siRNA + tubulin K40R mRNA-injected oocytes. Green, α-tubulin; red, DNA. b The incidence of abnormal spindles and misaligned chromosomes in control oocytes, Kif18a siRNA oocytes and Kif18a siRNA + tubulin K40R mRNA-injected oocytes. Data are presented as mean percentage (± SEM) of at least three independent experiments. *p
    Figure Legend Snippet: Spindle assembly and chromosome alignment demand hypoacetylation of tubulin K40 during meiosis. a Typical images of MI control oocytes, Kif18a siRNA oocytes and Kif18a siRNA + tubulin K40R mRNA-injected oocytes. Green, α-tubulin; red, DNA. b The incidence of abnormal spindles and misaligned chromosomes in control oocytes, Kif18a siRNA oocytes and Kif18a siRNA + tubulin K40R mRNA-injected oocytes. Data are presented as mean percentage (± SEM) of at least three independent experiments. *p

    Techniques Used: Injection

    11) Product Images from "Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation"

    Article Title: Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation

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

    doi:

    Mutations within the Pab1p-binding domain of Tif4631p and Tif4632p inhibit poly(A)-dependent translation. ( a ) Equal amounts of nuclease-treated translation extracts were programmed with [ 35 S]methionine and yeast mRNA with or without the cap analog 7m ). Translational rates of incorporation in the absence of the analog, relative to the wild-type extracts were: Tif4631-213p (88%), Tif4631-ΔN300p (115%), Tif4632-233 (70%)p, and Tif4632-ΔN300p (72%). ( b ) Equal amounts of translation extracts prepared from yeast strains harboring the indicated TIF4631 or TIF4632 ). Luciferase enzyme production was monitored by a luminescence assay. The data shown are representative of at least three independent assays. LUC mRNA lacking a cap and a poly(A) tail was not detectably translated in these extracts (data not shown).
    Figure Legend Snippet: Mutations within the Pab1p-binding domain of Tif4631p and Tif4632p inhibit poly(A)-dependent translation. ( a ) Equal amounts of nuclease-treated translation extracts were programmed with [ 35 S]methionine and yeast mRNA with or without the cap analog 7m ). Translational rates of incorporation in the absence of the analog, relative to the wild-type extracts were: Tif4631-213p (88%), Tif4631-ΔN300p (115%), Tif4632-233 (70%)p, and Tif4632-ΔN300p (72%). ( b ) Equal amounts of translation extracts prepared from yeast strains harboring the indicated TIF4631 or TIF4632 ). Luciferase enzyme production was monitored by a luminescence assay. The data shown are representative of at least three independent assays. LUC mRNA lacking a cap and a poly(A) tail was not detectably translated in these extracts (data not shown).

    Techniques Used: Binding Assay, Luciferase, Luminescence Assay

    12) Product Images from "VLPs Derived from the CCMV Plant Virus Can Directly Transfect and Deliver Heterologous Genes for Translation into Mammalian Cells"

    Article Title: VLPs Derived from the CCMV Plant Virus Can Directly Transfect and Deliver Heterologous Genes for Translation into Mammalian Cells

    Journal: BioMed Research International

    doi: 10.1155/2019/4630891

    mRNA-EGFP transcription. Capped (lane 3) and uncapped (lane 4) mRNAs that code for EGFP were synthesized along with a luciferase mRNA (control, lane 2). Their integrity was visually evaluated using electrophoresis agarose gels stained with GelRed™. Transcripts are compared with the ssRNA ladder (lane 1). Single gel experiment without cropping.
    Figure Legend Snippet: mRNA-EGFP transcription. Capped (lane 3) and uncapped (lane 4) mRNAs that code for EGFP were synthesized along with a luciferase mRNA (control, lane 2). Their integrity was visually evaluated using electrophoresis agarose gels stained with GelRed™. Transcripts are compared with the ssRNA ladder (lane 1). Single gel experiment without cropping.

    Techniques Used: Synthesized, Luciferase, Electrophoresis, Staining

    13) Product Images from "ALKBH5 suppresses malignancy of hepatocellular carcinoma via m6A-guided epigenetic inhibition of LYPD1"

    Article Title: ALKBH5 suppresses malignancy of hepatocellular carcinoma via m6A-guided epigenetic inhibition of LYPD1

    Journal: Molecular Cancer

    doi: 10.1186/s12943-020-01239-w

    ALKBH5 impairs the stability of LYPD1 mRNA via an IGF2BP1-m 6 A-dependent pattern. a m 6 A abundance on LYPD1 mRNA in negative control or ALKBH5-overexpressing HCCLM3 cells was plotted by the IGV. Green and pink colors show the m 6 A signals of input samples, while red and blue stand for signals of IP samples. The range of signals in all groups was normalized to a 0–560 scale. At the same position, m 6 A peaks of IP group over input group were recognized as the genuine m 6 A level. Black blocks below figure indicated the sites where the m 6 A level differed between two groups, and the most remarkable location was highlighted with a gray pane. b Relative enrichment of LYPD1 mRNA associated with ALKBH5 protein was identified by RIP assays using anti-IgG and anti-ALKBH5 antibodies. The IgG group was a negative control to preclude nonspecific binding. The Y axis represented the percent of input for each IP sample according to the formula: %Input =1/10*2 Ct [IP] – Ct [input] . c m 6 A modification of LYPD1 was detected by MeRIP-qPCR analysis using anti-IgG and anti-m 6 A antibodies. Relative m 6 A enrichment of LYPD1 mRNA for each IP group was normalized to input. Silencing of ALKBH5 induced an increase m 6 A abundance on LYPD1 compared with control group, while ALKBH5 overexpression led to the opposite result; d Graphical explanation for construction of luciferase reporters. The wild-type (full-length) or mutant (m 6 A motif mutated) sequence of LYPD1–3’UTR was inserted into a pcDNA3.1 vector between Firefly and Renilla elements. Relative luciferase activity was computed by the ratio of Firefly and Renilla luciferase values. e Relative luciferase activity of Huh7, MHCC97H and HCCLM3 cells transfected with the LYPD1-wild type or -mutated construct was measured, with normal or altered expression of ALKBH5; f ALKBH5-silenced or -overexpressed cells were treated with actinomycin D and harvested at 0, 3 and 6 h. RNA decay rate was determined to estimate the stability of LYPD1 (normalized to the expression at 0 h); g IGF2BP1 was knockdown in two HCC cells followed by the measurement of LYPD1 expression via qPCR; h RIP-qPCR validated that IGF2BP1 could bind to LYPD1 mRNA. Relative enrichment of LYPD1 mRNA in each group was showed with the normalization to input; i Rescue assays were employed to verify the impact of IGF2BP1 on ALKBH5-mediated modulation of LYPD1
    Figure Legend Snippet: ALKBH5 impairs the stability of LYPD1 mRNA via an IGF2BP1-m 6 A-dependent pattern. a m 6 A abundance on LYPD1 mRNA in negative control or ALKBH5-overexpressing HCCLM3 cells was plotted by the IGV. Green and pink colors show the m 6 A signals of input samples, while red and blue stand for signals of IP samples. The range of signals in all groups was normalized to a 0–560 scale. At the same position, m 6 A peaks of IP group over input group were recognized as the genuine m 6 A level. Black blocks below figure indicated the sites where the m 6 A level differed between two groups, and the most remarkable location was highlighted with a gray pane. b Relative enrichment of LYPD1 mRNA associated with ALKBH5 protein was identified by RIP assays using anti-IgG and anti-ALKBH5 antibodies. The IgG group was a negative control to preclude nonspecific binding. The Y axis represented the percent of input for each IP sample according to the formula: %Input =1/10*2 Ct [IP] – Ct [input] . c m 6 A modification of LYPD1 was detected by MeRIP-qPCR analysis using anti-IgG and anti-m 6 A antibodies. Relative m 6 A enrichment of LYPD1 mRNA for each IP group was normalized to input. Silencing of ALKBH5 induced an increase m 6 A abundance on LYPD1 compared with control group, while ALKBH5 overexpression led to the opposite result; d Graphical explanation for construction of luciferase reporters. The wild-type (full-length) or mutant (m 6 A motif mutated) sequence of LYPD1–3’UTR was inserted into a pcDNA3.1 vector between Firefly and Renilla elements. Relative luciferase activity was computed by the ratio of Firefly and Renilla luciferase values. e Relative luciferase activity of Huh7, MHCC97H and HCCLM3 cells transfected with the LYPD1-wild type or -mutated construct was measured, with normal or altered expression of ALKBH5; f ALKBH5-silenced or -overexpressed cells were treated with actinomycin D and harvested at 0, 3 and 6 h. RNA decay rate was determined to estimate the stability of LYPD1 (normalized to the expression at 0 h); g IGF2BP1 was knockdown in two HCC cells followed by the measurement of LYPD1 expression via qPCR; h RIP-qPCR validated that IGF2BP1 could bind to LYPD1 mRNA. Relative enrichment of LYPD1 mRNA in each group was showed with the normalization to input; i Rescue assays were employed to verify the impact of IGF2BP1 on ALKBH5-mediated modulation of LYPD1

    Techniques Used: Negative Control, Binding Assay, Modification, Real-time Polymerase Chain Reaction, Over Expression, Luciferase, Mutagenesis, Sequencing, Plasmid Preparation, Activity Assay, Transfection, Construct, Expressing

    14) Product Images from "p180 Promotes the Ribosome-Independent Localization of a Subset of mRNA to the Endoplasmic Reticulum"

    Article Title: p180 Promotes the Ribosome-Independent Localization of a Subset of mRNA to the Endoplasmic Reticulum

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001336

    Poly(A) transcripts associate with ER independently of ribosomes and translation. (A) A single digitonin-extracted COS-7 cell co-stained for poly(A) mRNA using poly(dT) FISH probes, and for the ER marker Trapα by immunofluorescence. Note the general co-localization between the mRNA (green) and Trapα (red). (B) The fluorescence intensity ( y -axis) of the poly(A) mRNA (green) and Trapα (red) along the arrow ( x -axis) in the overlay image in (A). Note the correlation between peaks in intensity of poly(A) mRNA and Trapα (black arrows). (C–D) COS-7 cells were treated with either DMSO, puromycin (“Puro”), homoharringtonine (“HHT”) for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for poly(A) mRNA using poly(dT) FISH probes, and then treated with RNase H (RNase H “+”) or control buffer (“Cont” or RNase H “−”) for 1 h at 37°C. Cells were imaged (C) and the fluorescence intensity of the ER and nucleus were quantified (D). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background normalized to the signal in the ER of DMSO/control treated cells. Note that in cells not treated with RNase H, the amount of mRNA bound to the ER decreased by about half in all the drug-treated cells as compared to DMSO-treated cells. In contrast RNase H treatment eliminated most of the ER fluorescence and the majority of the nuclear signal. All scale bars = 20 µm. (E) COS-7 cells were treated with control medium (DMSO), puromycin, or HHT for 15 min, then incubated in 35 S-methionine to label newly synthesized proteins for an additional 15 min. Cell lysates were collected and separated by SDS-PAGE. Total proteins were visualized by Coomassie blue stain, and newly synthesized proteins were detected by autoradiography. Molecular weight markers are indicated on the left (“Ladder”; 188 kD, 62 kD, 49 kD, 38 kD, 28 kD, 18 kD). (F) COS-7 cells were either treated with DMSO (“Cont”) or puromycin for 30 min, then extracted with digitonin (in the absence or presence of 20 mM EDTA). Cytoplasmic (“C”; i.e., non-ER) and ER (“ER”) fractions were separated by SDS-PAGE, then transferred to nitrocellulose, and immunoblotted with antibodies against the small ribosomal protein S6, the ER marker Trapα, and the cytoplasmic marker αtubulin. Note that most of the S6 protein is released from the membrane to the cytoplasmic fraction only after cells are treated with puromycin and then extracted with EDTA. (G) COS-7 and U2OS cells were treated either with cyclohexamide (“control”) and then extracted, or with puromycin for 30 min and then extracted in the presence of 20 mM EDTA. ER and cytoplasmic fractions were isolated as in (F) except that either cyclohexamide, or puromycin and EDTA, was present in all solutions. cDNA was synthesized from each fraction using poly(dT) primers and 32 P-dNTPs, and ratio of counts in the ER to total (cytoplasm+ER) were tabulated. Each bar represents the average and standard error of three independent experiments.
    Figure Legend Snippet: Poly(A) transcripts associate with ER independently of ribosomes and translation. (A) A single digitonin-extracted COS-7 cell co-stained for poly(A) mRNA using poly(dT) FISH probes, and for the ER marker Trapα by immunofluorescence. Note the general co-localization between the mRNA (green) and Trapα (red). (B) The fluorescence intensity ( y -axis) of the poly(A) mRNA (green) and Trapα (red) along the arrow ( x -axis) in the overlay image in (A). Note the correlation between peaks in intensity of poly(A) mRNA and Trapα (black arrows). (C–D) COS-7 cells were treated with either DMSO, puromycin (“Puro”), homoharringtonine (“HHT”) for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for poly(A) mRNA using poly(dT) FISH probes, and then treated with RNase H (RNase H “+”) or control buffer (“Cont” or RNase H “−”) for 1 h at 37°C. Cells were imaged (C) and the fluorescence intensity of the ER and nucleus were quantified (D). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background normalized to the signal in the ER of DMSO/control treated cells. Note that in cells not treated with RNase H, the amount of mRNA bound to the ER decreased by about half in all the drug-treated cells as compared to DMSO-treated cells. In contrast RNase H treatment eliminated most of the ER fluorescence and the majority of the nuclear signal. All scale bars = 20 µm. (E) COS-7 cells were treated with control medium (DMSO), puromycin, or HHT for 15 min, then incubated in 35 S-methionine to label newly synthesized proteins for an additional 15 min. Cell lysates were collected and separated by SDS-PAGE. Total proteins were visualized by Coomassie blue stain, and newly synthesized proteins were detected by autoradiography. Molecular weight markers are indicated on the left (“Ladder”; 188 kD, 62 kD, 49 kD, 38 kD, 28 kD, 18 kD). (F) COS-7 cells were either treated with DMSO (“Cont”) or puromycin for 30 min, then extracted with digitonin (in the absence or presence of 20 mM EDTA). Cytoplasmic (“C”; i.e., non-ER) and ER (“ER”) fractions were separated by SDS-PAGE, then transferred to nitrocellulose, and immunoblotted with antibodies against the small ribosomal protein S6, the ER marker Trapα, and the cytoplasmic marker αtubulin. Note that most of the S6 protein is released from the membrane to the cytoplasmic fraction only after cells are treated with puromycin and then extracted with EDTA. (G) COS-7 and U2OS cells were treated either with cyclohexamide (“control”) and then extracted, or with puromycin for 30 min and then extracted in the presence of 20 mM EDTA. ER and cytoplasmic fractions were isolated as in (F) except that either cyclohexamide, or puromycin and EDTA, was present in all solutions. cDNA was synthesized from each fraction using poly(dT) primers and 32 P-dNTPs, and ratio of counts in the ER to total (cytoplasm+ER) were tabulated. Each bar represents the average and standard error of three independent experiments.

