gene expression real time quantitative reverse transcription pcr qrt pcr analyses  (Roche)


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    Roche gene expression real time quantitative reverse transcription pcr qrt pcr analyses
    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by <t>qRT-PCR</t> and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.
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    1) Product Images from "Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits"

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01259

    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.
    Figure Legend Snippet: Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression of key ABA and sucrose biosynthetic genes VmNCED1 (A) , VmSS (B) , VmSPS1 (C) , VmSPS2 (D) , and VmSPS3 (E) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).
    Figure Legend Snippet: Effect of post-harvest ABA and sugar treatments on the expression of key ABA and sucrose biosynthetic genes VmNCED1 (A) , VmSS (B) , VmSPS1 (C) , VmSPS2 (D) , and VmSPS3 (E) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression of anthocyanin biosynthetic genes VmCHS (A) , VmCHI (B) , VmF3H (C) , VmF3 ′ H (D) , VmF3 ′ 5 ′ H (E) , VmDFR (F) , VmANS (G) , and VmUFGT (H) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).
    Figure Legend Snippet: Effect of post-harvest ABA and sugar treatments on the expression of anthocyanin biosynthetic genes VmCHS (A) , VmCHI (B) , VmF3H (C) , VmF3 ′ H (D) , VmF3 ′ 5 ′ H (E) , VmDFR (F) , VmANS (G) , and VmUFGT (H) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of VmNCED1 silencing on anthocyanin biosynthesis in ripening bilberry fruit. Green unripe fruits still attached to the bilberry plants were injected with VmNCED1 -VIGS vector or pBINTRA6 vector only (control). Arrows indicate injection sites. Fruits were evaluated 4 weeks after injection for color (A) , and the expression of VmNCED1 and the key anthocyanin biosynthetic genes in intact fruits as well as in green and red sectors of chimeric fruits (B) . Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SDs of three replicates.
    Figure Legend Snippet: Effect of VmNCED1 silencing on anthocyanin biosynthesis in ripening bilberry fruit. Green unripe fruits still attached to the bilberry plants were injected with VmNCED1 -VIGS vector or pBINTRA6 vector only (control). Arrows indicate injection sites. Fruits were evaluated 4 weeks after injection for color (A) , and the expression of VmNCED1 and the key anthocyanin biosynthetic genes in intact fruits as well as in green and red sectors of chimeric fruits (B) . Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SDs of three replicates.

    Techniques Used: Injection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Effect of pre-harvest treatment with ABA on bilberry fruit color (A) , anthocyanin content (B) , and expression of anthocyanin biosynthetic genes (C) . Unripe green berries attached to plants were sprayed with 0.5 mM ABA, 2 mM ABA or water (control). Fruit color and anthocyanin content was evaluated after 7 days from the beginning of the experiment. Total anthocyanin content is expressed as milligrams of cyanidin-3-glucoside equivalents g -1 FW. Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of four replicates. Asterisks indicate significant differences from control in Student’s t -Test ( P ≤ 0.05).
    Figure Legend Snippet: Effect of pre-harvest treatment with ABA on bilberry fruit color (A) , anthocyanin content (B) , and expression of anthocyanin biosynthetic genes (C) . Unripe green berries attached to plants were sprayed with 0.5 mM ABA, 2 mM ABA or water (control). Fruit color and anthocyanin content was evaluated after 7 days from the beginning of the experiment. Total anthocyanin content is expressed as milligrams of cyanidin-3-glucoside equivalents g -1 FW. Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of four replicates. Asterisks indicate significant differences from control in Student’s t -Test ( P ≤ 0.05).

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression cell wall modifying genes VmPE1 (A) , VmPE2 (B) , VmPL (C) , VmPG1 (D) , VmPG2 (E) , VmRGLyase (F) , Vm β GAL1 (G) , Vm β GAL2 (H) , VmXTH (I) , VmCEL (J) , VmXYL (K) , VmEXP1 (L) , VmEXP2 (M) , and VmEXP3 (N) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (200), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR after 4 days of the beginning of the experiment and normalized to VmGAPDH . Values represent means ± SEs of three replicates. PE , pectin esterase; PL , pectate lyase; PG , polygalacturonase; RGLyase , rhamnogalacturonate lyase; β GAL , β-galactosidase; XTH , xyloglucan endotransglycosylase/hydrolase; CEL , endo-β - 1,4 glucanase: XYL , β-xylosidase; EXP , expansin. Asterisks indicate significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001).
    Figure Legend Snippet: Effect of post-harvest ABA and sugar treatments on the expression cell wall modifying genes VmPE1 (A) , VmPE2 (B) , VmPL (C) , VmPG1 (D) , VmPG2 (E) , VmRGLyase (F) , Vm β GAL1 (G) , Vm β GAL2 (H) , VmXTH (I) , VmCEL (J) , VmXYL (K) , VmEXP1 (L) , VmEXP2 (M) , and VmEXP3 (N) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (200), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR after 4 days of the beginning of the experiment and normalized to VmGAPDH . Values represent means ± SEs of three replicates. PE , pectin esterase; PL , pectate lyase; PG , polygalacturonase; RGLyase , rhamnogalacturonate lyase; β GAL , β-galactosidase; XTH , xyloglucan endotransglycosylase/hydrolase; CEL , endo-β - 1,4 glucanase: XYL , β-xylosidase; EXP , expansin. Asterisks indicate significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001).

    Techniques Used: Expressing, Quantitative RT-PCR

    2) Product Images from "Single-stranded DNA binding protein Ssbp3 induces differentiation of mouse embryonic stem cells into trophoblast-like cells"

    Article Title: Single-stranded DNA binding protein Ssbp3 induces differentiation of mouse embryonic stem cells into trophoblast-like cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-016-0340-1

    Ssbp3 overexpression activates MAPK/Erk1/2 and TGF-β pathways. a Significantly enriched signaling pathways of all DEGs upon overexpression of Ssbp3 by KEGG pathway analysis. b , c qRT-PCR analysis for expression levels of upregulated genes related to MAPK/Erk/1/2 and TGF-β pathways in Ssbp3-overexpressing ESCs. The average mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as mean ± SD ( n = 3). * p
    Figure Legend Snippet: Ssbp3 overexpression activates MAPK/Erk1/2 and TGF-β pathways. a Significantly enriched signaling pathways of all DEGs upon overexpression of Ssbp3 by KEGG pathway analysis. b , c qRT-PCR analysis for expression levels of upregulated genes related to MAPK/Erk/1/2 and TGF-β pathways in Ssbp3-overexpressing ESCs. The average mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as mean ± SD ( n = 3). * p

