quantitative reverse transcription pcr qrt pcr assays  (Roche)

 
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    Structured Review

    Roche quantitative reverse transcription pcr qrt pcr assays
    Comparison of expression profiles of ten representative genes from module ‘Pink’ as measured by RNA-seq and <t>qRT-PCR.</t> The ten genes are assigned to the flavonoid/anthocyanin pathway in Fig. 4 , including five previously characterized and five uncharacterized genes. Columns represent expression determined by qRT-PCR (left y -axis), while lines represent expression by RNA-seq in RPKM values (right y -axis). The x -axis in each chart represents the four developmental stages (S1–S4). For qRT-PCR assay, the mean was calculated from three biological replicates each with three technical replicates ( n =9). Standard curves were used to calculate the number of target gene molecules per sample. These were then normalized relative to the expression of MdAct . For RNA-seq, each point is the mean of three biological replicates. Correlations between qRT-PCR and RNA-seq expressions were calculated and their associated P -values are indicated. Error bars show SD. This figure is available in colour at JXB online.
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    1) Product Images from "Transcriptome analysis of an apple (Malus × domestica) yellow fruit somatic mutation identifies a gene network module highly associated with anthocyanin and epigenetic regulation"

    Article Title: Transcriptome analysis of an apple (Malus × domestica) yellow fruit somatic mutation identifies a gene network module highly associated with anthocyanin and epigenetic regulation

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erv433

    Comparison of expression profiles of ten representative genes from module ‘Pink’ as measured by RNA-seq and qRT-PCR. The ten genes are assigned to the flavonoid/anthocyanin pathway in Fig. 4 , including five previously characterized and five uncharacterized genes. Columns represent expression determined by qRT-PCR (left y -axis), while lines represent expression by RNA-seq in RPKM values (right y -axis). The x -axis in each chart represents the four developmental stages (S1–S4). For qRT-PCR assay, the mean was calculated from three biological replicates each with three technical replicates ( n =9). Standard curves were used to calculate the number of target gene molecules per sample. These were then normalized relative to the expression of MdAct . For RNA-seq, each point is the mean of three biological replicates. Correlations between qRT-PCR and RNA-seq expressions were calculated and their associated P -values are indicated. Error bars show SD. This figure is available in colour at JXB online.
    Figure Legend Snippet: Comparison of expression profiles of ten representative genes from module ‘Pink’ as measured by RNA-seq and qRT-PCR. The ten genes are assigned to the flavonoid/anthocyanin pathway in Fig. 4 , including five previously characterized and five uncharacterized genes. Columns represent expression determined by qRT-PCR (left y -axis), while lines represent expression by RNA-seq in RPKM values (right y -axis). The x -axis in each chart represents the four developmental stages (S1–S4). For qRT-PCR assay, the mean was calculated from three biological replicates each with three technical replicates ( n =9). Standard curves were used to calculate the number of target gene molecules per sample. These were then normalized relative to the expression of MdAct . For RNA-seq, each point is the mean of three biological replicates. Correlations between qRT-PCR and RNA-seq expressions were calculated and their associated P -values are indicated. Error bars show SD. This figure is available in colour at JXB online.

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

    Relationships between anthocyanin contents and transcript levels of the ten representative genes from module ‘Pink’ in 14 Malus accessions of varying colours in fruit skin and flesh. The ten genes are the same as those used in Fig. 5 , which are listed in Table 1 as well as in Fig. 4 . For each accession, the expression was determined in two developmental stages immature (S2) and mature (S4) of skin (A and B) and flesh (C and D) tissues. Details of qRT-PCR analysis are as described in Fig. 5 . Anthocyanin levels are indicated by red lines. The x -axis in each chart is the same and represents the 14 Malus accessions as indicated by their fruit close-up views and names at the bottom panel, which are arranged in four groups (distinguished by colour): 1, yellow skin/white flesh; 2, red skin/white flesh; 3, red skin/red flesh; and 4, yellow skin/red flesh. The left y -axis represents relative expression levels determined by qRT-PCR, and the right y -axis represents anthocyanin content (µg g -1 dry weight). Each point stands for a mean ±SD ( n =3). Correlation coefficient values between gene expression profile and anthocyanin levels are presented above each gene legend correspondingly ( n =14, r 0.05 = 0.497, r 0.01 = 0.628).
    Figure Legend Snippet: Relationships between anthocyanin contents and transcript levels of the ten representative genes from module ‘Pink’ in 14 Malus accessions of varying colours in fruit skin and flesh. The ten genes are the same as those used in Fig. 5 , which are listed in Table 1 as well as in Fig. 4 . For each accession, the expression was determined in two developmental stages immature (S2) and mature (S4) of skin (A and B) and flesh (C and D) tissues. Details of qRT-PCR analysis are as described in Fig. 5 . Anthocyanin levels are indicated by red lines. The x -axis in each chart is the same and represents the 14 Malus accessions as indicated by their fruit close-up views and names at the bottom panel, which are arranged in four groups (distinguished by colour): 1, yellow skin/white flesh; 2, red skin/white flesh; 3, red skin/red flesh; and 4, yellow skin/red flesh. The left y -axis represents relative expression levels determined by qRT-PCR, and the right y -axis represents anthocyanin content (µg g -1 dry weight). Each point stands for a mean ±SD ( n =3). Correlation coefficient values between gene expression profile and anthocyanin levels are presented above each gene legend correspondingly ( n =14, r 0.05 = 0.497, r 0.01 = 0.628).