    Techniques Used: Staining, Fluorescence In Situ Hybridization, Marker, Immunofluorescence, Fluorescence, Incubation, Synthesized, SDS Page, Autoradiography, Molecular Weight, Isolation

    Over-expression of p180 can enhance the ribosome-independent association of t-ftz mRNA with the ER. (A) COS-7 cells were transfected with plasmids containing vector alone (“mock”), GFP-p180, or GFP-p180ΔLysΔRepeat. After 18–24 h cell lysates were collected, separated by SDS-PAGE, and immunoblotted for GFP, p180, or αtubulin. The position of molecular weight markers are indicated on the left and proteins are labeled on the right. Note that a high molecular weight band (denoted by an asterisk), which is positive for p180 and GFP, is detected in cells expressing GFP-p180ΔLysΔRepeat. We suspect that this is an aggregate of the over-expressed protein. (B–E, G–H) COS-7 cells were transfected with plasmids containing t-ftz gene alone (“mock”), or with various GFP-tagged genes as indicated. The cells were allowed to express t-ftz mRNA and GFP-tagged proteins for 18–24 h. Cells were then treated with either control media or HHT for 30 min to disrupt ribosomes, and then extracted, fixed, and stained for t-ftz mRNA using specific FISH probes. (B, H) The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified. Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C–D, G) Each row represents a single field of HHT-treated cells (30 min) that was imaged for t-ftz mRNA and GFP. Cells co-expressing t-ftz mRNA and the GFP-tagged protein are denoted by arrows, while cells that expressed only t-ftz are indicated by arrowheads. Scale bar = 20 µm. Note that t-ftz mRNA remains associated to the ER in cells over-expressing GFP-p180 (C, arrows), but not GFP-CLIMP63 (D, arrows) or in cells expressing t-ftz alone (C–D and G, arrowheads). Cells over-expressing GFP-p180ΔLysΔRepeat (G, arrows) show an intermediate phenotype. (E) COS-7 cells that were transfected with plasmids containing t-ftz gene alone (“mock”) or with various GFP-tagged genes were lysed, separated by SDS-PAGE, and immunoblotted for p180, GFP, CLIMP63, translocon components (Sec61β and Trapα), or αtubulin. (F) Domain architecture of the GFP-tagged p180 constructs. Both contain the CALR SSCR to mediate proper protein translocation (purple), GFP (green), the p180 luminal region which is 7 amino acids long, and the p180 single pass transmembrane domain (TMD, orange). The lysine-rich region (“Lys,” dark blue) and decapeptide repeat region (light blue) are present only in the GFP-p180 construct. Both end with the p180 coil-coil domain (red).
    Figure Legend Snippet: Over-expression of p180 can enhance the ribosome-independent association of t-ftz mRNA with the ER. (A) COS-7 cells were transfected with plasmids containing vector alone (“mock”), GFP-p180, or GFP-p180ΔLysΔRepeat. After 18–24 h cell lysates were collected, separated by SDS-PAGE, and immunoblotted for GFP, p180, or αtubulin. The position of molecular weight markers are indicated on the left and proteins are labeled on the right. Note that a high molecular weight band (denoted by an asterisk), which is positive for p180 and GFP, is detected in cells expressing GFP-p180ΔLysΔRepeat. We suspect that this is an aggregate of the over-expressed protein. (B–E, G–H) COS-7 cells were transfected with plasmids containing t-ftz gene alone (“mock”), or with various GFP-tagged genes as indicated. The cells were allowed to express t-ftz mRNA and GFP-tagged proteins for 18–24 h. Cells were then treated with either control media or HHT for 30 min to disrupt ribosomes, and then extracted, fixed, and stained for t-ftz mRNA using specific FISH probes. (B, H) The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified. Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C–D, G) Each row represents a single field of HHT-treated cells (30 min) that was imaged for t-ftz mRNA and GFP. Cells co-expressing t-ftz mRNA and the GFP-tagged protein are denoted by arrows, while cells that expressed only t-ftz are indicated by arrowheads. Scale bar = 20 µm. Note that t-ftz mRNA remains associated to the ER in cells over-expressing GFP-p180 (C, arrows), but not GFP-CLIMP63 (D, arrows) or in cells expressing t-ftz alone (C–D and G, arrowheads). Cells over-expressing GFP-p180ΔLysΔRepeat (G, arrows) show an intermediate phenotype. (E) COS-7 cells that were transfected with plasmids containing t-ftz gene alone (“mock”) or with various GFP-tagged genes were lysed, separated by SDS-PAGE, and immunoblotted for p180, GFP, CLIMP63, translocon components (Sec61β and Trapα), or αtubulin. (F) Domain architecture of the GFP-tagged p180 constructs. Both contain the CALR SSCR to mediate proper protein translocation (purple), GFP (green), the p180 luminal region which is 7 amino acids long, and the p180 single pass transmembrane domain (TMD, orange). The lysine-rich region (“Lys,” dark blue) and decapeptide repeat region (light blue) are present only in the GFP-p180 construct. Both end with the p180 coil-coil domain (red).

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, SDS Page, Molecular Weight, Labeling, Expressing, Staining, Fluorescence In Situ Hybridization, Fluorescence, Construct, Translocation Assay

    ALPP and CALR , but not t-ftz or INSL3 , mRNA remain associated with the ER independently of ribosomes and translation. (A–E) COS-7 cells were transfected with plasmids containing either the t-ftz (A), INSL3 (A–B), ALPP (A, C), cyto-ALPP (a version of ALPP lacking signal sequence and transmembrane domain coding regions; A, D–E), or CALR (A) genes and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (“Cont”), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels B–D for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused INSL3 mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing cyto-ALPP mRNA. cyto-ALPP mRNA was visualized by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of cyto-ALPP mRNA (red) and Trapα (green) in the overlay. All scale bars = 20 µm.
    Figure Legend Snippet: ALPP and CALR , but not t-ftz or INSL3 , mRNA remain associated with the ER independently of ribosomes and translation. (A–E) COS-7 cells were transfected with plasmids containing either the t-ftz (A), INSL3 (A–B), ALPP (A, C), cyto-ALPP (a version of ALPP lacking signal sequence and transmembrane domain coding regions; A, D–E), or CALR (A) genes and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (“Cont”), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels B–D for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused INSL3 mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing cyto-ALPP mRNA. cyto-ALPP mRNA was visualized by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of cyto-ALPP mRNA (red) and Trapα (green) in the overlay. All scale bars = 20 µm.

    Techniques Used: Transfection, Sequencing, Staining, Fluorescence In Situ Hybridization, Fluorescence, Expressing, Immunofluorescence

    The initial ER-targeting of ALPP and CALR , but not t-ftz or INSL3 , mRNA occurs independently of translation or ribosomes. (A–B) COS-7 cells were pretreated with DMSO (“Control”) or HHT for 15 min, then microinjected with plasmids containing either the ALPP , INSL3 , t-ftz , or CALR genes and allowed to express mRNA for 2 h in the presence of DMSO or HHT. To label the microinjected cells, Alexa488-conjugated 70 kD dextran was co-injected (see insets in A). The cells were then extracted with digitonin, fixed, stained for mRNA using specific FISH probes, and imaged (A). The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified (B). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C) COS-7 cells were pretreated with HHT for 15 min, then microinjected with plasmids containing the ALPP gene. Cells were then incubated for 2 h in the presence of HHT, then extracted with digitonin, fixed, and then co-stained for ALPP mRNA by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of ALPP mRNA (red) and Trapα (green). All scale bars = 20 µm.
    Figure Legend Snippet: The initial ER-targeting of ALPP and CALR , but not t-ftz or INSL3 , mRNA occurs independently of translation or ribosomes. (A–B) COS-7 cells were pretreated with DMSO (“Control”) or HHT for 15 min, then microinjected with plasmids containing either the ALPP , INSL3 , t-ftz , or CALR genes and allowed to express mRNA for 2 h in the presence of DMSO or HHT. To label the microinjected cells, Alexa488-conjugated 70 kD dextran was co-injected (see insets in A). The cells were then extracted with digitonin, fixed, stained for mRNA using specific FISH probes, and imaged (A). The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified (B). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C) COS-7 cells were pretreated with HHT for 15 min, then microinjected with plasmids containing the ALPP gene. Cells were then incubated for 2 h in the presence of HHT, then extracted with digitonin, fixed, and then co-stained for ALPP mRNA by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of ALPP mRNA (red) and Trapα (green). All scale bars = 20 µm.

    Techniques Used: Injection, Staining, Fluorescence In Situ Hybridization, Fluorescence, Incubation, Immunofluorescence

    p180 is required for the ER-association of mRNA. (A–H) U2OS cells were infected with specific shRNAs against p180 (shRNA clones B9 and B10), kinectin or CLIMP63, or with control lentivirus (“Cont”). (A–B) Cell lysates were separated by SDS-PAGE and immunoblotted for p180, CLIMP63, kinectin, αtubulin, Trapα, and Sec61β. (C–E) Cells depleted of p180 (Clone B9; C, E) or kinectin (D), or infected with control lentivirus (“cont’; C–E), were treated with control media (no drug, “ND”) or HHT for 30 min, then extracted with digitonin and stained for poly(A) mRNA using poly(dT) FISH probes. (C–D) For each cell the total level of ER-associated poly (A) FISH signal (normalized from the background (0), to the brightest cell in the entire experiment (1); y -axis) was plotted against cell size (pixels squared, x -axis). For each data set a regression line was plotted and the coefficient of determination (R 2 ) was indicated. (E) The ratio of ER to nuclear poly(A) fluorescence was quantified and normalized. Each bar represents the average and standard error of five independent experiments, each consisting of the average of > 30 cells. (F–H) Cells were depleted of p180 or kinectin with specific shRNAs, or infected with control lentivirus (“Cont”), then transfected with plasmids containing either the ALPP (F–G) or CALR (H) gene. The cells were allowed to express mRNA for 18–24 h, then treated with control media (no drug, “ND”) or HHT for 30 min, and then extracted with digitonin. Cells were then fixed, stained for mRNA using specific FISH probes against the exogenous mRNA, and imaged. Nuclei are outlined with blue dotted lines. Scale bar = 20 µm. (G–H) The fluorescence intensity on the ER and nucleus were quantified. Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background.
    Figure Legend Snippet: p180 is required for the ER-association of mRNA. (A–H) U2OS cells were infected with specific shRNAs against p180 (shRNA clones B9 and B10), kinectin or CLIMP63, or with control lentivirus (“Cont”). (A–B) Cell lysates were separated by SDS-PAGE and immunoblotted for p180, CLIMP63, kinectin, αtubulin, Trapα, and Sec61β. (C–E) Cells depleted of p180 (Clone B9; C, E) or kinectin (D), or infected with control lentivirus (“cont’; C–E), were treated with control media (no drug, “ND”) or HHT for 30 min, then extracted with digitonin and stained for poly(A) mRNA using poly(dT) FISH probes. (C–D) For each cell the total level of ER-associated poly (A) FISH signal (normalized from the background (0), to the brightest cell in the entire experiment (1); y -axis) was plotted against cell size (pixels squared, x -axis). For each data set a regression line was plotted and the coefficient of determination (R 2 ) was indicated. (E) The ratio of ER to nuclear poly(A) fluorescence was quantified and normalized. Each bar represents the average and standard error of five independent experiments, each consisting of the average of > 30 cells. (F–H) Cells were depleted of p180 or kinectin with specific shRNAs, or infected with control lentivirus (“Cont”), then transfected with plasmids containing either the ALPP (F–G) or CALR (H) gene. The cells were allowed to express mRNA for 18–24 h, then treated with control media (no drug, “ND”) or HHT for 30 min, and then extracted with digitonin. Cells were then fixed, stained for mRNA using specific FISH probes against the exogenous mRNA, and imaged. Nuclei are outlined with blue dotted lines. Scale bar = 20 µm. (G–H) The fluorescence intensity on the ER and nucleus were quantified. Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background.

    Techniques Used: Infection, shRNA, SDS Page, Staining, Fluorescence In Situ Hybridization, Fluorescence, Transfection

    15) Product Images from "Cotranslational signal independent SRP preloading during membrane targeting"

    Article Title: Cotranslational signal independent SRP preloading during membrane targeting

    Journal: Nature

    doi: 10.1038/nature19309

    Translation and the role of SRP a, Distributions of RNA-seq SRP enrichment scores from secretory protein transcripts (SS, TMD, SS-TMD, or TA), with or without puromycin treatment. Included ORFs have at least 2-fold SRP enrichment without puromycin. b, The prt1-1 allele prevents initiation at non-permissive temperatures. Translational run-off removes all ribosomes from transcripts. b, Transcripts are retained on the membrane though binding of the RNC to the translocon. It is also possible that mRNA binding proteins at the ER bind transcripts. c, Distributions of RNA-seq membrane enrichment scores of secretory protein transcripts (n = 584). d, After mRNA export, a pioneer round of targeting directs secretory transcripts to the ER membrane. SRP is specifically pre-recruited transcripts that will present a functional targeting signal. Upon emergence of an SS or TMD, SRP directs RNCs to the ER membrane. Once at the ER membrane, transcripts are retained over multiple rounds of translation.
    Figure Legend Snippet: Translation and the role of SRP a, Distributions of RNA-seq SRP enrichment scores from secretory protein transcripts (SS, TMD, SS-TMD, or TA), with or without puromycin treatment. Included ORFs have at least 2-fold SRP enrichment without puromycin. b, The prt1-1 allele prevents initiation at non-permissive temperatures. Translational run-off removes all ribosomes from transcripts. b, Transcripts are retained on the membrane though binding of the RNC to the translocon. It is also possible that mRNA binding proteins at the ER bind transcripts. c, Distributions of RNA-seq membrane enrichment scores of secretory protein transcripts (n = 584). d, After mRNA export, a pioneer round of targeting directs secretory transcripts to the ER membrane. SRP is specifically pre-recruited transcripts that will present a functional targeting signal. Upon emergence of an SS or TMD, SRP directs RNCs to the ER membrane. Once at the ER membrane, transcripts are retained over multiple rounds of translation.

    Techniques Used: RNA Sequencing Assay, Binding Assay, Functional Assay

    The role translation in membrane enrichment a, Lysates were treated with puromycin prior to membrane fractionation. mRNA recovered from the soluble and membrane fractions were used for RNA-seq b, Membrane enrichment of secretory protein transcripts (SS, TMD, SS-TMD, or TA, n = 729) following puromycin treatment of lysates.
    Figure Legend Snippet: The role translation in membrane enrichment a, Lysates were treated with puromycin prior to membrane fractionation. mRNA recovered from the soluble and membrane fractions were used for RNA-seq b, Membrane enrichment of secretory protein transcripts (SS, TMD, SS-TMD, or TA, n = 729) following puromycin treatment of lysates.