    Techniques Used: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation

    Teratomas derived from Ssbp3-overexpressing ESCs contain hemorrhage. a Gross appearance of teratomas derived from control cells or Ssbp3-overexpressing ESCs ( upper panel ). The number of teratomas examined is presented in the table ( lower panel ). b The net weight of teratomas derived from control cells and Ssbp3-overexpressing ESCs. c Cross-section of teratomas derived from control cells ( upper panel ) or Ssbp3-overexpressing ESCs ( lower panel ). d Histology of teratomas derived from control cells ( upper panel ) or Ssbp3-overexpressing ESCs ( lower panel ) showing tissue complexity (hematoxylin and eosin staining). Arrowheads mark the endoderm ( black ), mesoderm ( blue ), and ectoderm ( green ) cells. e Hematoxylin and eosin-stained images for sections of a teratoma derived from Ssbp3-overexpressing ESCs. The trophoblast cluster ( arrow heads , left panel ) and trophoblast giant cells with the enlarged nuclei ( arrow heads , right panel ) are indicated. f qRT-PCR analysis for expression levels of trophoblast-specific markers in teratomas derived from control cells or Ssbp3-overexpressing ESCs. The average mRNA level in teratomas derived from control cells was set at 1.0. Data are shown as mean ± SD ( n = 3). * p
    Figure Legend Snippet: Teratomas derived from Ssbp3-overexpressing ESCs contain hemorrhage. a Gross appearance of teratomas derived from control cells or Ssbp3-overexpressing ESCs ( upper panel ). The number of teratomas examined is presented in the table ( lower panel ). b The net weight of teratomas derived from control cells and Ssbp3-overexpressing ESCs. c Cross-section of teratomas derived from control cells ( upper panel ) or Ssbp3-overexpressing ESCs ( lower panel ). d Histology of teratomas derived from control cells ( upper panel ) or Ssbp3-overexpressing ESCs ( lower panel ) showing tissue complexity (hematoxylin and eosin staining). Arrowheads mark the endoderm ( black ), mesoderm ( blue ), and ectoderm ( green ) cells. e Hematoxylin and eosin-stained images for sections of a teratoma derived from Ssbp3-overexpressing ESCs. The trophoblast cluster ( arrow heads , left panel ) and trophoblast giant cells with the enlarged nuclei ( arrow heads , right panel ) are indicated. f qRT-PCR analysis for expression levels of trophoblast-specific markers in teratomas derived from control cells or Ssbp3-overexpressing ESCs. The average mRNA level in teratomas derived from control cells was set at 1.0. Data are shown as mean ± SD ( n = 3). * p

    Techniques Used: Derivative Assay, Staining, Quantitative RT-PCR, Expressing

    Forced expression of Ssbp3 induces differentiation of mouse ESCs with a trophoblast-like gene expression pattern. a Western blotting of Ssbp3 protein levels in ESCs transfected with a vector, or an Ssbp3 plasmid. Twenty-four hours after transfection, ESCs were selected by puromycin for an additional 72 h. b Morphology changes and AKP staining of ESCs overexpressing Ssbp3. c – g Expression levels of pluripotency and lineage markers in ESCs overexpressing Ssbp3 determined by qRT-PCR analyses. Pluripotency markers ( c ), trophoblast markers ( d ), primitive endoderm and endoderm markers ( e ), mesoderm markers ( f ), and ectoderm markers ( g ). The average mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as mean ± SD ( n = 3). * p
    Figure Legend Snippet: Forced expression of Ssbp3 induces differentiation of mouse ESCs with a trophoblast-like gene expression pattern. a Western blotting of Ssbp3 protein levels in ESCs transfected with a vector, or an Ssbp3 plasmid. Twenty-four hours after transfection, ESCs were selected by puromycin for an additional 72 h. b Morphology changes and AKP staining of ESCs overexpressing Ssbp3. c – g Expression levels of pluripotency and lineage markers in ESCs overexpressing Ssbp3 determined by qRT-PCR analyses. Pluripotency markers ( c ), trophoblast markers ( d ), primitive endoderm and endoderm markers ( e ), mesoderm markers ( f ), and ectoderm markers ( g ). The average mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as mean ± SD ( n = 3). * p

    Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation, ALP Assay, Staining, Quantitative RT-PCR

    Ssbp3 depletion attenuates the activation of trophoblast gene expression induced by downregulation of Oct4 in mouse ESCs. a The morphology of ZHBTc4 cells after treatment with Tc. Differentiation was triggered by Tc-mediated downregulation of Oct4. b Expression levels of Ssbp3 during differentiation of the ZHBTc4 cell line were determined by qRT-PCR analysis. The average mRNA level in ZHBTc4 cells cultured without Tc was set at 1.0. Data are shown as mean ± SD ( n = 3). * p
    Figure Legend Snippet: Ssbp3 depletion attenuates the activation of trophoblast gene expression induced by downregulation of Oct4 in mouse ESCs. a The morphology of ZHBTc4 cells after treatment with Tc. Differentiation was triggered by Tc-mediated downregulation of Oct4. b Expression levels of Ssbp3 during differentiation of the ZHBTc4 cell line were determined by qRT-PCR analysis. The average mRNA level in ZHBTc4 cells cultured without Tc was set at 1.0. Data are shown as mean ± SD ( n = 3). * p

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Cell Culture

    Overexpression of Ssbp3 induces a trophoblast-like transcriptional program. a Heatmap of the DEGs induced by Ssbp3 overexpression in ESCs (fold change > 2). Green and red values represent fold changes for down- and upregulation, respectively. Heatmap in the right panel shows the top 30 upregulated genes in detail. b Venn diagram showing the overlap of the DEGs induced by Ssbp3 ( green ), Gata3 ( blue ), or Cdx2 ( orange ) overexpression, with the number of genes indicated. Out of 1880 DEGs induced by Ssbp3, 1141 DEGs were shared with Gata3 or Cdx2. c Significantly enriched GO terms of the 1141 DEGs shared between Ssbp3 and Cdx2 or between Ssbp3 and Gata3. d , e qRT-PCR analysis for expression levels of trophoblast-specific markers in Cdx2 and Elf5 stable knockdown cell lines 96 h after Ssbp3 overexpression. The average mRNA level in stable cell line expressing shNT was set at 1.0. Data are shown as mean ± SD ( n = 3). * p
    Figure Legend Snippet: Overexpression of Ssbp3 induces a trophoblast-like transcriptional program. a Heatmap of the DEGs induced by Ssbp3 overexpression in ESCs (fold change > 2). Green and red values represent fold changes for down- and upregulation, respectively. Heatmap in the right panel shows the top 30 upregulated genes in detail. b Venn diagram showing the overlap of the DEGs induced by Ssbp3 ( green ), Gata3 ( blue ), or Cdx2 ( orange ) overexpression, with the number of genes indicated. Out of 1880 DEGs induced by Ssbp3, 1141 DEGs were shared with Gata3 or Cdx2. c Significantly enriched GO terms of the 1141 DEGs shared between Ssbp3 and Cdx2 or between Ssbp3 and Gata3. d , e qRT-PCR analysis for expression levels of trophoblast-specific markers in Cdx2 and Elf5 stable knockdown cell lines 96 h after Ssbp3 overexpression. The average mRNA level in stable cell line expressing shNT was set at 1.0. Data are shown as mean ± SD ( n = 3). * p

    Techniques Used: Over Expression, Quantitative RT-PCR, Expressing, Stable Transfection

    Ssbp3 depletion weakens the trophoblast gene expression induced by BMP4 and bFGF treatment in ESCs. a The morphology of E14T cells after treatment with bone BMP4 and bFGF at the indicated time points. b Expression levels of Ssbp3 gradually increased in E14T cells treated with BMP4 and bFGF. The expression levels of Ssbp3 were determined by qRT-PCR analysis. The average mRNA level in E14T cells cultured without treatment was set at 1.0. Data are shown as mean ± SD ( n = 3). * p
    Figure Legend Snippet: Ssbp3 depletion weakens the trophoblast gene expression induced by BMP4 and bFGF treatment in ESCs. a The morphology of E14T cells after treatment with bone BMP4 and bFGF at the indicated time points. b Expression levels of Ssbp3 gradually increased in E14T cells treated with BMP4 and bFGF. The expression levels of Ssbp3 were determined by qRT-PCR analysis. The average mRNA level in E14T cells cultured without treatment was set at 1.0. Data are shown as mean ± SD ( n = 3). * p