    Techniques Used: Expressing, Quantitative RT-PCR

    Diagram of the flavonoid/anthocyanin pathway assigned with 24 genes from the WGCNA module ‘Pink’. The proteins with names shown in blue and underlined are encoded by the 24 genes, including 12 previously characterized (in regular font) and 12 newly identified in this study (in bold font). Genes with IDs underlined were chosen for qRT-PCR assays. Please refer to the abbreviation section for the full names of proteins or genes abbreviated in the figure. This figure is available in colour at JXB online.
    Figure Legend Snippet: Diagram of the flavonoid/anthocyanin pathway assigned with 24 genes from the WGCNA module ‘Pink’. The proteins with names shown in blue and underlined are encoded by the 24 genes, including 12 previously characterized (in regular font) and 12 newly identified in this study (in bold font). Genes with IDs underlined were chosen for qRT-PCR assays. Please refer to the abbreviation section for the full names of proteins or genes abbreviated in the figure. This figure is available in colour at JXB online.

    Techniques Used: Quantitative RT-PCR

    2) Product Images from "Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas"

    Article Title: Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025594

    Relative expression of Cg-BigDef transcripts in oyster hemocytes by real-time quantitative PCR. a : Expression analysis of the three Cg-BigDef forms at 12 h post-stimulation with sterile sea water (white bars, SSW) and heat-killed bacteria (black bars, stimulation). Asterisks (*) indicate significant differences between conditions according to the Student's t-test ( p
    Figure Legend Snippet: Relative expression of Cg-BigDef transcripts in oyster hemocytes by real-time quantitative PCR. a : Expression analysis of the three Cg-BigDef forms at 12 h post-stimulation with sterile sea water (white bars, SSW) and heat-killed bacteria (black bars, stimulation). Asterisks (*) indicate significant differences between conditions according to the Student's t-test ( p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    3) Product Images from "Palmitate Promotes the Paracrine Effects of Macrophages on Vascular Smooth Muscle Cells: The Role of Bone Morphogenetic Proteins"

    Article Title: Palmitate Promotes the Paracrine Effects of Macrophages on Vascular Smooth Muscle Cells: The Role of Bone Morphogenetic Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029100

    BMP production by palmitate-stimulated macrophages. A. RAW 264.7 cells were treated with BSA control or palmitate (250 µM) for 4 hours and conditioned media were collected 20 hours later. BMP 2 and 4 secreted in the media were pulled down on heparin-Sepharose columns and detected by immunoblotting. B. RAW 264.7 cells were treated with BSA control or palmitate for 4 hours, and then incubated with fresh media for 20 hours. BMP2 and BMP4 mRNA levels were analyzed by real-time PCR. Data are illustrated in arbitrary unit relative control and represent the means ± SE from three independent experiments. *p
    Figure Legend Snippet: BMP production by palmitate-stimulated macrophages. A. RAW 264.7 cells were treated with BSA control or palmitate (250 µM) for 4 hours and conditioned media were collected 20 hours later. BMP 2 and 4 secreted in the media were pulled down on heparin-Sepharose columns and detected by immunoblotting. B. RAW 264.7 cells were treated with BSA control or palmitate for 4 hours, and then incubated with fresh media for 20 hours. BMP2 and BMP4 mRNA levels were analyzed by real-time PCR. Data are illustrated in arbitrary unit relative control and represent the means ± SE from three independent experiments. *p