    Techniques Used: Fractionation, RNA Sequencing Assay

    16) Product Images from "Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation"

    Article Title: Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation

    Journal: Bioengineered

    doi: 10.1080/21655979.2017.1313648

    PVSA Effect on Decoupled in vitro Transcription with Subsequent Translation. (A) A schematic illustrates in vitro transcription (IVT) and subsequent purification with isopropanol precipitation and in vitro translation. (B) Image of mRNA product from IVT after agarose gel electrophoresis and staining with ethidium bromide. Lane 1 is the nucleic acid marker of double stranded DNA with bands corresponding to 400, 500, 600, 700, and 800 base pairs from bottom to top. Lane 2 is the IVT product where no PVSA was added. Lane 3 is the IVT product with 5 mg/mL PVSA. The expected migration location for the 898 nucleotide long mRNA is shown by arrow and corresponds to ∼600 base pairs of double stranded DNA due to the mRNA's single stranded and only partially hybridized nature. (C) Relative GFP protein yields as translated with mRNA produced by IVT in the presence of 0, 5, or 10 mg/mL PVSA and after 0 or 7 d of storage (n = 6, error bars represent one standard deviation).
    Figure Legend Snippet: PVSA Effect on Decoupled in vitro Transcription with Subsequent Translation. (A) A schematic illustrates in vitro transcription (IVT) and subsequent purification with isopropanol precipitation and in vitro translation. (B) Image of mRNA product from IVT after agarose gel electrophoresis and staining with ethidium bromide. Lane 1 is the nucleic acid marker of double stranded DNA with bands corresponding to 400, 500, 600, 700, and 800 base pairs from bottom to top. Lane 2 is the IVT product where no PVSA was added. Lane 3 is the IVT product with 5 mg/mL PVSA. The expected migration location for the 898 nucleotide long mRNA is shown by arrow and corresponds to ∼600 base pairs of double stranded DNA due to the mRNA's single stranded and only partially hybridized nature. (C) Relative GFP protein yields as translated with mRNA produced by IVT in the presence of 0, 5, or 10 mg/mL PVSA and after 0 or 7 d of storage (n = 6, error bars represent one standard deviation).

    Techniques Used: In Vitro, Purification, Agarose Gel Electrophoresis, Staining, Marker, Migration, Produced, Standard Deviation

    17) Product Images from "The presence of multiple introns is essential for ERECTA expression in Arabidopsis"

    Article Title: The presence of multiple introns is essential for ERECTA expression in Arabidopsis

    Journal: RNA

    doi: 10.1261/rna.2825811

    The intronless ERECTA gene does not rescue the erecta knockout mutant and produces dramatically reduced amount of mRNA. ( A ) Complementation of erecta-105 with ER::gERECTA and ER::cERECTA . Bar, 3 mm. ( B – E ) The amount of ERECTA mRNA in ER::cERECTA
    Figure Legend Snippet: The intronless ERECTA gene does not rescue the erecta knockout mutant and produces dramatically reduced amount of mRNA. ( A ) Complementation of erecta-105 with ER::gERECTA and ER::cERECTA . Bar, 3 mm. ( B – E ) The amount of ERECTA mRNA in ER::cERECTA

    Techniques Used: Knock-Out, Mutagenesis

    cERECTA-RLUC mRNA has a significantly lower level of polyadenylation compared to gERECTA-RLUC . ( A ) Poly(A) tail-length assay of total RNA detected about 120 adenines (the amplified fragment is ∼400 bp long, of which 280 bp encodes a segment of
    Figure Legend Snippet: cERECTA-RLUC mRNA has a significantly lower level of polyadenylation compared to gERECTA-RLUC . ( A ) Poly(A) tail-length assay of total RNA detected about 120 adenines (the amplified fragment is ∼400 bp long, of which 280 bp encodes a segment of

    Techniques Used: Amplification

    The c ERECTA-RLUC gene produces 11–43 times less mRNA and 486–941 times less protein compared with the gERECTA-RLUC gene. ( A , B ) The amount of ERECTA mRNA in ER::cERECTA-RLUC plants is dramatically lower compared with the wild type and
    Figure Legend Snippet: The c ERECTA-RLUC gene produces 11–43 times less mRNA and 486–941 times less protein compared with the gERECTA-RLUC gene. ( A , B ) The amount of ERECTA mRNA in ER::cERECTA-RLUC plants is dramatically lower compared with the wild type and

    Techniques Used:

    18) Product Images from "Zebrafish wnt3 is Expressed in Developing Neural Tissue"

    Article Title: Zebrafish wnt3 is Expressed in Developing Neural Tissue

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    doi: 10.1002/dvdy.21977

    Zebrafish Wnt3 can activate the canonical Wnt pathway. Zebrafish heterozygous for a gfp transgene under the control of a Wnt/β-catenin-dependent promoter ( Tg ( TOP:GFP ) w25 /+ ) were outcrossed, and the resulting embryos were allowed to develop uninjected (left column), or injected with either 30 pg (middle column) or 70pg (right column) of wnt3 mRNA. At shield stage, embryos were fixed and processed by WISH for expression of either gfp (top row) or chd (bottom row) as indicated. Injected embryos showed a dose-dependent increase in canonical Wnt signaling-driven transcript abundance. 100% of embryos displayed the phenotypes depicted for each condition. Non-transgenic siblings provided an internal control for gfp in situs (not pictured).
    Figure Legend Snippet: Zebrafish Wnt3 can activate the canonical Wnt pathway. Zebrafish heterozygous for a gfp transgene under the control of a Wnt/β-catenin-dependent promoter ( Tg ( TOP:GFP ) w25 /+ ) were outcrossed, and the resulting embryos were allowed to develop uninjected (left column), or injected with either 30 pg (middle column) or 70pg (right column) of wnt3 mRNA. At shield stage, embryos were fixed and processed by WISH for expression of either gfp (top row) or chd (bottom row) as indicated. Injected embryos showed a dose-dependent increase in canonical Wnt signaling-driven transcript abundance. 100% of embryos displayed the phenotypes depicted for each condition. Non-transgenic siblings provided an internal control for gfp in situs (not pictured).

    Techniques Used: Injection, Expressing, Transgenic Assay

    19) Product Images from "Phenylpropionc acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity"

    Article Title: Phenylpropionc acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity

    Journal: bioRxiv

    doi: 10.1101/811984

    A. TAC mice were fed with PPA (0.4% in drinking water) or regular water (CTL) for 4 weeks. A group of mice were sacrificed after overnight fasting, and liver RNAs were isolated. The other group of mice received APAP (300 mg/kg intraperitoneally) after overnight fasting. The mice were sacrificed after 6 h, followed by liver RNA isolation. Transcriptome profiles were obtained by RNA-sequencing TAC mice. B. JAX or TAC mice were sacrificed after overnight fasting, followed by isolation of mitochondria, cytosol (Cyt) and microsomes. Mitochondrial and cytosolic proteins (10 μg/well) as well as microsomal proteins (1, 5, or 10 μg/well) were analyzed by western blot using antibodies detecting VDAC (voltage-dependent anion channel; mitochondria marker), α-tubulin (cytosol marker), and calnexin (microsome marker). C. Basal hepatic Cyp2e1 mRNA levels in PPA-supplemented mice were measured by qRT-PCR. D. Gut-microbiota transplanted mice were sacrificed after overnight fasting, and microsomal CYP2E1 and CYP1A2 levels were measured by western blot (left). APAP bioactivation levels (right) were determined by measuring APAP-GSH formation rate in the hepatic microsomes. E. Hepatic CYP2E1 activity levels were determined by measuring the rate of chlorzoxazone to 6-hydroxychlorzoxazone conversion rate in the hepatic microsomes. F. Mouse hepatic microsomes were incubated with chlorzoxazone (100 μM) in the presence of PPA (1 mM) or p -nitrophenol (PNP; 1 mM; a known CYP2E1 substrate as a competitive inhibitor) for 30 min, and the extent of 6-hydroxychlorzoxazone production was compared. All data are shown as mean ± S.D.
    Figure Legend Snippet: A. TAC mice were fed with PPA (0.4% in drinking water) or regular water (CTL) for 4 weeks. A group of mice were sacrificed after overnight fasting, and liver RNAs were isolated. The other group of mice received APAP (300 mg/kg intraperitoneally) after overnight fasting. The mice were sacrificed after 6 h, followed by liver RNA isolation. Transcriptome profiles were obtained by RNA-sequencing TAC mice. B. JAX or TAC mice were sacrificed after overnight fasting, followed by isolation of mitochondria, cytosol (Cyt) and microsomes. Mitochondrial and cytosolic proteins (10 μg/well) as well as microsomal proteins (1, 5, or 10 μg/well) were analyzed by western blot using antibodies detecting VDAC (voltage-dependent anion channel; mitochondria marker), α-tubulin (cytosol marker), and calnexin (microsome marker). C. Basal hepatic Cyp2e1 mRNA levels in PPA-supplemented mice were measured by qRT-PCR. D. Gut-microbiota transplanted mice were sacrificed after overnight fasting, and microsomal CYP2E1 and CYP1A2 levels were measured by western blot (left). APAP bioactivation levels (right) were determined by measuring APAP-GSH formation rate in the hepatic microsomes. E. Hepatic CYP2E1 activity levels were determined by measuring the rate of chlorzoxazone to 6-hydroxychlorzoxazone conversion rate in the hepatic microsomes. F. Mouse hepatic microsomes were incubated with chlorzoxazone (100 μM) in the presence of PPA (1 mM) or p -nitrophenol (PNP; 1 mM; a known CYP2E1 substrate as a competitive inhibitor) for 30 min, and the extent of 6-hydroxychlorzoxazone production was compared. All data are shown as mean ± S.D.

    Techniques Used: Mouse Assay, Isolation, RNA Sequencing Assay, Western Blot, Marker, Quantitative RT-PCR, Activity Assay, Incubation

    20) Product Images from "Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis"

    Article Title: Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06898-4

    Mettl3-mediated m 6 A modification in MSCs regulates Pth1r translation. a m 6 A MeRIP-Seq revealed that Pth1r has high enriched and specific m 6 A peak near its translation stop codon. b MeRIP-qPCR validation of Pth1r m 6 A peak specificity. c Heatmap of representative osteogenesis and adipogenesis associated genes. d GSEA showed decreased enrichment of PTH-regulated genes in Mettl3 -deficient MSCs. e qRT-PCR analysis of Pth1r expression. f Western blot analysis of Pth1r. WT : Mettl3 fl/fl , cKO : Prx1-Cre;Mettl3 fl/fl . g Translation efficiency of Pth1r. h PCR analysis of Pth1r mRNA in different polysome gradient fractions in the Mettl3 knockout and control cells. Hprt1 was used as a control. i Quantification of Pth1r mRNA relative distribution in the Mettl3 depleted and control cells. The band intensities in g were analyzed by Image J. The relative amount of Pth1r or Hprt1 mRNA in each fraction was calculated as percentage of the total. Then the relative distribution of Pth1r mRNA was plotted by normalizing the percentage of Pth1r mRNA to Hprt1 mRNA in each fraction. Results are from three independent experiments. Data are expressed as mean ± s.e.m.; * P
    Figure Legend Snippet: Mettl3-mediated m 6 A modification in MSCs regulates Pth1r translation. a m 6 A MeRIP-Seq revealed that Pth1r has high enriched and specific m 6 A peak near its translation stop codon. b MeRIP-qPCR validation of Pth1r m 6 A peak specificity. c Heatmap of representative osteogenesis and adipogenesis associated genes. d GSEA showed decreased enrichment of PTH-regulated genes in Mettl3 -deficient MSCs. e qRT-PCR analysis of Pth1r expression. f Western blot analysis of Pth1r. WT : Mettl3 fl/fl , cKO : Prx1-Cre;Mettl3 fl/fl . g Translation efficiency of Pth1r. h PCR analysis of Pth1r mRNA in different polysome gradient fractions in the Mettl3 knockout and control cells. Hprt1 was used as a control. i Quantification of Pth1r mRNA relative distribution in the Mettl3 depleted and control cells. The band intensities in g were analyzed by Image J. The relative amount of Pth1r or Hprt1 mRNA in each fraction was calculated as percentage of the total. Then the relative distribution of Pth1r mRNA was plotted by normalizing the percentage of Pth1r mRNA to Hprt1 mRNA in each fraction. Results are from three independent experiments. Data are expressed as mean ± s.e.m.; * P

    Techniques Used: Modification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Polymerase Chain Reaction, Knock-Out

    21) Product Images from "Ablation of adipocyte creatine transport impairs thermogenesis and causes diet-induced obesity"

    Article Title: Ablation of adipocyte creatine transport impairs thermogenesis and causes diet-induced obesity

    Journal: Nature metabolism

    doi: 10.1038/s42255-019-0035-x

    CRT expression in human adipocytes is negatively correlated with obesity and insulin resistance. a, mRNA abundance of GATM , GAMT , and CRT by RNA-sequencing ( n = 43). b, Pearson correlation of CRT mRNA expression with body mass index (BMI) ( n = 43). Black line, linear regression; gray curved lines, 95% confidence intervals. c, mRNA abundance of GATM , GAMT , and CRT in patients stratified as insulin resistant (IR) and insulin sensitive (IS) (IS, n = 16; IR, n = 27). d, BMI of IS patients with relatively high ( n = 9) or low ( n = 7) CRT mRNA abundance. Data are presented as mean ± s.e.m. of biologically independent samples. One-way ANOVA ( a ); pearson correlation ( b ); multiple two-tailed Student’s t -tests ( c ); two-tailed Student’s t -tests ( d ).
    Figure Legend Snippet: CRT expression in human adipocytes is negatively correlated with obesity and insulin resistance. a, mRNA abundance of GATM , GAMT , and CRT by RNA-sequencing ( n = 43). b, Pearson correlation of CRT mRNA expression with body mass index (BMI) ( n = 43). Black line, linear regression; gray curved lines, 95% confidence intervals. c, mRNA abundance of GATM , GAMT , and CRT in patients stratified as insulin resistant (IR) and insulin sensitive (IS) (IS, n = 16; IR, n = 27). d, BMI of IS patients with relatively high ( n = 9) or low ( n = 7) CRT mRNA abundance. Data are presented as mean ± s.e.m. of biologically independent samples. One-way ANOVA ( a ); pearson correlation ( b ); multiple two-tailed Student’s t -tests ( c ); two-tailed Student’s t -tests ( d ).