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

    3) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.
    Figure Legend Snippet: The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    4) Product Images from "Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice"

    Article Title: Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003755

    Treg secretion and function in both AE-WT and AE- fgl2 -/- mice after E . multilocularis infection, and in WT mice in response to recombinant FGL2/anti-FGL2-MAb blockade. (A) Spleen was taken from non-infected WT and AE-WT mice, CD4 + Teffs, CD8 + T cells, CD4 + CD25 + Tregs and APCs were isolated by FACS cell sorting, fgl2 mRNA levels were determined by qRT-PCR. (B) Representative images of Foxp3 mean fluorescence intensity (MFI) from AE-WT and AE- fgl2 -/- mice, and non-infected mice as controls. (C) Frequency of Foxp3 + T cells within CD4 + CD25 + T cells in PECs and spleen cells from AE-WT and AE- fgl2 -/- mice at 1 month and 4 months post-infection. (D) Foxp3 and IL-10 gene expression in PECs during E . multilocularis infection (measured by qRT-PCR). AU: arbitrary units. Graphs show the mean±SD. Data represent mean±SD of three independent experiments of a total 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. (E) 0, 1, and 5 μg/mL of recombinant FGL2 and 1μg/mL of anti-FGL2-MAb were added to primary spleen cells from non-infected WT mice, or spleen cells stimulated with ConA or vesicle fluid (VF). Relative expression levels of Foxp3 + /CD4 + CD25 + were determined by flow cytometry. (F) CD4 + CD25 + Tregs (suppressor cells) and CD4 + CD25 - Teff cells (responder cells) were isolated from spleen cells of both non-infected and infected AE-WT mice by FACS. The two cell populations were co-cultured at a ratio of 1:1 (suppressor: responder) in the presence of APCs and ConA (2 μg/mL), rFGL2 (1 μg/mL), ConA (2 μg/mL) + rFGL2 (1 μg/mL), or anti-FGL2-MAb (1 μg/mL); cell proliferation was measured using BrdU ELISA. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Expression of Foxp3 + /CD4+CD25 + was normalized with negative control (cells without rFGL2 and anti-FGL2-MAb (negative control) were considered as base line, e.g. as 1.0). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. * P
    Figure Legend Snippet: Treg secretion and function in both AE-WT and AE- fgl2 -/- mice after E . multilocularis infection, and in WT mice in response to recombinant FGL2/anti-FGL2-MAb blockade. (A) Spleen was taken from non-infected WT and AE-WT mice, CD4 + Teffs, CD8 + T cells, CD4 + CD25 + Tregs and APCs were isolated by FACS cell sorting, fgl2 mRNA levels were determined by qRT-PCR. (B) Representative images of Foxp3 mean fluorescence intensity (MFI) from AE-WT and AE- fgl2 -/- mice, and non-infected mice as controls. (C) Frequency of Foxp3 + T cells within CD4 + CD25 + T cells in PECs and spleen cells from AE-WT and AE- fgl2 -/- mice at 1 month and 4 months post-infection. (D) Foxp3 and IL-10 gene expression in PECs during E . multilocularis infection (measured by qRT-PCR). AU: arbitrary units. Graphs show the mean±SD. Data represent mean±SD of three independent experiments of a total 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. (E) 0, 1, and 5 μg/mL of recombinant FGL2 and 1μg/mL of anti-FGL2-MAb were added to primary spleen cells from non-infected WT mice, or spleen cells stimulated with ConA or vesicle fluid (VF). Relative expression levels of Foxp3 + /CD4 + CD25 + were determined by flow cytometry. (F) CD4 + CD25 + Tregs (suppressor cells) and CD4 + CD25 - Teff cells (responder cells) were isolated from spleen cells of both non-infected and infected AE-WT mice by FACS. The two cell populations were co-cultured at a ratio of 1:1 (suppressor: responder) in the presence of APCs and ConA (2 μg/mL), rFGL2 (1 μg/mL), ConA (2 μg/mL) + rFGL2 (1 μg/mL), or anti-FGL2-MAb (1 μg/mL); cell proliferation was measured using BrdU ELISA. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Expression of Foxp3 + /CD4+CD25 + was normalized with negative control (cells without rFGL2 and anti-FGL2-MAb (negative control) were considered as base line, e.g. as 1.0). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. * P

    Techniques Used: Mouse Assay, Infection, Recombinant, Isolation, FACS, Quantitative RT-PCR, Fluorescence, Expressing, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control

    Serum FGL2 levels in E . multilocularis infected mice, effect of IL-17A on FGL2 secretion in spleen cells, and effects of FGL2 on parasite load and proliferation in E . multilocularis infected mice. (A) Serum levels of FGL2 in infected AE-WT mice at different stages of infection, as compared to non-infected control mice (Control). (B) Different concentrations of recombinant IL-17A (0, 0.5, 1, 2 μg/mL) or anti-IL-17A MAbs (1 μg/ml) were added to primary spleen cells isolated from non-infected WT mice (4 months p.i.). FGL2-levels in culture supernatants were determined by ELISA. (C) Parasite load in AE-WT and AE- fgl2 -/- mice assessed by wet weight measurement at 1 month and 4 months post- infection. (D) Representative images of E . multilocularis infection in AE-WT and AE- fgl2 -/- mice at 4 months p.i.; arrows point at intraperitoneal metacestode tissue/lesions. (E) Parasite cell proliferation in AE-WT versus AE- fgl2 -/- assessed by em14-3-3 qRT-PCR at 4 months p.i, upon comparison to the constitutively expressed house-keeping Em II/3-10 gene. Data represent mean ± SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. * P
    Figure Legend Snippet: Serum FGL2 levels in E . multilocularis infected mice, effect of IL-17A on FGL2 secretion in spleen cells, and effects of FGL2 on parasite load and proliferation in E . multilocularis infected mice. (A) Serum levels of FGL2 in infected AE-WT mice at different stages of infection, as compared to non-infected control mice (Control). (B) Different concentrations of recombinant IL-17A (0, 0.5, 1, 2 μg/mL) or anti-IL-17A MAbs (1 μg/ml) were added to primary spleen cells isolated from non-infected WT mice (4 months p.i.). FGL2-levels in culture supernatants were determined by ELISA. (C) Parasite load in AE-WT and AE- fgl2 -/- mice assessed by wet weight measurement at 1 month and 4 months post- infection. (D) Representative images of E . multilocularis infection in AE-WT and AE- fgl2 -/- mice at 4 months p.i.; arrows point at intraperitoneal metacestode tissue/lesions. (E) Parasite cell proliferation in AE-WT versus AE- fgl2 -/- assessed by em14-3-3 qRT-PCR at 4 months p.i, upon comparison to the constitutively expressed house-keeping Em II/3-10 gene. Data represent mean ± SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. * P