    Techniques Used: Incubation, Real-time Polymerase Chain Reaction

    4) Product Images from "Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion"

    Article Title: Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077306

    Splenocytes and adipocytes differentially express pro-inflammatory markers. Differentiated 3T3-L1 adipocytes were co-cultured in direct contact with GFP-expressing murine splenocytes and activated by incubation with LPS (1 µg/mL) for 24 h. Cells were sorted for GFP-positive (splenocyte) and negative (adipocyte) cells by FACS. (A) Representative FACS is shown, with GFP-negative cells gated in R1 and GFP-positive cells gated in R2. (B) Quantitative real-time PCR (qRT-PCR) was used to measure adiponectin and F4/80 expression, specific markers for adipocytes and splenocytes, respectively, to confirm efficiency of cell sorting. (C) TNFα, IL-6 and MCP-1 mRNA expression levels were quantified by qRT-PCR in splenocytes and adipocytes following cell sorting to distinguish individual cytokine expression patterns. All qRT-PCR values were normalized to values obtained for 36B4, a ribosomal 60S subunit protein. Experimental points were measured in duplicate (B and C) for calculation of means and standard deviations. In (C), statistical significance of differing secretion levels between splenocytes and adipocytes was determined by an unpaired two-tailed Student's t-test. A significant effect was accepted when p
    Figure Legend Snippet: Splenocytes and adipocytes differentially express pro-inflammatory markers. Differentiated 3T3-L1 adipocytes were co-cultured in direct contact with GFP-expressing murine splenocytes and activated by incubation with LPS (1 µg/mL) for 24 h. Cells were sorted for GFP-positive (splenocyte) and negative (adipocyte) cells by FACS. (A) Representative FACS is shown, with GFP-negative cells gated in R1 and GFP-positive cells gated in R2. (B) Quantitative real-time PCR (qRT-PCR) was used to measure adiponectin and F4/80 expression, specific markers for adipocytes and splenocytes, respectively, to confirm efficiency of cell sorting. (C) TNFα, IL-6 and MCP-1 mRNA expression levels were quantified by qRT-PCR in splenocytes and adipocytes following cell sorting to distinguish individual cytokine expression patterns. All qRT-PCR values were normalized to values obtained for 36B4, a ribosomal 60S subunit protein. Experimental points were measured in duplicate (B and C) for calculation of means and standard deviations. In (C), statistical significance of differing secretion levels between splenocytes and adipocytes was determined by an unpaired two-tailed Student's t-test. A significant effect was accepted when p

    Techniques Used: Cell Culture, Expressing, Incubation, FACS, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

    Effects of paracrine factors and cell contact on IL-10 secretion and expression. (A) Differentiated 3T3-L1 adipocytes or wild type murine splenocytes were cultured alone (columns 1 and 2 or 3 and 4, respectively) or together with either no contact (columns 5 and 6) or direct contact (columns 7 and 8). Cells were incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h as indicated. Interleukin-10 (IL-10) in culture media was quantified by capture ELISA. (B) Differentiated 3T3-L1 adipocytes were co-cultured with no contact or direct contact with GFP-expressing murine splenocytes as in Figure 3 and activated by incubation with LPS (1 µg/mL) for 24 h. Splenocytes were sorted as GFP-positive cells by FACS and IL-10 mRNA expression was measured by qRT-PCR. qRT-PCR values were normalized to values obtained for 36B4. In (A) experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between all conditions was calculated using ANOVA followed by the post-hoc Bonferroni test. No statistical significance was found between any measured points (p > 0.05). For (B), experimental points were performed in duplicate, and a statistical comparison between no contact and direct contact was made using an unpaired two-tailed Student's t-test.
    Figure Legend Snippet: Effects of paracrine factors and cell contact on IL-10 secretion and expression. (A) Differentiated 3T3-L1 adipocytes or wild type murine splenocytes were cultured alone (columns 1 and 2 or 3 and 4, respectively) or together with either no contact (columns 5 and 6) or direct contact (columns 7 and 8). Cells were incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h as indicated. Interleukin-10 (IL-10) in culture media was quantified by capture ELISA. (B) Differentiated 3T3-L1 adipocytes were co-cultured with no contact or direct contact with GFP-expressing murine splenocytes as in Figure 3 and activated by incubation with LPS (1 µg/mL) for 24 h. Splenocytes were sorted as GFP-positive cells by FACS and IL-10 mRNA expression was measured by qRT-PCR. qRT-PCR values were normalized to values obtained for 36B4. In (A) experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between all conditions was calculated using ANOVA followed by the post-hoc Bonferroni test. No statistical significance was found between any measured points (p > 0.05). For (B), experimental points were performed in duplicate, and a statistical comparison between no contact and direct contact was made using an unpaired two-tailed Student's t-test.