    Techniques Used: Expressing, RNA Sequencing Assay, Two Tailed Test

    22) Product Images from "Targeted in vivo epigenome editing of H3K27me3"

    Article Title: Targeted in vivo epigenome editing of H3K27me3

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-019-0263-z

    H3K27me3 epigenome editing by dCas9-olEzh2(∆SET) and higher concentration injection. a – c dCas9-olEzh2(∆SET) analyses targeting Arhgap35 promoter. ChIP-qPCR using anti-FLAG antibody ( a ), ChIP-qPCR using anti-H3K27me3 antibody ( b ) and Arhgap35 mRNA expression fold change ( c ). d – e Arhgap35 mRNA expression fold change ( d ) and ChIP-qPCR using anti-H3K27me3 antibody ( e ) in higher concentration injection. f The epigenetic modification patterns around Kita , sgRNAs (blue bars) and ChIP-qPCR product (black bars) positions. H3K27me3 (red) and H3K27ac (blue) ChIP-seq [ 27 ], DNase I-seq (black) [ 28 ] and DNA methylation enrichment [ 34 ] at the blastula stage are shown for comparison. g – i dCas9-olEzh2(∆SET) analyses targeting Kita promoter. ChIP-qPCR using anti-FLAG antibody ( g ), ChIP-qPCR using anti-H3K27me3 antibody ( h ) and Kita mRNA expression fold change ( i ). In Fig. 4 c, e and i, after expression levels were normalized to that of beta-actin, fold changes (sample/no injection) were calculated. Light blue, gray, orange, red and pink bars in each bar graph represent no injection, sgRNAs/dCas9 injection, sgRNAs/dCas9-olEzh2 injection, sgRNAs/dCas9-olEzh2(∆SET)(350 nM) injection and sgRNAs/dCas9-olEzh2(∆SET)(550 nM) injection, respectively. (Tukey–Kramer test, * p
    Figure Legend Snippet: H3K27me3 epigenome editing by dCas9-olEzh2(∆SET) and higher concentration injection. a – c dCas9-olEzh2(∆SET) analyses targeting Arhgap35 promoter. ChIP-qPCR using anti-FLAG antibody ( a ), ChIP-qPCR using anti-H3K27me3 antibody ( b ) and Arhgap35 mRNA expression fold change ( c ). d – e Arhgap35 mRNA expression fold change ( d ) and ChIP-qPCR using anti-H3K27me3 antibody ( e ) in higher concentration injection. f The epigenetic modification patterns around Kita , sgRNAs (blue bars) and ChIP-qPCR product (black bars) positions. H3K27me3 (red) and H3K27ac (blue) ChIP-seq [ 27 ], DNase I-seq (black) [ 28 ] and DNA methylation enrichment [ 34 ] at the blastula stage are shown for comparison. g – i dCas9-olEzh2(∆SET) analyses targeting Kita promoter. ChIP-qPCR using anti-FLAG antibody ( g ), ChIP-qPCR using anti-H3K27me3 antibody ( h ) and Kita mRNA expression fold change ( i ). In Fig. 4 c, e and i, after expression levels were normalized to that of beta-actin, fold changes (sample/no injection) were calculated. Light blue, gray, orange, red and pink bars in each bar graph represent no injection, sgRNAs/dCas9 injection, sgRNAs/dCas9-olEzh2 injection, sgRNAs/dCas9-olEzh2(∆SET)(350 nM) injection and sgRNAs/dCas9-olEzh2(∆SET)(550 nM) injection, respectively. (Tukey–Kramer test, * p

    Techniques Used: Concentration Assay, Injection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Modification, DNA Methylation Assay

    H3K27me3 epigenome editing by dCas9-olEzh2 targeting hypomethylated promoters. a Schematic of dCas9, dCas9-olEzh2 and dCas9-olEzh2(∆SET) constructs and H3K27me3 induction caused by dCas9-olEzh2. b Schematic view of the dCas9-olEzh2 epigenome editing and injection experiments. sgRNA and mRNA were injected at the one-cell stage (stage 2). ChIP-qPCR was performed using the late blastula embryos (stage 11, 8 h after injection). RT-qPCR was performed using the pre-early gastrula embryos (stage 12, 10 h after injection), because ZGA occurs at the late blastula (stage 11) in medaka. c , g , k , n The epigenetic modification patterns around Arhgap35, Kita , Nanos3 and Dcx , sgRNAs (blue bars) and ChIP-qPCR product (black bars) positions. H3K27me3 (red) and H3K27ac (blue) ChIP-seq [ 27 ], DNase I-seq (black) [ 28 ] and DNA methylation [ 34 ] enrichment at the blastula stage are shown. d , e , h , i , l , m , o , p The results of ChIP-qPCR using anti-FLAG antibody ( d , h , l , o ) and anti-H3K27me3 antibody ( e , i , l , m ). H3K27me3 negative region (K27me3 NC) and H3K27me3 positive region (K27me3 PC) were used for ChIP control (described in Additional file 1 : Fig. S2). f , j Arhgap35 and Pfkfb4a mRNA expression fold change. After expression levels were normalized to that of beta-actin, fold changes (sample/no injection) were calculated. Light blue, gray and orange bars in each bar graph represent no injection, sgRNAs/dCas9 injection and sgRNAs/dCas9-olEzh2 injection, respectively. (Tukey–Kramer test and only in Fig. 1f, j Student’s t test, * p
    Figure Legend Snippet: H3K27me3 epigenome editing by dCas9-olEzh2 targeting hypomethylated promoters. a Schematic of dCas9, dCas9-olEzh2 and dCas9-olEzh2(∆SET) constructs and H3K27me3 induction caused by dCas9-olEzh2. b Schematic view of the dCas9-olEzh2 epigenome editing and injection experiments. sgRNA and mRNA were injected at the one-cell stage (stage 2). ChIP-qPCR was performed using the late blastula embryos (stage 11, 8 h after injection). RT-qPCR was performed using the pre-early gastrula embryos (stage 12, 10 h after injection), because ZGA occurs at the late blastula (stage 11) in medaka. c , g , k , n The epigenetic modification patterns around Arhgap35, Kita , Nanos3 and Dcx , sgRNAs (blue bars) and ChIP-qPCR product (black bars) positions. H3K27me3 (red) and H3K27ac (blue) ChIP-seq [ 27 ], DNase I-seq (black) [ 28 ] and DNA methylation [ 34 ] enrichment at the blastula stage are shown. d , e , h , i , l , m , o , p The results of ChIP-qPCR using anti-FLAG antibody ( d , h , l , o ) and anti-H3K27me3 antibody ( e , i , l , m ). H3K27me3 negative region (K27me3 NC) and H3K27me3 positive region (K27me3 PC) were used for ChIP control (described in Additional file 1 : Fig. S2). f , j Arhgap35 and Pfkfb4a mRNA expression fold change. After expression levels were normalized to that of beta-actin, fold changes (sample/no injection) were calculated. Light blue, gray and orange bars in each bar graph represent no injection, sgRNAs/dCas9 injection and sgRNAs/dCas9-olEzh2 injection, respectively. (Tukey–Kramer test and only in Fig. 1f, j Student’s t test, * p

    Techniques Used: Construct, Injection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Modification, DNA Methylation Assay, Expressing

    23) Product Images from "Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system"

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08345-4

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d
    Figure Legend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d

    Techniques Used: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization

    24) Product Images from "Proteasome-mediated regulation of Cdhr1a by Siah1 modulates photoreceptor development and survival in zebrafish"

    Article Title: Proteasome-mediated regulation of Cdhr1a by Siah1 modulates photoreceptor development and survival in zebrafish

    Journal: bioRxiv

    doi: 10.1101/2020.05.15.098350

    Cone photoreceptor development relies on sufficient levels of Cdhr1a. Confocal stacks of heat shocked (HS) Tg[ TαC :GFP] (wildtype) and Tg[ hsp70 :siah1]/Tg[ TαC :GFP] (siah1) embryos or those injected with cdhr1a mRNA or cdhr1a LMA mRNA were analyzed in 3D for GFP fluorescence ( A-D ). Injection of both wildtype and the LMA variant of cdhr1a mRNA resulted in numbers of GFP+ cone cells comparable to that of wildtype ( E ). Scale bar = 50 μm.
    Figure Legend Snippet: Cone photoreceptor development relies on sufficient levels of Cdhr1a. Confocal stacks of heat shocked (HS) Tg[ TαC :GFP] (wildtype) and Tg[ hsp70 :siah1]/Tg[ TαC :GFP] (siah1) embryos or those injected with cdhr1a mRNA or cdhr1a LMA mRNA were analyzed in 3D for GFP fluorescence ( A-D ). Injection of both wildtype and the LMA variant of cdhr1a mRNA resulted in numbers of GFP+ cone cells comparable to that of wildtype ( E ). Scale bar = 50 μm.

    Techniques Used: Injection, Fluorescence, Variant Assay

    Rod photoreceptor development relies on sufficient levels of Cdhr1a. Retinal cryosections from Tg[ hsp70 :siah1]/Tg[ XOPS :GFP] (siah1), injected with wildtype cdhr1a or cdhr1a LMA mRNA were heat shocked (HS) and analyzed for immature and mature rod cells using IHC for 4C12 (red) ( A-C’ ). Injection of both cdhr1a and cdhr1a LMA mRNA increased the number of immature and mature rod cells compared to siah1 HS alone ( D ). Retinal cryosections from Tg[ hsp70 :siah1]/Tg[ XOPS :GFP] (siah1), injected with wildtype cdhr1a or cdhr1a LMA mRNA were heat shocked (HS) and analyzed for GFP signal (green) ( E-G’ ). Injection of both cdhr1a and cdhr1a LMA mRNA increased the number of GFP+ rod cells compared to siah1 HS alone, with cdhr1a LMA giving a significantly stronger response ( H ). DNA was stained with DAPI (blue). Scale bar = 50μm. L: lens, ONL: outer nuclear layer, INL: Inner nuclear layer, GCL: ganglion cell layer, D: Dorsal and V: Ventral.
    Figure Legend Snippet: Rod photoreceptor development relies on sufficient levels of Cdhr1a. Retinal cryosections from Tg[ hsp70 :siah1]/Tg[ XOPS :GFP] (siah1), injected with wildtype cdhr1a or cdhr1a LMA mRNA were heat shocked (HS) and analyzed for immature and mature rod cells using IHC for 4C12 (red) ( A-C’ ). Injection of both cdhr1a and cdhr1a LMA mRNA increased the number of immature and mature rod cells compared to siah1 HS alone ( D ). Retinal cryosections from Tg[ hsp70 :siah1]/Tg[ XOPS :GFP] (siah1), injected with wildtype cdhr1a or cdhr1a LMA mRNA were heat shocked (HS) and analyzed for GFP signal (green) ( E-G’ ). Injection of both cdhr1a and cdhr1a LMA mRNA increased the number of GFP+ rod cells compared to siah1 HS alone, with cdhr1a LMA giving a significantly stronger response ( H ). DNA was stained with DAPI (blue). Scale bar = 50μm. L: lens, ONL: outer nuclear layer, INL: Inner nuclear layer, GCL: ganglion cell layer, D: Dorsal and V: Ventral.

    Techniques Used: Injection, Immunohistochemistry, Staining

    25) Product Images from "Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy"

    Article Title: Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-11-166

    mRNA-electroporated moDCs and LCs retain eGFP expression after cryopreservation. Immature (□) and partially-matured (▤) moDCs and LCs were electroporated with eGFP mRNA and cryopreserved. After thawing, eGFP expression for each experimental group was measured by flow cytometry and compared with pre-cryopreservation eGFP expression values to determine percent retention of eGFP expression. Pooled data are shown for each experimental group (mean ± SD, n = 3 independent experiments, ns = not significant).
    Figure Legend Snippet: mRNA-electroporated moDCs and LCs retain eGFP expression after cryopreservation. Immature (□) and partially-matured (▤) moDCs and LCs were electroporated with eGFP mRNA and cryopreserved. After thawing, eGFP expression for each experimental group was measured by flow cytometry and compared with pre-cryopreservation eGFP expression values to determine percent retention of eGFP expression. Pooled data are shown for each experimental group (mean ± SD, n = 3 independent experiments, ns = not significant).

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Optimization of mRNA-electroporation conditions. Immature moDCs (A-C) and partially-matured LCs (D-F) were electroporated with eGFP-encoding mRNA under different conditions of set voltage, number of electroporation pulses, or amount of mRNA. Only one of the three parameters was varied in any given set of tested conditions, as summarized in each panel. Cells were assessed for viability (○) by trypan blue exclusion and transfection efficiency (▤) by flow cytometry.
    Figure Legend Snippet: Optimization of mRNA-electroporation conditions. Immature moDCs (A-C) and partially-matured LCs (D-F) were electroporated with eGFP-encoding mRNA under different conditions of set voltage, number of electroporation pulses, or amount of mRNA. Only one of the three parameters was varied in any given set of tested conditions, as summarized in each panel. Cells were assessed for viability (○) by trypan blue exclusion and transfection efficiency (▤) by flow cytometry.