    Techniques Used: Infection, Mouse Assay, Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    5) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ) downstream of the insertion sites was detected by RT–PCR analysis, with the combinations of primers EMP12-F, EMP12-F2, EMP12-F3, and EMP12-R. PPR motifs (P) of EMP12 are predicted by TPRpred ( https://toolkit.tuebingen.mpg.de/#/tools/tprpred ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.
    Figure Legend Snippet: The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ) downstream of the insertion sites was detected by RT–PCR analysis, with the combinations of primers EMP12-F, EMP12-F2, EMP12-F3, and EMP12-R. PPR motifs (P) of EMP12 are predicted by TPRpred ( https://toolkit.tuebingen.mpg.de/#/tools/tprpred ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Light Microscopy

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    7) Product Images from "Regulation of endothelial Fas expression as a mechanism of promotion of vascular integrity by mural cells in tumors"

    Article Title: Regulation of endothelial Fas expression as a mechanism of promotion of vascular integrity by mural cells in tumors

    Journal: Cancer Science

    doi: 10.1111/cas.13216

    si RNA ‐mediated knockdown of endothelial Fas recapitulated the reduction in hemorrhage seen in the presence of 10T1/2 cells in vivo . (a) Representative HE stain images of the E, E‐Fas KD , and E+M condition xenografts. The slides were also stained for PECAM ‐1 (brown). Yellow arrows indicate hemorrhagic vessels identified by extravasated red blood cells ( RBC s). Scale bars = 50 μm. (b) Quantification of hemorrhagic vessels as seen in (a). The addition of 10T1/2 cells markedly reduced hemorrhage compared to MS ‐1 cells alone. The knockdown of Fas in MS ‐1 cells recapitulated the reduction of hemorrhage to levels comparable to that seen in the E+M xenografts. (c) Fas expression in MS ‐1 cells was quantified by qRT ‐ PCR to confirm successful knockdown by si RNA . Data represented as mean ± SD in all graphs. *** P
    Figure Legend Snippet: si RNA ‐mediated knockdown of endothelial Fas recapitulated the reduction in hemorrhage seen in the presence of 10T1/2 cells in vivo . (a) Representative HE stain images of the E, E‐Fas KD , and E+M condition xenografts. The slides were also stained for PECAM ‐1 (brown). Yellow arrows indicate hemorrhagic vessels identified by extravasated red blood cells ( RBC s). Scale bars = 50 μm. (b) Quantification of hemorrhagic vessels as seen in (a). The addition of 10T1/2 cells markedly reduced hemorrhage compared to MS ‐1 cells alone. The knockdown of Fas in MS ‐1 cells recapitulated the reduction of hemorrhage to levels comparable to that seen in the E+M xenografts. (c) Fas expression in MS ‐1 cells was quantified by qRT ‐ PCR to confirm successful knockdown by si RNA . Data represented as mean ± SD in all graphs. *** P

    Techniques Used: In Vivo, H&E Stain, Staining, Mass Spectrometry, Expressing, Quantitative RT-PCR

    Fas gene expression and susceptibility to Fas‐dependent apoptosis of MS ‐1 cells were decreased in the presence of 10T1/2 cells in vitro . (a) Representative image of the in vitro coculture of Mile Sven 1 ( MS ‐1) endothelial cells (green) and 10T1/2 mural progenitor cells (red) stably expressing enhanced green fluorescent protein ( eGFP ) and DsRed, respectively. Scale bar = 100 μm. (b) Successful segregation of MS ‐1 (E, for endothelial cells) and 10T1/2 cells (M, for mural cells) after coculture by fluorescence‐activated cell sorting ( FACS ) was confirmed by quantifying eGFP and DsRed mRNA expression which, respectively, is exclusively expressed in MS ‐1 and 10T1/2 cells by quantitative reverse transcription‐polymerase chain reaction ( qRT ‐ PCR ). (c) Fas mRNA expression was down‐regulated in MS ‐1 cells separated by FACS after coculture with 10T1/2 cells compared to monocultured MS ‐1 cells. (d) Endothelial susceptibility to apoptosis was determined by comparing the change in the apoptosis index upon the addition of Jo2, a Fas agonist antibody, compared to isotype control. Though the apoptotic index at baseline was not significantly different with the presence of 10T1/2 cells, a clear suppression of apoptosis was seen in the cocultured MS ‐1 cells upon Jo2 stimulation. Data represented as mean ± SD in all graphs. ** P
    Figure Legend Snippet: Fas gene expression and susceptibility to Fas‐dependent apoptosis of MS ‐1 cells were decreased in the presence of 10T1/2 cells in vitro . (a) Representative image of the in vitro coculture of Mile Sven 1 ( MS ‐1) endothelial cells (green) and 10T1/2 mural progenitor cells (red) stably expressing enhanced green fluorescent protein ( eGFP ) and DsRed, respectively. Scale bar = 100 μm. (b) Successful segregation of MS ‐1 (E, for endothelial cells) and 10T1/2 cells (M, for mural cells) after coculture by fluorescence‐activated cell sorting ( FACS ) was confirmed by quantifying eGFP and DsRed mRNA expression which, respectively, is exclusively expressed in MS ‐1 and 10T1/2 cells by quantitative reverse transcription‐polymerase chain reaction ( qRT ‐ PCR ). (c) Fas mRNA expression was down‐regulated in MS ‐1 cells separated by FACS after coculture with 10T1/2 cells compared to monocultured MS ‐1 cells. (d) Endothelial susceptibility to apoptosis was determined by comparing the change in the apoptosis index upon the addition of Jo2, a Fas agonist antibody, compared to isotype control. Though the apoptotic index at baseline was not significantly different with the presence of 10T1/2 cells, a clear suppression of apoptosis was seen in the cocultured MS ‐1 cells upon Jo2 stimulation. Data represented as mean ± SD in all graphs. ** P

    Techniques Used: Expressing, Mass Spectrometry, In Vitro, Stable Transfection, Fluorescence, FACS, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    8) Product Images from "Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells"