    Techniques Used: Expressing, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, FACS, Quantitative RT-PCR, Two Tailed Test

    5) Product Images from "TNF-α induces human neural progenitor cell survival after oxygen–glucose deprivation by activating the NF-κB pathway"

    Article Title: TNF-α induces human neural progenitor cell survival after oxygen–glucose deprivation by activating the NF-κB pathway

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0033-1

    Effect of TNF-α treatment on the expression of survival-related genes in hNPCs. a Heatmap of NGS data analysis of transcripts encoding genes associated with anti-apoptotic and antioxidant biological pathways. FPKM-normalized values of each gene were used and converted to their log 10 values to generate the graphs. Heatmaps were generated using the cummeRbund R package. Asterisks in heatmaps denote genes discussed in the text. b qRT-PCR analysis of anti-apoptotic and antioxidant genes showing key factors (such as cIAP2 and SOD2, respectively) whose expression was significantly increased by TNF-α treatment. TNF-α-induced cIAP2 and SOD2 mRNA expression was eliminated by the NF-κB inhibitor BAY11-7082. ** P
    Figure Legend Snippet: Effect of TNF-α treatment on the expression of survival-related genes in hNPCs. a Heatmap of NGS data analysis of transcripts encoding genes associated with anti-apoptotic and antioxidant biological pathways. FPKM-normalized values of each gene were used and converted to their log 10 values to generate the graphs. Heatmaps were generated using the cummeRbund R package. Asterisks in heatmaps denote genes discussed in the text. b qRT-PCR analysis of anti-apoptotic and antioxidant genes showing key factors (such as cIAP2 and SOD2, respectively) whose expression was significantly increased by TNF-α treatment. TNF-α-induced cIAP2 and SOD2 mRNA expression was eliminated by the NF-κB inhibitor BAY11-7082. ** P

    Techniques Used: Expressing, Next-Generation Sequencing, Generated, Quantitative RT-PCR

    6) Product Images from "Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion"

    Article Title: Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077306

    Rates of cytokine secretion and overall levels are affected by direct contact between adipocytes and splenocytes. Differentiated 3T3-L1 adipocytes and isolated murine splenocytes were co-cultured with no contact (separated by a 0.4 µm transwell filter system) (dashed lines) or with direct contact (solid lines) and incubated with LPS (1 µg/mL) for 0, 8, 24 and 48 h. Cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were measured in culture media following these incubation times by capture ELISA. All experimental points were performed in triplicate.
    Figure Legend Snippet: Rates of cytokine secretion and overall levels are affected by direct contact between adipocytes and splenocytes. Differentiated 3T3-L1 adipocytes and isolated murine splenocytes were co-cultured with no contact (separated by a 0.4 µm transwell filter system) (dashed lines) or with direct contact (solid lines) and incubated with LPS (1 µg/mL) for 0, 8, 24 and 48 h. Cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were measured in culture media following these incubation times by capture ELISA. All experimental points were performed in triplicate.