    Techniques Used: Electroporation, Transfection, Flow Cytometry, Cytometry

    mRNA electroporation induces the maturation and activation of LCs to a greater magnitude than for moDCs. Immature moDCs and LCs were electroporated with eGFP mRNA and then cultured with (+) or without (−) standard maturation-inducing inflammatory cytokines. After 24 hours, cells in each experimental group were compared with pre-electroporation controls by flow cytometry for the expression of phenotypic markers of DC maturation and activation, based on the upregulation of (A) CD83, (B) CD80, and (C) CD86, respectively. Representative dot plots of eGFP mRNA-electroporated moDCs and LCs from one of three independent experiments are shown in the top row. Pooled data for each experimental group (mean ± SD, n = 3 independent experiments) are shown in the bottom row (gray bar = pre-electroporation control, white bar = post-electroporation without inflammatory cytokines, black bar = post-electroporation with inflammatory cytokines). * P
    Figure Legend Snippet: mRNA electroporation induces the maturation and activation of LCs to a greater magnitude than for moDCs. Immature moDCs and LCs were electroporated with eGFP mRNA and then cultured with (+) or without (−) standard maturation-inducing inflammatory cytokines. After 24 hours, cells in each experimental group were compared with pre-electroporation controls by flow cytometry for the expression of phenotypic markers of DC maturation and activation, based on the upregulation of (A) CD83, (B) CD80, and (C) CD86, respectively. Representative dot plots of eGFP mRNA-electroporated moDCs and LCs from one of three independent experiments are shown in the top row. Pooled data for each experimental group (mean ± SD, n = 3 independent experiments) are shown in the bottom row (gray bar = pre-electroporation control, white bar = post-electroporation without inflammatory cytokines, black bar = post-electroporation with inflammatory cytokines). * P

    Techniques Used: Electroporation, Activation Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Cell recovery and viability of moDCs and LCs after electroporation and cryopreservation. Immature (□) and partially-matured (▤) moDCs and LCs were electroporated with eGFP-encoding mRNA, without purification by HLA-DR selection a priori. (A) Immediate post-electroporation viable cell recovery relative to the total number of cells electroporated was assessed by trypan blue exclusion (mean ± SD, n = 4 independent experiments). (B) Surviving cells from (A) were returned to culture, and cell viability was assessed 24 hours after electroporation, relative to initial number of cells returned to culture after electroporation (mean ± SD, n = 4 independent experiments). (C) After at least 24 hours in culture, cells from (B) were cryopreserved. Upon subsequent thawing, immediate post-thaw viability was assessed relative to initial number of cells frozen (mean ± SD, n = 4 independent experiments). (D) After 24 hours in culture, thawed cells from (C) were reassessed for viability (mean ± SD, n = 4 independent experiments).
    Figure Legend Snippet: Cell recovery and viability of moDCs and LCs after electroporation and cryopreservation. Immature (□) and partially-matured (▤) moDCs and LCs were electroporated with eGFP-encoding mRNA, without purification by HLA-DR selection a priori. (A) Immediate post-electroporation viable cell recovery relative to the total number of cells electroporated was assessed by trypan blue exclusion (mean ± SD, n = 4 independent experiments). (B) Surviving cells from (A) were returned to culture, and cell viability was assessed 24 hours after electroporation, relative to initial number of cells returned to culture after electroporation (mean ± SD, n = 4 independent experiments). (C) After at least 24 hours in culture, cells from (B) were cryopreserved. Upon subsequent thawing, immediate post-thaw viability was assessed relative to initial number of cells frozen (mean ± SD, n = 4 independent experiments). (D) After 24 hours in culture, thawed cells from (C) were reassessed for viability (mean ± SD, n = 4 independent experiments).

    Techniques Used: Electroporation, Purification, Selection

    mRNA-electroporated moDCs and LCs remain potent inducers of allogeneic T cell proliferation, including after cryopreservation and thawing. (A) MoDCs and LCs, as indicated on the x axes, were electroporated with WT1 mRNA (○), eGFP mRNA (△), or mock-electroporated with no mRNA (□). After electroporation, DCs were terminally matured for 24 hours and then cultured with allogeneic T cells for five days in allogeneic MLRs. DC:T ratios ranged from 1:10 to 1:1000. [3H]TdR uptake by proliferating allogeneic T cells over the final 8 hours of culture was measured as an index of DC immunogenicity (triplicate means ± SEM, n = 3 independent experiments). (B) To assess preservation of allo-stimulatory capacity in MLRs after cryopreservation and thawing, WT1 mRNA-electroporated moDCs (△) and LCs (○) were compared with non-cryopreserved mature moDCs (▤). Culture conditions were otherwise exactly the same as in (A) . T cell proliferation was measured by a colorimetric assay (triplicate means ± SEM, n = 3 independent experiments). Dotted line represents T cells alone.
    Figure Legend Snippet: mRNA-electroporated moDCs and LCs remain potent inducers of allogeneic T cell proliferation, including after cryopreservation and thawing. (A) MoDCs and LCs, as indicated on the x axes, were electroporated with WT1 mRNA (○), eGFP mRNA (△), or mock-electroporated with no mRNA (□). After electroporation, DCs were terminally matured for 24 hours and then cultured with allogeneic T cells for five days in allogeneic MLRs. DC:T ratios ranged from 1:10 to 1:1000. [3H]TdR uptake by proliferating allogeneic T cells over the final 8 hours of culture was measured as an index of DC immunogenicity (triplicate means ± SEM, n = 3 independent experiments). (B) To assess preservation of allo-stimulatory capacity in MLRs after cryopreservation and thawing, WT1 mRNA-electroporated moDCs (△) and LCs (○) were compared with non-cryopreserved mature moDCs (▤). Culture conditions were otherwise exactly the same as in (A) . T cell proliferation was measured by a colorimetric assay (triplicate means ± SEM, n = 3 independent experiments). Dotted line represents T cells alone.

    Techniques Used: Electroporation, Cell Culture, Preserving, Colorimetric Assay

    The maturation status of moDCs and LCs affects mRNA-electroporation transfection efficiency. Immature (□) and partially-matured (▤) moDCs (A, C) and LCs (B, D) were electroporated with eGFP-encoding mRNA. The transfection efficiency for each experimental group was assessed by flow cytometry at the indicated time points. Pooled data (mean ± SD, n = 6 independent experiments) are shown for moDCs (A) and LCs (B) . * P
    Figure Legend Snippet: The maturation status of moDCs and LCs affects mRNA-electroporation transfection efficiency. Immature (□) and partially-matured (▤) moDCs (A, C) and LCs (B, D) were electroporated with eGFP-encoding mRNA. The transfection efficiency for each experimental group was assessed by flow cytometry at the indicated time points. Pooled data (mean ± SD, n = 6 independent experiments) are shown for moDCs (A) and LCs (B) . * P

    Techniques Used: Electroporation, Transfection, Flow Cytometry, Cytometry

    26) Product Images from "Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology"

    Article Title: Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology

    Journal: Scientific Reports

    doi: 10.1038/srep05290

    Strategy to generate NRG Fah knockout mice. (a) Schematic illustration of IVF and pronuclear microinjection. Female NRG mice are superovulated with PMSG and hCG followed by oocyte collection. Sperm is collected from male NRG mice. The oocytes and sperm are incubated to generate fertilized eggs and embryos, which are then microinjected with gRNA, Cas9 mRNA and ssDNA in the pronucleus. The injected embryos are then transferred into pseudopregnant surrogate mothers. Mouse pups are genotyped. (b) Mechanism of gRNA, Cas9 mRNA, and ssDNA mediated Fah gene knockout. Cas9 mRNA is translated into Cas9 protein after microinjection. The Cas9/gRNA complex binds the genomic DNA and generates DSBs. The ssDNA contains homologous sequence spanning the double strand break sites with ~50 bp on each side, two stop codons and a BamHI site. The ssDNA can be used as a template for homologous recombination to introduce the stop codons and BamHI site. The mouse is drawn by the authors (F.L. and L.S.), using Adobe Photoshop and Adobe Illustrator.
    Figure Legend Snippet: Strategy to generate NRG Fah knockout mice. (a) Schematic illustration of IVF and pronuclear microinjection. Female NRG mice are superovulated with PMSG and hCG followed by oocyte collection. Sperm is collected from male NRG mice. The oocytes and sperm are incubated to generate fertilized eggs and embryos, which are then microinjected with gRNA, Cas9 mRNA and ssDNA in the pronucleus. The injected embryos are then transferred into pseudopregnant surrogate mothers. Mouse pups are genotyped. (b) Mechanism of gRNA, Cas9 mRNA, and ssDNA mediated Fah gene knockout. Cas9 mRNA is translated into Cas9 protein after microinjection. The Cas9/gRNA complex binds the genomic DNA and generates DSBs. The ssDNA contains homologous sequence spanning the double strand break sites with ~50 bp on each side, two stop codons and a BamHI site. The ssDNA can be used as a template for homologous recombination to introduce the stop codons and BamHI site. The mouse is drawn by the authors (F.L. and L.S.), using Adobe Photoshop and Adobe Illustrator.

    Techniques Used: Knock-Out, Mouse Assay, Incubation, Injection, Gene Knockout, Sequencing, Homologous Recombination, Introduce

    Genotyping and sequence analysis of founder mice. (a) PCR results of founder mice. Mice from two microinjection sessions were genotyped by PCR amplification and BamHI digestion. PCR and BamHI digestion products were electrophoresed in 1.2% argorose gel. Mice from the first injection session (wild type Cas9 mRNA) were labeled #1-1 to #1-10, and mice from the second injection session (mutant D10A Cas9 mRNA) were labeled #2-1 to #2-10. Wild type mouse genomic DNA was used as negative control (WT con). Cropped gels are shown. Full-length gels are provided for review in the supplementary file . (b) Sequence analysis of the mutated Fah alleles. The wild type sequence of exon5 is shown on top, with the encoded amino acids indicated above the sequence. The gRNA targeting sites are shown in red letters and PAM NGGs in turquoise. The mutant alleles of each mouse are labeled with A and B, following the mouse ID number. The indels for each mutated allele from different mice are shown in the middle. The deleted sequences are marked in gray, and the inserted sequences are shown below the wild type sequence. The deletion or insertion length of each indel is shown on the right. The Fah mutant alleles with the knock-in sequence are shown in the lower panel. Only one sequence is shown for the stop codon (@) and BamHI insertion. Indel, insertion and deletion; in, insertion; del, deletion.
    Figure Legend Snippet: Genotyping and sequence analysis of founder mice. (a) PCR results of founder mice. Mice from two microinjection sessions were genotyped by PCR amplification and BamHI digestion. PCR and BamHI digestion products were electrophoresed in 1.2% argorose gel. Mice from the first injection session (wild type Cas9 mRNA) were labeled #1-1 to #1-10, and mice from the second injection session (mutant D10A Cas9 mRNA) were labeled #2-1 to #2-10. Wild type mouse genomic DNA was used as negative control (WT con). Cropped gels are shown. Full-length gels are provided for review in the supplementary file . (b) Sequence analysis of the mutated Fah alleles. The wild type sequence of exon5 is shown on top, with the encoded amino acids indicated above the sequence. The gRNA targeting sites are shown in red letters and PAM NGGs in turquoise. The mutant alleles of each mouse are labeled with A and B, following the mouse ID number. The indels for each mutated allele from different mice are shown in the middle. The deleted sequences are marked in gray, and the inserted sequences are shown below the wild type sequence. The deletion or insertion length of each indel is shown on the right. The Fah mutant alleles with the knock-in sequence are shown in the lower panel. Only one sequence is shown for the stop codon (@) and BamHI insertion. Indel, insertion and deletion; in, insertion; del, deletion.

    Techniques Used: Sequencing, Mouse Assay, Polymerase Chain Reaction, Amplification, Injection, Labeling, Mutagenesis, Negative Control, Knock-In

    27) Product Images from "Integrating transcriptome and microRNA analysis identifies genes and microRNAs for AHO-induced systemic acquired resistance in N. tabacum"

    Article Title: Integrating transcriptome and microRNA analysis identifies genes and microRNAs for AHO-induced systemic acquired resistance in N. tabacum

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12249-y

    Expression of miRNAs and their target transcripts. Image ( a ) shows the DEMs abundance change in RNA-seq analysis with AHO treatment. Image ( b ) represents the potential regulation between miRNAs and their target genes. The T shape end and arrow represent the opposite or same regulation trend between miRNA and mRNA. Image ( c ) shows the DEGs level in RNA-seq analysis. Image ( d ) and ( e ) shows the relative expression levels of miRNA and mRNA transcript assessed by qRT-PCR, respectively. The relative level of qRT-PCR calculated by the delta-delta-CT method. Data is presented as mean ± standard error.
    Figure Legend Snippet: Expression of miRNAs and their target transcripts. Image ( a ) shows the DEMs abundance change in RNA-seq analysis with AHO treatment. Image ( b ) represents the potential regulation between miRNAs and their target genes. The T shape end and arrow represent the opposite or same regulation trend between miRNA and mRNA. Image ( c ) shows the DEGs level in RNA-seq analysis. Image ( d ) and ( e ) shows the relative expression levels of miRNA and mRNA transcript assessed by qRT-PCR, respectively. The relative level of qRT-PCR calculated by the delta-delta-CT method. Data is presented as mean ± standard error.

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    28) Product Images from "Integrating transcriptome and microRNA analysis identifies genes and microRNAs for AHO-induced systemic acquired resistance in N. tabacum"

    Article Title: Integrating transcriptome and microRNA analysis identifies genes and microRNAs for AHO-induced systemic acquired resistance in N. tabacum

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12249-y

    Expression of miRNAs and their target transcripts. Image ( a ) shows the DEMs abundance change in RNA-seq analysis with AHO treatment. Image ( b ) represents the potential regulation between miRNAs and their target genes. The T shape end and arrow represent the opposite or same regulation trend between miRNA and mRNA. Image ( c ) shows the DEGs level in RNA-seq analysis. Image ( d ) and ( e ) shows the relative expression levels of miRNA and mRNA transcript assessed by qRT-PCR, respectively. The relative level of qRT-PCR calculated by the delta-delta-CT method. Data is presented as mean ± standard error.
    Figure Legend Snippet: Expression of miRNAs and their target transcripts. Image ( a ) shows the DEMs abundance change in RNA-seq analysis with AHO treatment. Image ( b ) represents the potential regulation between miRNAs and their target genes. The T shape end and arrow represent the opposite or same regulation trend between miRNA and mRNA. Image ( c ) shows the DEGs level in RNA-seq analysis. Image ( d ) and ( e ) shows the relative expression levels of miRNA and mRNA transcript assessed by qRT-PCR, respectively. The relative level of qRT-PCR calculated by the delta-delta-CT method. Data is presented as mean ± standard error.