    Article Title: Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4573-5

    Analysis of Dlg2 isoforms expressed in IFNβ/YFP + -producing pDCs. ( a ) Gene architecture and alternatively spliced transcripts of the Dlg2 gene. ( a , upper graph) Genomic position of Dlg2 gene is presented. Exons are shown as vertical bars and introns as thin horizontal lines. Introns and exons are drawn to scale. Smaller exons (less than 1 point line space) are not to scale. ( a , lower graph) Exons are shown as boxes and are drawn to scale. Exons are named or numbered as indicated. Alternative exon-exon-junctions are indicated with connecting lines. Grey boxes show protein coding regions whereas empty boxes represent untranslated mRNA regions. ( b ) RT-PCR of Dlg2 N-terminal isoforms in IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs (upper and middle panel). Flt3L cultures from six pooled IFNβ mob/mob mice were stimulated with CpG for 6 h and FACS-sorted for YFP + (pDC + ) and YFP — pDCs (pDC — ). Naïve brain from C57BL/6 N mice was used as positive and not reversely transcribed RNA from YFP + pDCs as negative controls (Control). Lower panel shows Gapdh and Ifnb expression in the respective cDNA samples indicating successful stimulation and sorting of pDCs as well as equal template amounts. ( c ) SH3-GUK linker isoforms of Dlg2 in RT-PCR. SH3-GUK region was amplified using cDNA samples as described above in ( b ). ( d ) Restriction analysis of Dlg2 clones generated after 5´-RACE PCR. Empty vector or selected 5´-RACE clones were digested either with Eco RI (left), Hin dIII (middle), or with both restriction enzymes (right). Lower panel shows the plasmid maps for the clones shown in the upper panel. ( e ) Exon-intron structure of the Dlg2 isoforms expressed in pDCs. Exons are shown as boxes and are drawn to scale as shown in A (lower part)
    Figure Legend Snippet: Analysis of Dlg2 isoforms expressed in IFNβ/YFP + -producing pDCs. ( a ) Gene architecture and alternatively spliced transcripts of the Dlg2 gene. ( a , upper graph) Genomic position of Dlg2 gene is presented. Exons are shown as vertical bars and introns as thin horizontal lines. Introns and exons are drawn to scale. Smaller exons (less than 1 point line space) are not to scale. ( a , lower graph) Exons are shown as boxes and are drawn to scale. Exons are named or numbered as indicated. Alternative exon-exon-junctions are indicated with connecting lines. Grey boxes show protein coding regions whereas empty boxes represent untranslated mRNA regions. ( b ) RT-PCR of Dlg2 N-terminal isoforms in IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs (upper and middle panel). Flt3L cultures from six pooled IFNβ mob/mob mice were stimulated with CpG for 6 h and FACS-sorted for YFP + (pDC + ) and YFP — pDCs (pDC — ). Naïve brain from C57BL/6 N mice was used as positive and not reversely transcribed RNA from YFP + pDCs as negative controls (Control). Lower panel shows Gapdh and Ifnb expression in the respective cDNA samples indicating successful stimulation and sorting of pDCs as well as equal template amounts. ( c ) SH3-GUK linker isoforms of Dlg2 in RT-PCR. SH3-GUK region was amplified using cDNA samples as described above in ( b ). ( d ) Restriction analysis of Dlg2 clones generated after 5´-RACE PCR. Empty vector or selected 5´-RACE clones were digested either with Eco RI (left), Hin dIII (middle), or with both restriction enzymes (right). Lower panel shows the plasmid maps for the clones shown in the upper panel. ( e ) Exon-intron structure of the Dlg2 isoforms expressed in pDCs. Exons are shown as boxes and are drawn to scale as shown in A (lower part)

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Mouse Assay, FACS, Expressing, Amplification, Clone Assay, Generated, Polymerase Chain Reaction, Plasmid Preparation

    High expression of DLG2 in IFNβ-producing pDCs. ( a ) Analysis of Dlg2 expression in splenic pDCs. IFNβ/YFP + and IFNβ/YFP — splenic pDCs were ex vivo FACS sorted from IFNβ mob/mob mice 6 h after i.v. injection with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. ( b ) Dlg2 expression in in vitro BM-derived pDCs. BM-derived DCs were generated from IFNβ mob/mob mice in Flt3L cultures. IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs were FACS sorted 6 h after stimulation with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. Data shown in ( a ) and ( b ) is fold mRNA quantity in IFNβ-producing pDCs relative to IFNβ non-producers. Data shown is mean ± standard error of mean (SEM) from four independent experiments (each sample is pooled from 12 ( a ) or 6 ( b ) mice. Statistical differences between IFNβ/YFP + and IFNβ/YFP — were analysed by two tailed, unpaired t test. ns: P > 0.05, *: P
    Figure Legend Snippet: High expression of DLG2 in IFNβ-producing pDCs. ( a ) Analysis of Dlg2 expression in splenic pDCs. IFNβ/YFP + and IFNβ/YFP — splenic pDCs were ex vivo FACS sorted from IFNβ mob/mob mice 6 h after i.v. injection with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. ( b ) Dlg2 expression in in vitro BM-derived pDCs. BM-derived DCs were generated from IFNβ mob/mob mice in Flt3L cultures. IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs were FACS sorted 6 h after stimulation with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. Data shown in ( a ) and ( b ) is fold mRNA quantity in IFNβ-producing pDCs relative to IFNβ non-producers. Data shown is mean ± standard error of mean (SEM) from four independent experiments (each sample is pooled from 12 ( a ) or 6 ( b ) mice. Statistical differences between IFNβ/YFP + and IFNβ/YFP — were analysed by two tailed, unpaired t test. ns: P > 0.05, *: P

    Techniques Used: Expressing, Ex Vivo, FACS, Mouse Assay, Injection, Quantitative RT-PCR, In Vitro, Derivative Assay, Generated, Two Tailed Test

    9) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ) downstream of the insertion sites was detected by RT–PCR analysis, with the combinations of primers EMP12-F, EMP12-F2, EMP12-F3, and EMP12-R. PPR motifs (P) of EMP12 are predicted by TPRpred ( https://toolkit.tuebingen.mpg.de/#/tools/tprpred ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.
    Figure Legend Snippet: The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ) downstream of the insertion sites was detected by RT–PCR analysis, with the combinations of primers EMP12-F, EMP12-F2, EMP12-F3, and EMP12-R. PPR motifs (P) of EMP12 are predicted by TPRpred ( https://toolkit.tuebingen.mpg.de/#/tools/tprpred ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Light Microscopy

    The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.
    Figure Legend Snippet: The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    10) Product Images from "Downregulation of Steroid Receptor Coactivator-2 Modulates Estrogen-Responsive Genes and Stimulates Proliferation of MCF-7 Breast Cancer Cells"

    Article Title: Downregulation of Steroid Receptor Coactivator-2 Modulates Estrogen-Responsive Genes and Stimulates Proliferation of MCF-7 Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070096

    PKA-mediated downregulation of SRC-2 changes mRNA expression of ER-target genes. Control MCF-7 cells (Ctr shRNA) and SRC-2 KD MCF-7 cells (SRC-2 shRNA) were grown in phenol red-free DMEM supplemented with charcoaled stripped FBS (5%) and 17β-estradiol (10 nM) for two days. The two cell lines were then treated with Vehicle or 8-CPT-cAMP (150 µM), IBMX (50 µM) and forskolin (10 µM) (cAMP) for 24 hours. The mRNA expression of selected genes was measured by qRT-PCR. The expression level of each gene is relative to TBP mRNA. The results presents mean values ± SE obtained from four-six independent qRT-PCRs.
    Figure Legend Snippet: PKA-mediated downregulation of SRC-2 changes mRNA expression of ER-target genes. Control MCF-7 cells (Ctr shRNA) and SRC-2 KD MCF-7 cells (SRC-2 shRNA) were grown in phenol red-free DMEM supplemented with charcoaled stripped FBS (5%) and 17β-estradiol (10 nM) for two days. The two cell lines were then treated with Vehicle or 8-CPT-cAMP (150 µM), IBMX (50 µM) and forskolin (10 µM) (cAMP) for 24 hours. The mRNA expression of selected genes was measured by qRT-PCR. The expression level of each gene is relative to TBP mRNA. The results presents mean values ± SE obtained from four-six independent qRT-PCRs.

    Techniques Used: Expressing, shRNA, Cycling Probe Technology, Quantitative RT-PCR

    11) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.
    Figure Legend Snippet: The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.
    Figure Legend Snippet: The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.

    Techniques Used: Mutagenesis, Expressing, Light Microscopy, Reverse Transcription Polymerase Chain Reaction

    The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.
    Figure Legend Snippet: The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    13) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.
    Figure Legend Snippet: The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.