    Techniques Used: Isolation, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

    Cell contact-mediated enhancement of IL-6 and MCP-1 secretion requires TNFα signaling. (A and B) Differentiated adipocytes or murine splenocytes (black bars, isolated from wild type mice; gray bars, isolated from TNFα -/- mice) were cultured alone (individual culture, columns 1 and 2) or in co-culture with no contact (columns 3 and 4) or direct contact (columns 5 and 6) as in Figure 1 . Cells were incubated in the absence (−) or presence (+) of LPS (1 µg/mL) for 24 h as indicated. (C and D) Wild type (WT) or TNFα -/- (TNFα KO) splenocytes were incubated with adipocytes with direct contact in the presence of LPS (1 µg/mL) and co-cultures were supplemented with 0, 300 pg/mL or 10 ng/mL purified murine TNFα as indicated. Secreted IL-6 (A and C) and MCP-1 (B and D) were quantified by capture ELISA. Experimental points were measured in triplicate (A and B) or duplicate (C and D) for calculation of means and standard deviations. For (A and B) comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact from WT and TNFα KO were calculated using ANOVA and followed by the post-hoc Bonferroni test. For C and D all experimental points were compared statistically by ANOVA followed by the Bonferroni post-hoc test, and differed significantly (p
    Figure Legend Snippet: Cell contact-mediated enhancement of IL-6 and MCP-1 secretion requires TNFα signaling. (A and B) Differentiated adipocytes or murine splenocytes (black bars, isolated from wild type mice; gray bars, isolated from TNFα -/- mice) were cultured alone (individual culture, columns 1 and 2) or in co-culture with no contact (columns 3 and 4) or direct contact (columns 5 and 6) as in Figure 1 . Cells were incubated in the absence (−) or presence (+) of LPS (1 µg/mL) for 24 h as indicated. (C and D) Wild type (WT) or TNFα -/- (TNFα KO) splenocytes were incubated with adipocytes with direct contact in the presence of LPS (1 µg/mL) and co-cultures were supplemented with 0, 300 pg/mL or 10 ng/mL purified murine TNFα as indicated. Secreted IL-6 (A and C) and MCP-1 (B and D) were quantified by capture ELISA. Experimental points were measured in triplicate (A and B) or duplicate (C and D) for calculation of means and standard deviations. For (A and B) comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact from WT and TNFα KO were calculated using ANOVA and followed by the post-hoc Bonferroni test. For C and D all experimental points were compared statistically by ANOVA followed by the Bonferroni post-hoc test, and differed significantly (p

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Co-Culture Assay, Incubation, Purification, Enzyme-linked Immunosorbent Assay

    Splenocytes and adipocytes differentially express pro-inflammatory markers. Differentiated 3T3-L1 adipocytes were co-cultured in direct contact with GFP-expressing murine splenocytes and activated by incubation with LPS (1 µg/mL) for 24 h. Cells were sorted for GFP-positive (splenocyte) and negative (adipocyte) cells by FACS. (A) Representative FACS is shown, with GFP-negative cells gated in R1 and GFP-positive cells gated in R2. (B) Quantitative real-time PCR (qRT-PCR) was used to measure adiponectin and F4/80 expression, specific markers for adipocytes and splenocytes, respectively, to confirm efficiency of cell sorting. (C) TNFα, IL-6 and MCP-1 mRNA expression levels were quantified by qRT-PCR in splenocytes and adipocytes following cell sorting to distinguish individual cytokine expression patterns. All qRT-PCR values were normalized to values obtained for 36B4, a ribosomal 60S subunit protein. Experimental points were measured in duplicate (B and C) for calculation of means and standard deviations. In (C), statistical significance of differing secretion levels between splenocytes and adipocytes was determined by an unpaired two-tailed Student's t-test. A significant effect was accepted when p
    Figure Legend Snippet: Splenocytes and adipocytes differentially express pro-inflammatory markers. Differentiated 3T3-L1 adipocytes were co-cultured in direct contact with GFP-expressing murine splenocytes and activated by incubation with LPS (1 µg/mL) for 24 h. Cells were sorted for GFP-positive (splenocyte) and negative (adipocyte) cells by FACS. (A) Representative FACS is shown, with GFP-negative cells gated in R1 and GFP-positive cells gated in R2. (B) Quantitative real-time PCR (qRT-PCR) was used to measure adiponectin and F4/80 expression, specific markers for adipocytes and splenocytes, respectively, to confirm efficiency of cell sorting. (C) TNFα, IL-6 and MCP-1 mRNA expression levels were quantified by qRT-PCR in splenocytes and adipocytes following cell sorting to distinguish individual cytokine expression patterns. All qRT-PCR values were normalized to values obtained for 36B4, a ribosomal 60S subunit protein. Experimental points were measured in duplicate (B and C) for calculation of means and standard deviations. In (C), statistical significance of differing secretion levels between splenocytes and adipocytes was determined by an unpaired two-tailed Student's t-test. A significant effect was accepted when p

    Techniques Used: Cell Culture, Expressing, Incubation, FACS, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