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    29) Product Images from "Intradermal Delivery of Synthetic mRNA Using Hollow Microneedles for Efficient and Rapid Production of Exogenous Proteins in Skin"

    Article Title: Intradermal Delivery of Synthetic mRNA Using Hollow Microneedles for Efficient and Rapid Production of Exogenous Proteins in Skin

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.03.005

    Detection of Luciferase Activity after Transfection of HEK923 Cells with Synthetic hGLuc mRNA 3 × 10 5 HEK293 cells were incubated for 4 hr at 37°C with 0.2, 0.5, or 1.5 μg mRNA complexed with Lipofectamine 2000. Then transfection complexes were discarded, and fresh medium was added to the cells. After 24 hr, the luciferase activity (RLUs) was detected in the supernatants and cell lysates. Cells treated with medium or medium and Lipofectamine 2000 (L2000) served as negative controls. Results are shown as mean ± SEM (n = 3). Statistical differences were determined using two-way ANOVA followed by Bonferroni’s multiple comparisons test (**p
    Figure Legend Snippet: Detection of Luciferase Activity after Transfection of HEK923 Cells with Synthetic hGLuc mRNA 3 × 10 5 HEK293 cells were incubated for 4 hr at 37°C with 0.2, 0.5, or 1.5 μg mRNA complexed with Lipofectamine 2000. Then transfection complexes were discarded, and fresh medium was added to the cells. After 24 hr, the luciferase activity (RLUs) was detected in the supernatants and cell lysates. Cells treated with medium or medium and Lipofectamine 2000 (L2000) served as negative controls. Results are shown as mean ± SEM (n = 3). Statistical differences were determined using two-way ANOVA followed by Bonferroni’s multiple comparisons test (**p

    Techniques Used: Luciferase, Activity Assay, Transfection, Incubation

    Delivery of Synthetic hGLuc mRNA into Porcine Skin Using Hollow Microneedles and Analysis of Protein Expression (1) Porcine skin detached from the outer side of pigs’ ears was trimmed and punched into 1-mm-thick pieces with 1.5-cm diameter and disinfected. The structure of the skin is shown schematically. S.c., stratum corneum ; E, epidermis; D, dermis. (2) Lipoplexes were generated by incubation of 1.5 μg hGLuc mRNA with 1.5 μl Lipofectamine 2000 in a total volume of 35 μL OptiMEM I reduced serum-free medium for 20 min at RT. The mixture was injected into the skin using MicronJet600 microneedles. (3) After washing with DPBS, the skin was transferred into a ThinCert insert, which served as a permeable barrier between the skin and the surrounding medium. (4) The skin was incubated air-exposed in 1.5 mL human endothelial cell culture medium in one well of a 12-well plate from 24 to 72 hr at 37°C and 5% CO 2 . The microinjected lipoplexes can enter the cells via endocytosis. After the release of mRNA into the cytosol, mRNA is translated by ribosomes into protein. (5) After the appropriate incubation time, the produced hGLuc protein is detected by luciferase assay in the surrounding medium as well as in the skin.
    Figure Legend Snippet: Delivery of Synthetic hGLuc mRNA into Porcine Skin Using Hollow Microneedles and Analysis of Protein Expression (1) Porcine skin detached from the outer side of pigs’ ears was trimmed and punched into 1-mm-thick pieces with 1.5-cm diameter and disinfected. The structure of the skin is shown schematically. S.c., stratum corneum ; E, epidermis; D, dermis. (2) Lipoplexes were generated by incubation of 1.5 μg hGLuc mRNA with 1.5 μl Lipofectamine 2000 in a total volume of 35 μL OptiMEM I reduced serum-free medium for 20 min at RT. The mixture was injected into the skin using MicronJet600 microneedles. (3) After washing with DPBS, the skin was transferred into a ThinCert insert, which served as a permeable barrier between the skin and the surrounding medium. (4) The skin was incubated air-exposed in 1.5 mL human endothelial cell culture medium in one well of a 12-well plate from 24 to 72 hr at 37°C and 5% CO 2 . The microinjected lipoplexes can enter the cells via endocytosis. After the release of mRNA into the cytosol, mRNA is translated by ribosomes into protein. (5) After the appropriate incubation time, the produced hGLuc protein is detected by luciferase assay in the surrounding medium as well as in the skin.

    Techniques Used: Expressing, Generated, Incubation, Injection, Cell Culture, Produced, Luciferase

    30) Product Images from "Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase"

    Article Title: Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2009111190

    FSK increases the AQP2 protein abundance in IMCD cells independent from transcriptional regulation. (A) IMCD cells were left untreated or treated with FSK (10 μM, 15 to 240 minutes). The AQP2 mRNA level was quantified by real-time PCR. Shown are
    Figure Legend Snippet: FSK increases the AQP2 protein abundance in IMCD cells independent from transcriptional regulation. (A) IMCD cells were left untreated or treated with FSK (10 μM, 15 to 240 minutes). The AQP2 mRNA level was quantified by real-time PCR. Shown are

    Techniques Used: Real-time Polymerase Chain Reaction

    31) Product Images from "A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2"

    Article Title: A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2

    Journal: Journal of Virology

    doi: 10.1128/JVI.02488-16

    Reovirus infection alters cellular mRNA splicing. (A) Venn diagram indicating differences in splicing based on MISO analysis of RNA-seq results. (B) Confirmation of differential expression of novel splicing variants using qRT-PCR. L929 cells were infected and stimulated with IFN-β using conditions identical to those used for RNA-seq. Transcripts with a specific exon skipped or spliced-in (as identified by RNA-seq) were quantitated by qRT-PCR for two representative genes. The results are expressed as a ratio of the two splicing events to capture the events comprising MISO analysis. The results of statistical analyses ( P
    Figure Legend Snippet: Reovirus infection alters cellular mRNA splicing. (A) Venn diagram indicating differences in splicing based on MISO analysis of RNA-seq results. (B) Confirmation of differential expression of novel splicing variants using qRT-PCR. L929 cells were infected and stimulated with IFN-β using conditions identical to those used for RNA-seq. Transcripts with a specific exon skipped or spliced-in (as identified by RNA-seq) were quantitated by qRT-PCR for two representative genes. The results are expressed as a ratio of the two splicing events to capture the events comprising MISO analysis. The results of statistical analyses ( P

    Techniques Used: Infection, RNA Sequencing Assay, Expressing, Quantitative RT-PCR

    32) Product Images from "Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade"

    Article Title: Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14979-6

    TARGET TF-perturbation assay captures direct targets of NLP7 based on TF regulation or TF binding in isolated root cells. a Schematic of the cell-based TARGET TF-perturbation system and experimental design 22 (see “Methods”). Root cells isolated from an nlp7 mutant 11 , 23 transfected with a 35S::NLP7-GR construct were allowed to express the TF–GR fusion protein and sequentially treated with (i) the nitrogen (N) signal transduced by the TF, (ii) cycloheximide (CHX) to block translation, allowing mRNA synthesis of only direct NLP7 targets, (iii) dexamethasone (DEX) to induce NLP7-GR nuclear import. Samples for NLP7 binding to targets as assayed by ChIP using anti-GR antibodies were collected after 0, 5, 10, 30, and 180 min of DEX-induced TF nuclear import. Genes whose expression is affected NLP7 nuclear import were assayed by RNA-seq (steady-state mRNA) or by affinity capture of de novo mRNA using 4tU 16 . b Representative examples of direct targets transcriptionally activated by NLP7. The expression of NRT2.1 , LBD37 , and CIPK8 is induced by +DEX as compared with −DEX treatment in both steady state and 4tU-enriched RNA-seq experiments (green bars). NLP7 binding was captured by ChIP-seq, and NLP7 peaks were identified by MACS2 using an input sample as a control (red bars) (see “Methods”). NLP7 peaks identified in whole roots by ChIP-chip (orange bars) 11 .
    Figure Legend Snippet: TARGET TF-perturbation assay captures direct targets of NLP7 based on TF regulation or TF binding in isolated root cells. a Schematic of the cell-based TARGET TF-perturbation system and experimental design 22 (see “Methods”). Root cells isolated from an nlp7 mutant 11 , 23 transfected with a 35S::NLP7-GR construct were allowed to express the TF–GR fusion protein and sequentially treated with (i) the nitrogen (N) signal transduced by the TF, (ii) cycloheximide (CHX) to block translation, allowing mRNA synthesis of only direct NLP7 targets, (iii) dexamethasone (DEX) to induce NLP7-GR nuclear import. Samples for NLP7 binding to targets as assayed by ChIP using anti-GR antibodies were collected after 0, 5, 10, 30, and 180 min of DEX-induced TF nuclear import. Genes whose expression is affected NLP7 nuclear import were assayed by RNA-seq (steady-state mRNA) or by affinity capture of de novo mRNA using 4tU 16 . b Representative examples of direct targets transcriptionally activated by NLP7. The expression of NRT2.1 , LBD37 , and CIPK8 is induced by +DEX as compared with −DEX treatment in both steady state and 4tU-enriched RNA-seq experiments (green bars). NLP7 binding was captured by ChIP-seq, and NLP7 peaks were identified by MACS2 using an input sample as a control (red bars) (see “Methods”). NLP7 peaks identified in whole roots by ChIP-chip (orange bars) 11 .

    Techniques Used: Binding Assay, Isolation, Mutagenesis, Transfection, Construct, Blocking Assay, Chromatin Immunoprecipitation, Expressing, RNA Sequencing Assay

    NLP7-direct targets are enriched in early N-responsive genes including secondary TFs. a Intersection of NLP7-direct and indirect targets detected in root cells with a time-series of N-response genes in whole roots 19 . The time points represent the just-in-time analysis 19 which binned genes based on the first time point at which their mRNA levels were affected by N treatment at 5, 10, 15, 20, 30, 45, 60, and 90 min. The significance ( p -value) of the intersection between NLP7 targets with each N-time point was calculated and −log10 ( p -value) was graphed. b Intersection of N-responsive secondary TFs regulated by NLP7 and genes belonging to each class of NLP7 binding. Size of overlap is listed in parentheses, and significance is indicated by yellow highlighting and asterisks (Fisher’s exact test, * p -value
    Figure Legend Snippet: NLP7-direct targets are enriched in early N-responsive genes including secondary TFs. a Intersection of NLP7-direct and indirect targets detected in root cells with a time-series of N-response genes in whole roots 19 . The time points represent the just-in-time analysis 19 which binned genes based on the first time point at which their mRNA levels were affected by N treatment at 5, 10, 15, 20, 30, 45, 60, and 90 min. The significance ( p -value) of the intersection between NLP7 targets with each N-time point was calculated and −log10 ( p -value) was graphed. b Intersection of N-responsive secondary TFs regulated by NLP7 and genes belonging to each class of NLP7 binding. Size of overlap is listed in parentheses, and significance is indicated by yellow highlighting and asterisks (Fisher’s exact test, * p -value

    Techniques Used: Binding Assay

    33) Product Images from "m6A modification of a 3′ UTR site reduces RME1 mRNA levels to promote meiosis"

    Article Title: m6A modification of a 3′ UTR site reduces RME1 mRNA levels to promote meiosis

    Journal: Nature Communications

    doi: 10.1038/s41467-019-11232-7

    IME4 m 6 A activity reduces RME1 mRNA levels to enable meiotic initiation. a Polysome profiles (absorbance at 254 nm vs. distance from the top of the tube) of mitotic cells during logarithmic growth and 2 h into meiosis. The locations of the 40S, 60S, monosome, and polysome peaks are indicated. Source data are provided in a Source Data file. b Polysome profiles of meiotic IME4 / IME4 and ime4-cat / ime4-cat cells, both in rme1-S288C / rme-S288C . The inset is a magnification of the polysome area with the number of ribosomes in each peak. The highlighted area marks the pooled fractions used for RNA-seq of polysome-associated mRNA. Source data are provided in a Source Data file. c RNA-seq quantifications of RME1 transcript from input mRNA prior to gradient fractionation (total mRNA) and pooled polysome fractions (polysomal RNA) from IME4 / IME4 and ime4-cat / ime4-cat meiotic cells. Individual values, means, and s.d. from three biological replicates. Two-way analysis of variance p values are indicated. Source data are provided in a Source Data file. d Rank order plot of mRNA levels in total RNA from ime4-cat / ime4-cat and IME4 / IME4 meiotic cells. Means from three biological replicates. Source data are provided in a Source Data file. e Left: Quantitative reverse transcription PCR (RT-qPCR) quantification of IME1 expression in RME1-Δ / RME1-Δ during meiosis in IME4 / IME4 and ime4-Δ / ime4-Δ cells. Middle: RT-qPCR quantification of IME1 expression in rme1-S288C / rme1-S288C during meiosis in IME4 / IME4 and ime4-Δ / ime4-Δ cells. Right: RT-qPCR quantification of IME1 expression in rme1-S288C / rme1-S288C during meiosis in IME4 / IME4 and ime4-cat / ime4-cat cells. Means and individual values from three biological replicates. Source data are provided in a Source Data file. f As in e above but with measurements of IME2 expression. Means and individual values from three biological replicates. Source data are provided in a Source Data file
    Figure Legend Snippet: IME4 m 6 A activity reduces RME1 mRNA levels to enable meiotic initiation. a Polysome profiles (absorbance at 254 nm vs. distance from the top of the tube) of mitotic cells during logarithmic growth and 2 h into meiosis. The locations of the 40S, 60S, monosome, and polysome peaks are indicated. Source data are provided in a Source Data file. b Polysome profiles of meiotic IME4 / IME4 and ime4-cat / ime4-cat cells, both in rme1-S288C / rme-S288C . The inset is a magnification of the polysome area with the number of ribosomes in each peak. The highlighted area marks the pooled fractions used for RNA-seq of polysome-associated mRNA. Source data are provided in a Source Data file. c RNA-seq quantifications of RME1 transcript from input mRNA prior to gradient fractionation (total mRNA) and pooled polysome fractions (polysomal RNA) from IME4 / IME4 and ime4-cat / ime4-cat meiotic cells. Individual values, means, and s.d. from three biological replicates. Two-way analysis of variance p values are indicated. Source data are provided in a Source Data file. d Rank order plot of mRNA levels in total RNA from ime4-cat / ime4-cat and IME4 / IME4 meiotic cells. Means from three biological replicates. Source data are provided in a Source Data file. e Left: Quantitative reverse transcription PCR (RT-qPCR) quantification of IME1 expression in RME1-Δ / RME1-Δ during meiosis in IME4 / IME4 and ime4-Δ / ime4-Δ cells. Middle: RT-qPCR quantification of IME1 expression in rme1-S288C / rme1-S288C during meiosis in IME4 / IME4 and ime4-Δ / ime4-Δ cells. Right: RT-qPCR quantification of IME1 expression in rme1-S288C / rme1-S288C during meiosis in IME4 / IME4 and ime4-cat / ime4-cat cells. Means and individual values from three biological replicates. Source data are provided in a Source Data file. f As in e above but with measurements of IME2 expression. Means and individual values from three biological replicates. Source data are provided in a Source Data file