    Techniques Used: Mutagenesis, Expressing, Light Microscopy, Reverse Transcription Polymerase Chain Reaction

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    14) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ) downstream of the insertion sites was detected by RT–PCR analysis, with the combinations of primers EMP12-F, EMP12-F2, EMP12-F3, and EMP12-R. PPR motifs (P) of EMP12 are predicted by TPRpred ( https://toolkit.tuebingen.mpg.de/#/tools/tprpred ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.
    Figure Legend Snippet: The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ) downstream of the insertion sites was detected by RT–PCR analysis, with the combinations of primers EMP12-F, EMP12-F2, EMP12-F3, and EMP12-R. PPR motifs (P) of EMP12 are predicted by TPRpred ( https://toolkit.tuebingen.mpg.de/#/tools/tprpred ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Light Microscopy

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    15) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    16) Product Images from "Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation"

    Article Title: Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-016-0785-5

    Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)
    Figure Legend Snippet: Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of different light conditions on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)
    Figure Legend Snippet: Effect of different light conditions on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)

    Techniques Used: Expressing, Quantitative RT-PCR, Labeling

    Effect of different light conditions on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)
    Figure Legend Snippet: Effect of different light conditions on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)

    Techniques Used: Expressing, Quantitative RT-PCR, Labeling

    Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)
    Figure Legend Snippet: Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) during bilberry fruit development. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry
    Figure Legend Snippet: Expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) during bilberry fruit development. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of VmCCD1 gene during bilberry fruit development. The relative expression of the gene was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry
    Figure Legend Snippet: Expression of VmCCD1 gene during bilberry fruit development. The relative expression of the gene was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry

    Techniques Used: Expressing, Quantitative RT-PCR

    17) Product Images from "Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells"

    Article Title: Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4573-5

    Analysis of Dlg2 isoforms expressed in IFNβ/YFP + -producing pDCs. ( a ) Gene architecture and alternatively spliced transcripts of the Dlg2 gene. ( a , upper graph) Genomic position of Dlg2 gene is presented. Exons are shown as vertical bars and introns as thin horizontal lines. Introns and exons are drawn to scale. Smaller exons (less than 1 point line space) are not to scale. ( a , lower graph) Exons are shown as boxes and are drawn to scale. Exons are named or numbered as indicated. Alternative exon-exon-junctions are indicated with connecting lines. Grey boxes show protein coding regions whereas empty boxes represent untranslated mRNA regions. ( b ) RT-PCR of Dlg2 N-terminal isoforms in IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs (upper and middle panel). Flt3L cultures from six pooled IFNβ mob/mob mice were stimulated with CpG for 6 h and FACS-sorted for YFP + (pDC + ) and YFP — pDCs (pDC — ). Naïve brain from C57BL/6 N mice was used as positive and not reversely transcribed RNA from YFP + pDCs as negative controls (Control). Lower panel shows Gapdh and Ifnb expression in the respective cDNA samples indicating successful stimulation and sorting of pDCs as well as equal template amounts. ( c ) SH3-GUK linker isoforms of Dlg2 in RT-PCR. SH3-GUK region was amplified using cDNA samples as described above in ( b ). ( d ) Restriction analysis of Dlg2 clones generated after 5´-RACE PCR. Empty vector or selected 5´-RACE clones were digested either with Eco RI (left), Hin dIII (middle), or with both restriction enzymes (right). Lower panel shows the plasmid maps for the clones shown in the upper panel. ( e ) Exon-intron structure of the Dlg2 isoforms expressed in pDCs. Exons are shown as boxes and are drawn to scale as shown in A (lower part)
    Figure Legend Snippet: Analysis of Dlg2 isoforms expressed in IFNβ/YFP + -producing pDCs. ( a ) Gene architecture and alternatively spliced transcripts of the Dlg2 gene. ( a , upper graph) Genomic position of Dlg2 gene is presented. Exons are shown as vertical bars and introns as thin horizontal lines. Introns and exons are drawn to scale. Smaller exons (less than 1 point line space) are not to scale. ( a , lower graph) Exons are shown as boxes and are drawn to scale. Exons are named or numbered as indicated. Alternative exon-exon-junctions are indicated with connecting lines. Grey boxes show protein coding regions whereas empty boxes represent untranslated mRNA regions. ( b ) RT-PCR of Dlg2 N-terminal isoforms in IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs (upper and middle panel). Flt3L cultures from six pooled IFNβ mob/mob mice were stimulated with CpG for 6 h and FACS-sorted for YFP + (pDC + ) and YFP — pDCs (pDC — ). Naïve brain from C57BL/6 N mice was used as positive and not reversely transcribed RNA from YFP + pDCs as negative controls (Control). Lower panel shows Gapdh and Ifnb expression in the respective cDNA samples indicating successful stimulation and sorting of pDCs as well as equal template amounts. ( c ) SH3-GUK linker isoforms of Dlg2 in RT-PCR. SH3-GUK region was amplified using cDNA samples as described above in ( b ). ( d ) Restriction analysis of Dlg2 clones generated after 5´-RACE PCR. Empty vector or selected 5´-RACE clones were digested either with Eco RI (left), Hin dIII (middle), or with both restriction enzymes (right). Lower panel shows the plasmid maps for the clones shown in the upper panel. ( e ) Exon-intron structure of the Dlg2 isoforms expressed in pDCs. Exons are shown as boxes and are drawn to scale as shown in A (lower part)

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Mouse Assay, FACS, Expressing, Amplification, Clone Assay, Generated, Polymerase Chain Reaction, Plasmid Preparation

    High expression of DLG2 in IFNβ-producing pDCs. ( a ) Analysis of Dlg2 expression in splenic pDCs. IFNβ/YFP + and IFNβ/YFP — splenic pDCs were ex vivo FACS sorted from IFNβ mob/mob mice 6 h after i.v. injection with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. ( b ) Dlg2 expression in in vitro BM-derived pDCs. BM-derived DCs were generated from IFNβ mob/mob mice in Flt3L cultures. IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs were FACS sorted 6 h after stimulation with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. Data shown in ( a ) and ( b ) is fold mRNA quantity in IFNβ-producing pDCs relative to IFNβ non-producers. Data shown is mean ± standard error of mean (SEM) from four independent experiments (each sample is pooled from 12 ( a ) or 6 ( b ) mice. Statistical differences between IFNβ/YFP + and IFNβ/YFP — were analysed by two tailed, unpaired t test. ns: P > 0.05, *: P
    Figure Legend Snippet: High expression of DLG2 in IFNβ-producing pDCs. ( a ) Analysis of Dlg2 expression in splenic pDCs. IFNβ/YFP + and IFNβ/YFP — splenic pDCs were ex vivo FACS sorted from IFNβ mob/mob mice 6 h after i.v. injection with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. ( b ) Dlg2 expression in in vitro BM-derived pDCs. BM-derived DCs were generated from IFNβ mob/mob mice in Flt3L cultures. IFNβ/YFP + and IFNβ/YFP — BM-derived pDCs were FACS sorted 6 h after stimulation with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. Data shown in ( a ) and ( b ) is fold mRNA quantity in IFNβ-producing pDCs relative to IFNβ non-producers. Data shown is mean ± standard error of mean (SEM) from four independent experiments (each sample is pooled from 12 ( a ) or 6 ( b ) mice. Statistical differences between IFNβ/YFP + and IFNβ/YFP — were analysed by two tailed, unpaired t test. ns: P > 0.05, *: P

    Techniques Used: Expressing, Ex Vivo, FACS, Mouse Assay, Injection, Quantitative RT-PCR, In Vitro, Derivative Assay, Generated, Two Tailed Test

    18) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.
    Figure Legend Snippet: The maize Emp12 gene is involved in embryogenesis and endosperm development. (A) A self-pollinated ear segregating for emp12-673 mutant kernels at 15 days after pollination (DAP). Arrows show the emp maize kernels. Scale bar=0.5 cm. (B) The dried kernels of emp12-673 mutants and the wild type (WT). Scale bar=2 mm. (C) The embryo and endosperm of emp12-673 mutant and WT kernels at 12 DAP. Arrows indicate the embryo (Em). Scale bar=2 mm. (D) Schematic diagram of the Emp12 gene and its protein structure, showing the Mu insertion sites of emp12-673 and emp12-20 . The expression of full-length and partial Emp12 ( Emp12' and Emp12'' ). (E–H) Light microscopy of cytological sections of WT (E, G) and emp12-673 mutant kernels (F, H) are longitudinally sectioned early at 12 DAP (E, F) and late at 16 DAP (G, H). En, endosperm; Em, embryo; per, pericarp; sc, scutellum; su, suspensor; col, coleoptile; ep, embryo proper; sam, shoot apical meristem; ram, root apical meristem. Scale bar=1 mm. (I) RT–PCR analysis of full-length Emp12 and truncated Emp12' and Emp12'' expression indicated in (D) was performed in the emp12-673 and emp12-20 mutants and WT siblings at 12 DAP, with normalization by Ubiquitin primers.

    Techniques Used: Mutagenesis, Expressing, Light Microscopy, Reverse Transcription Polymerase Chain Reaction

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    19) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.
    Figure Legend Snippet: The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    20) Product Images from "The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize"

    Article Title: The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

    The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.
    Figure Legend Snippet: The emp 12 mutants only affect expression of nad2 in the mitochondria. Total RNA was extracted from fresh maize kernels at 12 DAP and reverse transcribed using hexamer primers. RT–PCR analysis was performed by using three biological replicates and was normalized to Ubiquitin . (A) Transcript analysis of nad genes in emp12 mutant alleles. (B) Expression levels of mitochondrial transcripts were quantified by qRT-PCR analysis. The transcript abundance was plotted as emp12 /wild-type log2 ratios using Ubiquitin for normalization.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR

    Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.
    Figure Legend Snippet: Emp12 is required for intron 1, intron 2, and intron 4 splicing of mitochondrial nad2. (A) qRT-PCR analysis of all group II introns in maize mitochondrial genes. Total RNA was isolated from the emp12-673 and emp12-20 mutant kernels at 12 DAP. Values represent the log2 ratio of spliced to unspliced forms for each transcript in the mutants compared with WT maize kernels. Each value is the mean of at least three biological replicates. (B) Structure of the maize nad2 gene. Exons are shown as filled gray boxes. The closed and open lines stand for cis - and trans -introns. Primers (F1+R1, F2+R2, F3+R3, F4+R4, and F1+R4) indicate the PCR products by using flanking exon–exon primers as described previously ( Xiu et al. , 2016 ). E1–E5, exon1–exon5. (C) RT–PCR analysis of the intron splicing of nad2 introns using exon–exon primers as indicated in (B). Arrows and asterisks indicate the spliced and unspliced PCR products, respectively.

    Techniques Used: Quantitative RT-PCR, Isolation, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

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    Sequencing:

    Article Title: Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models
    Article Snippet: .. Quantitative real-time PCR The identity of selected candidate genes was verified by sequencing of the corresponding array clones (Sequiserve, Vaterstetten, Germany). cDNA of independently treated AtT-20 cells was synthesized with SuperScript II-reverse transcriptase (Invitrogen) primed with oligo(dT) primers using 1 μg of total RNA according to the manufacturer's instructions. cDNA of untreated and 100 nM CRH-treated AtT-20 cells was analyzed by quantitative real-time PCR (qRT-PCR) using the LightCycler® FastStart DNA MasterPLUS SYBR Green I reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer's instructions and different oligonucleotide primers (see Table ). .. The experiments were performed in duplicates in the LightCycler® 2.0 instrument (Roche Diagnostics, Mannheim, Germany) with the following PCR settings: initial denaturation at 95°C for 10 min; 40 cycles of denaturation (95°C for tD = 10 sec), annealing (TA = 56-65°C for tA = 4-5 sec) and elongation (72°C, tE = 7-13 sec).

    Real-time Polymerase Chain Reaction:

    Article Title: Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models
    Article Snippet: .. Quantitative real-time PCR The identity of selected candidate genes was verified by sequencing of the corresponding array clones (Sequiserve, Vaterstetten, Germany). cDNA of independently treated AtT-20 cells was synthesized with SuperScript II-reverse transcriptase (Invitrogen) primed with oligo(dT) primers using 1 μg of total RNA according to the manufacturer's instructions. cDNA of untreated and 100 nM CRH-treated AtT-20 cells was analyzed by quantitative real-time PCR (qRT-PCR) using the LightCycler® FastStart DNA MasterPLUS SYBR Green I reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer's instructions and different oligonucleotide primers (see Table ). .. The experiments were performed in duplicates in the LightCycler® 2.0 instrument (Roche Diagnostics, Mannheim, Germany) with the following PCR settings: initial denaturation at 95°C for 10 min; 40 cycles of denaturation (95°C for tD = 10 sec), annealing (TA = 56-65°C for tA = 4-5 sec) and elongation (72°C, tE = 7-13 sec).

    Article Title: Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone
    Article Snippet: .. Real-time PCR (qRT-PCR) was carried out in a 25 μ L volume container with SYBR Green PCR Master Mix (Roche Diagnostics). .. The template source was either 5 ng cDNA or purified DNA standard.

    Article Title: Regulation of Nucleotide Excision Repair by Nuclear Lamin B1
    Article Snippet: .. The qRT–PCR analyses were carried out with a LightCycler 480 Real-Time PCR instrument (Roche) using the LightCycler 480 SYBR Green Master Kit (Roche). .. Relative expression analysis was carried out using the LightCycler 480 Real-Time PCR software with GAPDH serving as the reference gene.

    Article Title: Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion
    Article Snippet: .. Levels of F4/80, IL-6, adiponectin, MCP-1, TNFα and IL-10 were measured by quantitative Real-Time PCR (qRT-PCR), which was carried out using the LightCycler 480 SYBR Green I Master Mix chemistry (Roche Diagnostics, Indianapolis, IN) and analyzed on the LightCycler 480 instrument (Roche Diagnostics, Indianapolis, IN). ..

    Software:

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits
    Article Snippet: .. Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States). .. The transcript abundance of the genes was detected using a LightCycler® SYBR Green I Master qPCR Kit (Roche).