    Techniques Used: Activity Assay, RNA Sequencing Assay, Fractionation, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    RME1 methylation mutants are defective in meiotic DNA replication. a rme1-S288C and rme1-10 have the same promoter and coding sequence, but they differ in their 3′ untranslated region: rme1-10 has a single A to T substitution of the +129 methylated A (marked with an m over it) in the methylation consensus motif (yellow). b RNA-seq quantifications of RME1 mRNA. RME1 mRNA from input RNA prior to gradient fractionation (total mRNA) and polysome-associated RNA (polysomal RNA) from IME4 / IME4 rme1-S288C / rme1-S288C and IME4 / IME4 rme1-10 / rme1-10 cells incubated in SPO media for 5 h. Individual values, means, and s.d. from three biological replicates. Two-way analysis of variance p values are indicated. Because this experiment was performed together with the one shown in Fig. 2c , the results for RME1 mRNA in the IME4 background in Fig. 2c are re-plotted in this panel for the rme1-S288C background. Source data are provided in a Source Data file. c RNA-seq data from rme1-S288C homozygotes and rme1-10 homozygotes incubated for 5 h in SPO media. Data from three biological replicates are presented as normalized counts in rme1-10 , divided by normalized counts in rme1-S288C . d Flow cytometric analysis of DNA content over a meiotic time course in IME4 / IME4 rme1-10 / rme1-10 compared to IME4 / IME4 rme1-S288C / rme1-S288C and ime4-cat / ime4-cat rme1-S288C / rme1-S288C . Cells were incubated for 24 h at 30 °C or 37 °C, as indicated. Means, individual values, and s.d. from three biological replicates. * p = 0.011, ** p = 0.034, two-tailed t test. Source data are provided in a Source Data file
    Figure Legend Snippet: RME1 methylation mutants are defective in meiotic DNA replication. a rme1-S288C and rme1-10 have the same promoter and coding sequence, but they differ in their 3′ untranslated region: rme1-10 has a single A to T substitution of the +129 methylated A (marked with an m over it) in the methylation consensus motif (yellow). b RNA-seq quantifications of RME1 mRNA. RME1 mRNA from input RNA prior to gradient fractionation (total mRNA) and polysome-associated RNA (polysomal RNA) from IME4 / IME4 rme1-S288C / rme1-S288C and IME4 / IME4 rme1-10 / rme1-10 cells incubated in SPO media for 5 h. Individual values, means, and s.d. from three biological replicates. Two-way analysis of variance p values are indicated. Because this experiment was performed together with the one shown in Fig. 2c , the results for RME1 mRNA in the IME4 background in Fig. 2c are re-plotted in this panel for the rme1-S288C background. Source data are provided in a Source Data file. c RNA-seq data from rme1-S288C homozygotes and rme1-10 homozygotes incubated for 5 h in SPO media. Data from three biological replicates are presented as normalized counts in rme1-10 , divided by normalized counts in rme1-S288C . d Flow cytometric analysis of DNA content over a meiotic time course in IME4 / IME4 rme1-10 / rme1-10 compared to IME4 / IME4 rme1-S288C / rme1-S288C and ime4-cat / ime4-cat rme1-S288C / rme1-S288C . Cells were incubated for 24 h at 30 °C or 37 °C, as indicated. Means, individual values, and s.d. from three biological replicates. * p = 0.011, ** p = 0.034, two-tailed t test. Source data are provided in a Source Data file

    Techniques Used: Methylation, Sequencing, RNA Sequencing Assay, Fractionation, Incubation, Two Tailed Test

    34) Product Images from "RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit"

    Article Title: RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit

    Journal: eLife

    doi: 10.7554/eLife.27082

    Strains lacking functional Bre5 show decreased co-transcriptional splicing. ( A ) RT-qPCR analysis of endogenous, polyadenylated and intron-containing transcripts using primer pairs over the 3’ splice site expressed relative to non-intron-containing PGI1 mRNA, in a wild type strain and in a strain lacking Bre5. The histogram shows the mean of three replicates with standard error, with the value in the BRE5 strain set to 1. The reduced level of unspliced poly(A) + shows increased efficiency of cotranscriptional splicing associated with RNAPII that has reached the 3’ end of the transcription unit. ( B ) Analysis of in-vivo RNA- binding activity of Bre5 with point mutants in the RRM. The top panel shows the recovery of radio-labelled RNA that was bound to Bre5 following in vivo crosslinking and multi-step, denaturing purification and separation by SDS-PAGE as described for CRAC analyses. The lower panel shows a western blot using an anti-TAP antibody against the tagged Bre5. ( C ) RT-qPCR analysis of endogenous unspliced, polyadenylated transcripts using primer pairs over the 3’ splice site expressed relative to non-intron-containing PGI1 mRNA. The histogram in a wild type HTP-tagged Bre5 strain and in strains carrying the point mutations in the RRM. The histogram shows the mean of three experiments with standard error, with the value in the strain expressing wild type Bre5 set to 1. The reduced level of unspliced, poly(A) + RNA in the mutant strains is interpreted as showing a requirement for a functional RRM in Bre5. 10.7554/eLife.27082.016 Source data for Figure 3A . 10.7554/eLife.27082.017 Source data for Figure 3C .
    Figure Legend Snippet: Strains lacking functional Bre5 show decreased co-transcriptional splicing. ( A ) RT-qPCR analysis of endogenous, polyadenylated and intron-containing transcripts using primer pairs over the 3’ splice site expressed relative to non-intron-containing PGI1 mRNA, in a wild type strain and in a strain lacking Bre5. The histogram shows the mean of three replicates with standard error, with the value in the BRE5 strain set to 1. The reduced level of unspliced poly(A) + shows increased efficiency of cotranscriptional splicing associated with RNAPII that has reached the 3’ end of the transcription unit. ( B ) Analysis of in-vivo RNA- binding activity of Bre5 with point mutants in the RRM. The top panel shows the recovery of radio-labelled RNA that was bound to Bre5 following in vivo crosslinking and multi-step, denaturing purification and separation by SDS-PAGE as described for CRAC analyses. The lower panel shows a western blot using an anti-TAP antibody against the tagged Bre5. ( C ) RT-qPCR analysis of endogenous unspliced, polyadenylated transcripts using primer pairs over the 3’ splice site expressed relative to non-intron-containing PGI1 mRNA. The histogram in a wild type HTP-tagged Bre5 strain and in strains carrying the point mutations in the RRM. The histogram shows the mean of three experiments with standard error, with the value in the strain expressing wild type Bre5 set to 1. The reduced level of unspliced, poly(A) + RNA in the mutant strains is interpreted as showing a requirement for a functional RRM in Bre5. 10.7554/eLife.27082.016 Source data for Figure 3A . 10.7554/eLife.27082.017 Source data for Figure 3C .

    Techniques Used: Functional Assay, Quantitative RT-PCR, In Vivo, RNA Binding Assay, Activity Assay, Purification, SDS Page, Western Blot, Expressing, Mutagenesis

    35) Product Images from "Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation"

    Article Title: Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation

    Journal: Genes & Development

    doi: 10.1101/gad.285312.116

    Simultaneous deletion of HSD11B1 substantially rescues the metabolic phenotype of the AdLKO mice. ( A ) mRNA levels of HSD11B1 in the control and LSD1-deficient BAT. (AdLHKO) Heterozygous LSD1 knockout. Mean ± SEM. n = 5. (***) P
    Figure Legend Snippet: Simultaneous deletion of HSD11B1 substantially rescues the metabolic phenotype of the AdLKO mice. ( A ) mRNA levels of HSD11B1 in the control and LSD1-deficient BAT. (AdLHKO) Heterozygous LSD1 knockout. Mean ± SEM. n = 5. (***) P

    Techniques Used: Mouse Assay, Knock-Out

    36) Product Images from "Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis"

    Article Title: Aphid feeding induces the relaxation of epigenetic control and the associated regulation of the defense response in Arabidopsis

    Journal: bioRxiv

    doi: 10.1101/2020.01.24.916783

    Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.
    Figure Legend Snippet: Changes induced at TE expression by aphid feeding. A. Volcano plot depicting TE mRNA-seq expression in the comparison aphid vs control RNA samples. Dots colored in red indicated genes with significant upregulation. B. Volcano plot depicting gene expression in the comparison aphid vs control PARE samples. Dots colored in red indicated genes with significant upregulation. C. Global sRNAs profiles of control and stressed samples. D. TE-derived sRNA profiles of control and stressed samples. E. Relative accumulation of 21,22 and 24 nt sRNAs in control (C) and aphid infested samples (Mper) for TEs of different sizes. Values shown are relative to control, where accumulation values for each sRNA category were set to 1. F. Venn diagram showing the overlap between the TE populations identified from each of the different RNA sequencing analyses. G. Venn diagram depicting the overlap of TEs upregulated two fold in the PARE sequencing data and TEs losing or gaining two fold 24 nt sRNAs. G. Screenshot of a genome browser showing the accumulation of PARE reads and 24 nt sRNAs in control and aphid samples for two of the TEs upregulated in the PARE libraries and showing a decrease of 24 nt sRNA accumulation.

    Techniques Used: Expressing, Derivative Assay, RNA Sequencing Assay, Sequencing

    37) Product Images from "piRNA-like small RNAs mark extended 3’UTRs present in germ and somatic cells"

    Article Title: piRNA-like small RNAs mark extended 3’UTRs present in germ and somatic cells

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1662-6

    Somatic small RNAs align to xUTRs defined in the testis. a and b . Density scatterplots of poly-A mRNA expression (x-axis, data from ENCODE) versus expression of novel small RNAs (y-axis, from this project) at genes with xUTRs. The intensity of the blue color is proportional to the number of genes with a given level of poly-A mRNA and small RNA expression, measured as log (RPKM); dots in areas of lowest regional density represent individual genes. Plots are generated with smoothScatter in R. 212 testis xUTRs have both mRNA and smRNA reads in liver ( a ), and 264 in spleen ( b ); read densities of smRNA and mRNA at these clusters are positively correlated (top right quadrant). Many testis xUTRs have mRNA reads in these tissues but lack smRNAs (bottom right quadrant). c Somatic small RNAs aligning to testis xUTRs are not piRNA-like. Length distribution of somatic small RNAs aligning to testis xUTRs in liver (black line) and spleen (grey line): the peak at 23 nt indicates that somatic smRNAs are shorter than adult testis piRNAs. d Novel small RNAs aligning to testis xUTRs are enriched for 5’ A in liver and spleen. Background is the base frequency across these unannotated 3’ UTR regions
    Figure Legend Snippet: Somatic small RNAs align to xUTRs defined in the testis. a and b . Density scatterplots of poly-A mRNA expression (x-axis, data from ENCODE) versus expression of novel small RNAs (y-axis, from this project) at genes with xUTRs. The intensity of the blue color is proportional to the number of genes with a given level of poly-A mRNA and small RNA expression, measured as log (RPKM); dots in areas of lowest regional density represent individual genes. Plots are generated with smoothScatter in R. 212 testis xUTRs have both mRNA and smRNA reads in liver ( a ), and 264 in spleen ( b ); read densities of smRNA and mRNA at these clusters are positively correlated (top right quadrant). Many testis xUTRs have mRNA reads in these tissues but lack smRNAs (bottom right quadrant). c Somatic small RNAs aligning to testis xUTRs are not piRNA-like. Length distribution of somatic small RNAs aligning to testis xUTRs in liver (black line) and spleen (grey line): the peak at 23 nt indicates that somatic smRNAs are shorter than adult testis piRNAs. d Novel small RNAs aligning to testis xUTRs are enriched for 5’ A in liver and spleen. Background is the base frequency across these unannotated 3’ UTR regions

    Techniques Used: Expressing, RNA Expression, Generated

    piRNA clusters mark extended 3’ UTRs. Potential precursors of extended 3’ UTR piRNA clusters are detectable as 3’ UTR isoforms in testis and in somatic tissues. a Representative example of a piRNA cluster that extends beyond an annotated 3’ UTR. The annotated 3’UTR is shown in purple and the piRNA cluster is shown in red. Individual piRNA alignments are in blue (sense strand) and red (antisense strand) at the bottom. Genomic features are visualized using the Integrative Genomics Viewer [ 51 ]. b Unannotated portions of 3’ UTR piRNA clusters are enriched for polyA+ RNA expression when compared to randomly selected unannotated regions. We used an adult testis RNA-Seq dataset [ 18 ] to determine expression of the unannotated portions of the extended 3’ UTR clusters (dark blue) or random intergenic regions of equal size (light blue). This indicates that the unannotated extended 3’UTRs are transcribed in the testis. c Expression of the unannotated portion of extended 3’ UTR clusters (X-axis) compared with expression of the corresponding annotated mRNA (Y-axis) in the adult testis RNA-Seq dataset [ 18 ]. The annotated portions of genes are generally expressed at higher RPKM than the unannotated portion of the 3’UTR. d Schematic of Northern blot probes, RT-PCR primers, and PCR amplicons. e Northern blot of Pdpr with an exonic probe (top) shows bands consistent with the length of the annotated mRNA, plus a band consistent with addition of the unannotated region corresponding to the piRNA cluster. The bands are visible only in brain. Probing of the same blot with the unannotated 3’UTR probe (bottom) shows a single band in the same position as the upper band in the exonic probe blot. f Representative RT-PCR results of the three Pdpr amplicons shown schematically in D and marked to the right, with (+) and without (−) reverse transcriptase during the cDNA synthesis step (top). g Summary of RT-PCR products obtained from 10 transcripts with extended 3’ UTR piRNA clusters. For each gene testis, brain, spleen, and liver were tested. All transcripts show evidence of an extended 3’UTR in testis and most also have the extended 3’ UTR in the other tissues as well
    Figure Legend Snippet: piRNA clusters mark extended 3’ UTRs. Potential precursors of extended 3’ UTR piRNA clusters are detectable as 3’ UTR isoforms in testis and in somatic tissues. a Representative example of a piRNA cluster that extends beyond an annotated 3’ UTR. The annotated 3’UTR is shown in purple and the piRNA cluster is shown in red. Individual piRNA alignments are in blue (sense strand) and red (antisense strand) at the bottom. Genomic features are visualized using the Integrative Genomics Viewer [ 51 ]. b Unannotated portions of 3’ UTR piRNA clusters are enriched for polyA+ RNA expression when compared to randomly selected unannotated regions. We used an adult testis RNA-Seq dataset [ 18 ] to determine expression of the unannotated portions of the extended 3’ UTR clusters (dark blue) or random intergenic regions of equal size (light blue). This indicates that the unannotated extended 3’UTRs are transcribed in the testis. c Expression of the unannotated portion of extended 3’ UTR clusters (X-axis) compared with expression of the corresponding annotated mRNA (Y-axis) in the adult testis RNA-Seq dataset [ 18 ]. The annotated portions of genes are generally expressed at higher RPKM than the unannotated portion of the 3’UTR. d Schematic of Northern blot probes, RT-PCR primers, and PCR amplicons. e Northern blot of Pdpr with an exonic probe (top) shows bands consistent with the length of the annotated mRNA, plus a band consistent with addition of the unannotated region corresponding to the piRNA cluster. The bands are visible only in brain. Probing of the same blot with the unannotated 3’UTR probe (bottom) shows a single band in the same position as the upper band in the exonic probe blot. f Representative RT-PCR results of the three Pdpr amplicons shown schematically in D and marked to the right, with (+) and without (−) reverse transcriptase during the cDNA synthesis step (top). g Summary of RT-PCR products obtained from 10 transcripts with extended 3’ UTR piRNA clusters. For each gene testis, brain, spleen, and liver were tested. All transcripts show evidence of an extended 3’UTR in testis and most also have the extended 3’ UTR in the other tissues as well

    Techniques Used: RNA Expression, RNA Sequencing Assay, Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    38) Product Images from "Immunomodulation of intracranial melanoma in response to blood-tumor barrier opening with focused ultrasound"

    Article Title: Immunomodulation of intracranial melanoma in response to blood-tumor barrier opening with focused ultrasound

    Journal: Theranostics

    doi: 10.7150/thno.47983

    Bulk RNA sequencing reveals that FUS+MBs mediated BBB/BTB opening elicits increased proinflammatory gene expression. A,B: Volcano plots showing significantly upregulated and downregulated genes in FUS+MBs treated tumors compared to sham at (A) 6 and (B) 24 h post treatment. C,D,E: Log2 fold change of FUS+MBs treated vs. sham tumors at 6 hours and 24 hours post treatment. Data is displayed for selected mRNA transcripts related to (C) inflammatory cytokines, chemokines, and vascular cell adhesion molecules (D) pattern recognition receptors and signaling molecules and (E) MHC class I antigen presentation and processing. *adj p
    Figure Legend Snippet: Bulk RNA sequencing reveals that FUS+MBs mediated BBB/BTB opening elicits increased proinflammatory gene expression. A,B: Volcano plots showing significantly upregulated and downregulated genes in FUS+MBs treated tumors compared to sham at (A) 6 and (B) 24 h post treatment. C,D,E: Log2 fold change of FUS+MBs treated vs. sham tumors at 6 hours and 24 hours post treatment. Data is displayed for selected mRNA transcripts related to (C) inflammatory cytokines, chemokines, and vascular cell adhesion molecules (D) pattern recognition receptors and signaling molecules and (E) MHC class I antigen presentation and processing. *adj p

    Techniques Used: RNA Sequencing Assay, Expressing

    39) Product Images from "Unexpected Diversity and Expression of Avian Endogenous Retroviruses"

    Article Title: Unexpected Diversity and Expression of Avian Endogenous Retroviruses

    Journal: mBio

    doi: 10.1128/mBio.00344-12

    Expression of endogenous proviral chains in CEFs and various chicken tissues. (A) mRNA-seq reads were uniquely aligned with the genome. The mRNA-seq reads mapping to any given endogenous proviral chain were counted and plotted on a log scale. The x axis represents unique endogenous proviruses identified by ReTe, with increasing scores from left to right. (B) RNA-seq data from various chicken tissues ( 33 ) were aligned using TopHat ( 47 ), and transcripts were constructed using Cufflinks ( 48 ). Transcripts that overlap ERV coordinates were extracted, and the number of fragments per kilobase per million (FPKM) was plotted against ERV chains. On the x axis, from left to right, are unique ERV chains with increasing ReTe chain scores.
    Figure Legend Snippet: Expression of endogenous proviral chains in CEFs and various chicken tissues. (A) mRNA-seq reads were uniquely aligned with the genome. The mRNA-seq reads mapping to any given endogenous proviral chain were counted and plotted on a log scale. The x axis represents unique endogenous proviruses identified by ReTe, with increasing scores from left to right. (B) RNA-seq data from various chicken tissues ( 33 ) were aligned using TopHat ( 47 ), and transcripts were constructed using Cufflinks ( 48 ). Transcripts that overlap ERV coordinates were extracted, and the number of fragments per kilobase per million (FPKM) was plotted against ERV chains. On the x axis, from left to right, are unique ERV chains with increasing ReTe chain scores.

    Techniques Used: Expressing, RNA Sequencing Assay, Construct

    40) Product Images from "Integrating transcriptome and microRNA analysis identifies genes and microRNAs for AHO-induced systemic acquired resistance in N. tabacum"

    Article Title: Integrating transcriptome and microRNA analysis identifies genes and microRNAs for AHO-induced systemic acquired resistance in N. tabacum

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12249-y

    Expression of miRNAs and their target transcripts. Image ( a ) shows the DEMs abundance change in RNA-seq analysis with AHO treatment. Image ( b ) represents the potential regulation between miRNAs and their target genes. The T shape end and arrow represent the opposite or same regulation trend between miRNA and mRNA. Image ( c ) shows the DEGs level in RNA-seq analysis. Image ( d ) and ( e ) shows the relative expression levels of miRNA and mRNA transcript assessed by qRT-PCR, respectively. The relative level of qRT-PCR calculated by the delta-delta-CT method. Data is presented as mean ± standard error.
    Figure Legend Snippet: Expression of miRNAs and their target transcripts. Image ( a ) shows the DEMs abundance change in RNA-seq analysis with AHO treatment. Image ( b ) represents the potential regulation between miRNAs and their target genes. The T shape end and arrow represent the opposite or same regulation trend between miRNA and mRNA. Image ( c ) shows the DEGs level in RNA-seq analysis. Image ( d ) and ( e ) shows the relative expression levels of miRNA and mRNA transcript assessed by qRT-PCR, respectively. The relative level of qRT-PCR calculated by the delta-delta-CT method. Data is presented as mean ± standard error.

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

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    The NEBNext Globin rRNA Depletion Kits Human Mouse Rat employ the NEBNext RNase H based RNA depletion workflow to deplete globin mRNA cytoplasmic rRNA and mitochondrial rRNA from human mouse
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    Normalized <t>LAC1</t> expression in low, intermediate and high nitrosative stress conditions. The abundance of LAC1 <t>mRNA</t> transcripts was quantified in two representative strains from the study population using RT-qPCR. In both strains, the normalized LAC1 expression was correlated with the corresponding level of melanin production. LAC1 expression was induced in intermediate nitrosative stress in comparison to low and high nitrosative stress.
    Lac1 Mrna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs snap25 endogenous mrna transcripts
    Regulation of hnRNP K binding to the KARRE by forskolin and its direct competition with U2AF65. ( A ) Western blot of hnRNP K proteins pulled down from PC12 nuclear extracts using the biotin-RNA probes in Figure 2 B. Et: vehicle ethanol, Fsk: forskolin (10 µM). ( B ) UV cross-linking immunoprecipitation of hnRNP K in forskolin-treated PC12 nuclear extracts with the 3′ splice site of exon 5a and its sensitivity to pretreatment by PPase. The wild type RNA probe sequence is the same as in Figure 2 B except that it is without biotin. The upper panel is a phosphorimage and the lower a Western blot for the hnRNP K protein in the same SDS-PAGE gel. ( C ) (Upper panel) a phosphorimage of recombinant His-hnRNP K incubated with active or heat-inactivated PKA in the presence of [32P-γ]ATP in in vitro kinase assay. Lower panel is a Western bot image of the same gel showing equal loading of His-hnRNP K. ( D ) HnRNP K interacts with the endogenous <t>Snap25</t> <t>pre-mRNA</t> transcript. Above the gel is a diagram of the PCR target pre-mRNA region of Snap25, with thin lines as introns, boxes as exons and arrowheads as locations of PCR primers. The agarose gel shows the RT-PCR products from RNA samples isolated from the nuclear extracts of forskolin-treated PC12 cells, or from immunoprecipitates using anti-hnRNP K (anti-K) or protein G beads. Each RNA sample was treated with DNase I and one of them also with RNase (A + T1) as indicated. a: a band insensitive to either DNase or RNase treatment, probably nonspecific product from the PCR primers. ( E ) Western blots of hnRNP K and U2AF65 proteins pulled down from HeLa nuclear extracts with increasing amount of His-hnRNP K added, using the wild type biotin-RNA probe in Figure 2 B. The blot was first probed with anti-hnRNP K antibody, stripped with SDS buffer and then reprobed with anti-U2AF65. ( F ) UV cross-linking of hnRNP K and U2AF65 to the 3′ splice site of exon 5a. The hnRNP K consensus motifs (underlined) and its C to G mutations (italicized) are shown above the denaturing PAGE gel. b: a protein band enhanced by the mutation, likely preferring the G tracts in the mutant, at similar size as hnRNP F/H.
    Snap25 Endogenous Mrna Transcripts, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs mrna
    Poly(A) tail reduction of specific maternal <t>mRNA</t> in aged eggs. Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates <t>RNaseH/oligo(dT)</t> 20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.
    Mrna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 700 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna/product/New England Biolabs
    Average 94 stars, based on 700 article reviews
    Price from $9.99 to $1999.99
    mrna - by Bioz Stars, 2020-09
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    Image Search Results


    Normalized LAC1 expression in low, intermediate and high nitrosative stress conditions. The abundance of LAC1 mRNA transcripts was quantified in two representative strains from the study population using RT-qPCR. In both strains, the normalized LAC1 expression was correlated with the corresponding level of melanin production. LAC1 expression was induced in intermediate nitrosative stress in comparison to low and high nitrosative stress.

    Journal: Scientific Reports

    Article Title: Genetic Factors and Genotype-Environment Interactions Contribute to Variation in Melanin Production in the Fungal Pathogen Cryptococcus neoformans

    doi: 10.1038/s41598-018-27813-3

    Figure Lengend Snippet: Normalized LAC1 expression in low, intermediate and high nitrosative stress conditions. The abundance of LAC1 mRNA transcripts was quantified in two representative strains from the study population using RT-qPCR. In both strains, the normalized LAC1 expression was correlated with the corresponding level of melanin production. LAC1 expression was induced in intermediate nitrosative stress in comparison to low and high nitrosative stress.

    Article Snippet: Quantification of LAC1 mRNA in the samples was achieved through RT-qPCR using Luna Universal One-Step RT-qPCR kit (NEB).

    Techniques: Expressing, Quantitative RT-PCR

    Regulation of hnRNP K binding to the KARRE by forskolin and its direct competition with U2AF65. ( A ) Western blot of hnRNP K proteins pulled down from PC12 nuclear extracts using the biotin-RNA probes in Figure 2 B. Et: vehicle ethanol, Fsk: forskolin (10 µM). ( B ) UV cross-linking immunoprecipitation of hnRNP K in forskolin-treated PC12 nuclear extracts with the 3′ splice site of exon 5a and its sensitivity to pretreatment by PPase. The wild type RNA probe sequence is the same as in Figure 2 B except that it is without biotin. The upper panel is a phosphorimage and the lower a Western blot for the hnRNP K protein in the same SDS-PAGE gel. ( C ) (Upper panel) a phosphorimage of recombinant His-hnRNP K incubated with active or heat-inactivated PKA in the presence of [32P-γ]ATP in in vitro kinase assay. Lower panel is a Western bot image of the same gel showing equal loading of His-hnRNP K. ( D ) HnRNP K interacts with the endogenous Snap25 pre-mRNA transcript. Above the gel is a diagram of the PCR target pre-mRNA region of Snap25, with thin lines as introns, boxes as exons and arrowheads as locations of PCR primers. The agarose gel shows the RT-PCR products from RNA samples isolated from the nuclear extracts of forskolin-treated PC12 cells, or from immunoprecipitates using anti-hnRNP K (anti-K) or protein G beads. Each RNA sample was treated with DNase I and one of them also with RNase (A + T1) as indicated. a: a band insensitive to either DNase or RNase treatment, probably nonspecific product from the PCR primers. ( E ) Western blots of hnRNP K and U2AF65 proteins pulled down from HeLa nuclear extracts with increasing amount of His-hnRNP K added, using the wild type biotin-RNA probe in Figure 2 B. The blot was first probed with anti-hnRNP K antibody, stripped with SDS buffer and then reprobed with anti-U2AF65. ( F ) UV cross-linking of hnRNP K and U2AF65 to the 3′ splice site of exon 5a. The hnRNP K consensus motifs (underlined) and its C to G mutations (italicized) are shown above the denaturing PAGE gel. b: a protein band enhanced by the mutation, likely preferring the G tracts in the mutant, at similar size as hnRNP F/H.

    Journal: Nucleic Acids Research

    Article Title: Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation

    doi: 10.1093/nar/gks504

    Figure Lengend Snippet: Regulation of hnRNP K binding to the KARRE by forskolin and its direct competition with U2AF65. ( A ) Western blot of hnRNP K proteins pulled down from PC12 nuclear extracts using the biotin-RNA probes in Figure 2 B. Et: vehicle ethanol, Fsk: forskolin (10 µM). ( B ) UV cross-linking immunoprecipitation of hnRNP K in forskolin-treated PC12 nuclear extracts with the 3′ splice site of exon 5a and its sensitivity to pretreatment by PPase. The wild type RNA probe sequence is the same as in Figure 2 B except that it is without biotin. The upper panel is a phosphorimage and the lower a Western blot for the hnRNP K protein in the same SDS-PAGE gel. ( C ) (Upper panel) a phosphorimage of recombinant His-hnRNP K incubated with active or heat-inactivated PKA in the presence of [32P-γ]ATP in in vitro kinase assay. Lower panel is a Western bot image of the same gel showing equal loading of His-hnRNP K. ( D ) HnRNP K interacts with the endogenous Snap25 pre-mRNA transcript. Above the gel is a diagram of the PCR target pre-mRNA region of Snap25, with thin lines as introns, boxes as exons and arrowheads as locations of PCR primers. The agarose gel shows the RT-PCR products from RNA samples isolated from the nuclear extracts of forskolin-treated PC12 cells, or from immunoprecipitates using anti-hnRNP K (anti-K) or protein G beads. Each RNA sample was treated with DNase I and one of them also with RNase (A + T1) as indicated. a: a band insensitive to either DNase or RNase treatment, probably nonspecific product from the PCR primers. ( E ) Western blots of hnRNP K and U2AF65 proteins pulled down from HeLa nuclear extracts with increasing amount of His-hnRNP K added, using the wild type biotin-RNA probe in Figure 2 B. The blot was first probed with anti-hnRNP K antibody, stripped with SDS buffer and then reprobed with anti-U2AF65. ( F ) UV cross-linking of hnRNP K and U2AF65 to the 3′ splice site of exon 5a. The hnRNP K consensus motifs (underlined) and its C to G mutations (italicized) are shown above the denaturing PAGE gel. b: a protein band enhanced by the mutation, likely preferring the G tracts in the mutant, at similar size as hnRNP F/H.

    Article Snippet: Some cultures were pretreated with 10 µM of H89 (N -[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride, Sigma, #B1427) for 10 min. To differentiate products of Snap25 endogenous mRNA transcripts from exons 5a and 5b, we digested 1.0 µl of 32 P-labelled-PCR products with AvaII and NdeI restriction enzymes in buffer 4 (New England Biolabs) in a 10 -µl reaction at 37°C for 1 h and run in 6% denaturing polyacrylamide gel electrophoresis (PAGE) gel.

    Techniques: Binding Assay, Western Blot, Cross-linking Immunoprecipitation, Sequencing, SDS Page, Recombinant, Incubation, In Vitro, Kinase Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Isolation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Poly(A) tail reduction of specific maternal mRNA in aged eggs. Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT) 20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.

    Journal: PLoS ONE

    Article Title: Aging of Xenopus tropicalis Eggs Leads to Deadenylation of a Specific Set of Maternal mRNAs and Loss of Developmental Potential

    doi: 10.1371/journal.pone.0013532

    Figure Lengend Snippet: Poly(A) tail reduction of specific maternal mRNA in aged eggs. Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT) 20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.

    Article Snippet: To remove all poly(A) tails, mRNA was treated with RNaseH (New England Biolabs) and 0.3 µg oligo(dT)20 for 30 min at 37°C in a 10 µl volume prior to ligation.

    Techniques: Ligation, Polymerase Chain Reaction, Marker, Sequencing