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    Roche gene expression real time quantitative reverse transcription pcr qrt pcr analyses
    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by <t>qRT-PCR</t> and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.
    Gene Expression Real Time Quantitative Reverse Transcription Pcr Qrt Pcr Analyses, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression of key ABA and sucrose biosynthetic genes VmNCED1 (A) , VmSS (B) , VmSPS1 (C) , VmSPS2 (D) , and VmSPS3 (E) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of post-harvest ABA and sugar treatments on the expression of key ABA and sucrose biosynthetic genes VmNCED1 (A) , VmSS (B) , VmSPS1 (C) , VmSPS2 (D) , and VmSPS3 (E) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression of anthocyanin biosynthetic genes VmCHS (A) , VmCHI (B) , VmF3H (C) , VmF3 ′ H (D) , VmF3 ′ 5 ′ H (E) , VmDFR (F) , VmANS (G) , and VmUFGT (H) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of post-harvest ABA and sugar treatments on the expression of anthocyanin biosynthetic genes VmCHS (A) , VmCHI (B) , VmF3H (C) , VmF3 ′ H (D) , VmF3 ′ 5 ′ H (E) , VmDFR (F) , VmANS (G) , and VmUFGT (H) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of VmNCED1 silencing on anthocyanin biosynthesis in ripening bilberry fruit. Green unripe fruits still attached to the bilberry plants were injected with VmNCED1 -VIGS vector or pBINTRA6 vector only (control). Arrows indicate injection sites. Fruits were evaluated 4 weeks after injection for color (A) , and the expression of VmNCED1 and the key anthocyanin biosynthetic genes in intact fruits as well as in green and red sectors of chimeric fruits (B) . Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SDs of three replicates.

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of VmNCED1 silencing on anthocyanin biosynthesis in ripening bilberry fruit. Green unripe fruits still attached to the bilberry plants were injected with VmNCED1 -VIGS vector or pBINTRA6 vector only (control). Arrows indicate injection sites. Fruits were evaluated 4 weeks after injection for color (A) , and the expression of VmNCED1 and the key anthocyanin biosynthetic genes in intact fruits as well as in green and red sectors of chimeric fruits (B) . Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SDs of three replicates.

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Injection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Effect of pre-harvest treatment with ABA on bilberry fruit color (A) , anthocyanin content (B) , and expression of anthocyanin biosynthetic genes (C) . Unripe green berries attached to plants were sprayed with 0.5 mM ABA, 2 mM ABA or water (control). Fruit color and anthocyanin content was evaluated after 7 days from the beginning of the experiment. Total anthocyanin content is expressed as milligrams of cyanidin-3-glucoside equivalents g -1 FW. Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of four replicates. Asterisks indicate significant differences from control in Student’s t -Test ( P ≤ 0.05).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of pre-harvest treatment with ABA on bilberry fruit color (A) , anthocyanin content (B) , and expression of anthocyanin biosynthetic genes (C) . Unripe green berries attached to plants were sprayed with 0.5 mM ABA, 2 mM ABA or water (control). Fruit color and anthocyanin content was evaluated after 7 days from the beginning of the experiment. Total anthocyanin content is expressed as milligrams of cyanidin-3-glucoside equivalents g -1 FW. Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of four replicates. Asterisks indicate significant differences from control in Student’s t -Test ( P ≤ 0.05).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression cell wall modifying genes VmPE1 (A) , VmPE2 (B) , VmPL (C) , VmPG1 (D) , VmPG2 (E) , VmRGLyase (F) , Vm β GAL1 (G) , Vm β GAL2 (H) , VmXTH (I) , VmCEL (J) , VmXYL (K) , VmEXP1 (L) , VmEXP2 (M) , and VmEXP3 (N) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (200), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR after 4 days of the beginning of the experiment and normalized to VmGAPDH . Values represent means ± SEs of three replicates. PE , pectin esterase; PL , pectate lyase; PG , polygalacturonase; RGLyase , rhamnogalacturonate lyase; β GAL , β-galactosidase; XTH , xyloglucan endotransglycosylase/hydrolase; CEL , endo-β - 1,4 glucanase: XYL , β-xylosidase; EXP , expansin. Asterisks indicate significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of post-harvest ABA and sugar treatments on the expression cell wall modifying genes VmPE1 (A) , VmPE2 (B) , VmPL (C) , VmPG1 (D) , VmPG2 (E) , VmRGLyase (F) , Vm β GAL1 (G) , Vm β GAL2 (H) , VmXTH (I) , VmCEL (J) , VmXYL (K) , VmEXP1 (L) , VmEXP2 (M) , and VmEXP3 (N) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (200), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR after 4 days of the beginning of the experiment and normalized to VmGAPDH . Values represent means ± SEs of three replicates. PE , pectin esterase; PL , pectate lyase; PG , polygalacturonase; RGLyase , rhamnogalacturonate lyase; β GAL , β-galactosidase; XTH , xyloglucan endotransglycosylase/hydrolase; CEL , endo-β - 1,4 glucanase: XYL , β-xylosidase; EXP , expansin. Asterisks indicate significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)

    Journal: BMC Plant Biology

    Article Title: Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

    doi: 10.1186/s12870-016-0785-5

    Figure Lengend Snippet: Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)

    Article Snippet: Relative quantification of gene expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of different light conditions on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)

    Journal: BMC Plant Biology

    Article Title: Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

    doi: 10.1186/s12870-016-0785-5

    Figure Lengend Snippet: Effect of different light conditions on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)

    Article Snippet: Relative quantification of gene expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Expressing, Quantitative RT-PCR, Labeling

    Effect of different light conditions on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)

    Journal: BMC Plant Biology

    Article Title: Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

    doi: 10.1186/s12870-016-0785-5

    Figure Lengend Snippet: Effect of different light conditions on the expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Columns labeled with different letters indicate statistically significant differences ( P ≤ 0.05, one-way ANOVA with post hoc comparisons)

    Article Snippet: Relative quantification of gene expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Expressing, Quantitative RT-PCR, Labeling

    Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)

    Journal: BMC Plant Biology

    Article Title: Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

    doi: 10.1186/s12870-016-0785-5

    Figure Lengend Snippet: Effect of 16/8 h photoperiodic white light treatment on the expression of carotenoid cleavage genes VmCCD1 ( a ) and VmNCED1 ( b ) in unripe (stage S3) and ripe (stage S5) bilberry fruits. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate statistically significant differences from respective control (dark treatment) in Student’s t -Test ( P ≤ 0.05)

    Article Snippet: Relative quantification of gene expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Expressing, Quantitative RT-PCR

    Expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) during bilberry fruit development. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry

    Journal: BMC Plant Biology

    Article Title: Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

    doi: 10.1186/s12870-016-0785-5

    Figure Lengend Snippet: Expression of carotenoid biosynthetic genes VmPSY ( a ), VmPDS ( b ), VmZDS ( c ), VmCRTISO ( d ), VmLCYB ( e ), VmBCH ( f ), VmLCYE ( g ) and VmCYP450-BCH ( h ) during bilberry fruit development. The relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry

    Article Snippet: Relative quantification of gene expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Expressing, Quantitative RT-PCR

    Expression of VmCCD1 gene during bilberry fruit development. The relative expression of the gene was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry

    Journal: BMC Plant Biology

    Article Title: Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

    doi: 10.1186/s12870-016-0785-5

    Figure Lengend Snippet: Expression of VmCCD1 gene during bilberry fruit development. The relative expression of the gene was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of at least three replicates. S1–S5 indicates the fruit developmental stages from flower to fully ripe berry

    Article Snippet: Relative quantification of gene expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Expressing, Quantitative RT-PCR