    Direct contact between splenocytes and adipocytes alters secreted levels of inflammatory cytokines. Differentiated 3T3-L1 adipocytes (columns 1 and 2) or isolated murine splenocytes (columns 3 and 4) were cultured alone or together with either direct contact (columns 7 and 8) or no contact (cells separated by a 0.4 µm transwell filter system; columns 5 and 6). Cells were additionally incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h. Secreted cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were quantified by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact were calculated using ANOVA and followed by the post-hoc Bonferroni test. A significant effect was accepted when p
    Figure Legend Snippet: Direct contact between splenocytes and adipocytes alters secreted levels of inflammatory cytokines. Differentiated 3T3-L1 adipocytes (columns 1 and 2) or isolated murine splenocytes (columns 3 and 4) were cultured alone or together with either direct contact (columns 7 and 8) or no contact (cells separated by a 0.4 µm transwell filter system; columns 5 and 6). Cells were additionally incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h. Secreted cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were quantified by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact were calculated using ANOVA and followed by the post-hoc Bonferroni test. A significant effect was accepted when p

    Techniques Used: Isolation, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

    NF-κB intracellular signaling pathway participates in the paracrine and cell contact-mediated enhancement of IL-6 and MCP-1 secretion. (A) Isolated splenocytes were incubated with LPS (1 µg/mL) in the absence (-) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of TNFα were measured by capture ELISA. (B and C) Differentiated 3T3-L1 adipocytes were co-cultured with isolated splenocytes with either no contact or direct contact as indicated. Cells were stimulated with LPS (1 µg/mL) in the absence (−) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of IL-6 (B) or MCP-1 (C) were measured by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. In (A) an unpaired two-tailed Student's t test was used to compare Bay11-7082 treated and untreated LPS-stimulated splenocytes. Comparisons between co-cultures (B and C) were calculated using ANOVA followed by the post-hoc Bonferroni test.
    Figure Legend Snippet: NF-κB intracellular signaling pathway participates in the paracrine and cell contact-mediated enhancement of IL-6 and MCP-1 secretion. (A) Isolated splenocytes were incubated with LPS (1 µg/mL) in the absence (-) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of TNFα were measured by capture ELISA. (B and C) Differentiated 3T3-L1 adipocytes were co-cultured with isolated splenocytes with either no contact or direct contact as indicated. Cells were stimulated with LPS (1 µg/mL) in the absence (−) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of IL-6 (B) or MCP-1 (C) were measured by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. In (A) an unpaired two-tailed Student's t test was used to compare Bay11-7082 treated and untreated LPS-stimulated splenocytes. Comparisons between co-cultures (B and C) were calculated using ANOVA followed by the post-hoc Bonferroni test.

    Techniques Used: Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

    7) Product Images from "Characterisation of peripheral and central components of the rat monoiodoacetate model of Osteoarthritis"

    Article Title: Characterisation of peripheral and central components of the rat monoiodoacetate model of Osteoarthritis

    Journal: Osteoarthritis and Cartilage

    doi: 10.1016/j.joca.2018.12.017

    Shows mRNA expression of glia cells and cytokines in the lumbar dorsal horn of EP and LP MIA animals (EP n = 4, EPS n = 4, LP n = 4, LPS n = 4). A) mRNA expression of Iba1 and glial fibrillary acid protein (GFAP) normalized to sham. B) mRNA expression of pro-inflammatory cytokines normalized to sham. C) There is a weak rise in Iba1 mRNA expression in LP MIA animals. D) GFAP mRNA levels remain similar between MIA and sham groups. E) There is a significant increase in IL-1β mRNA expression in LP MIA animals. F) IL-6 mRNA levels remain similar between MIA and sham groups. G) TNFα mRNA levels remain similar between MIA and sham groups (A–B: for mRNA expression normalized to sham controls a Kruskall–Wallis test was used, C–G: for mRNA expression expressed as 2-ΔCT values an independent samples t -test was used. * P
    Figure Legend Snippet: Shows mRNA expression of glia cells and cytokines in the lumbar dorsal horn of EP and LP MIA animals (EP n = 4, EPS n = 4, LP n = 4, LPS n = 4). A) mRNA expression of Iba1 and glial fibrillary acid protein (GFAP) normalized to sham. B) mRNA expression of pro-inflammatory cytokines normalized to sham. C) There is a weak rise in Iba1 mRNA expression in LP MIA animals. D) GFAP mRNA levels remain similar between MIA and sham groups. E) There is a significant increase in IL-1β mRNA expression in LP MIA animals. F) IL-6 mRNA levels remain similar between MIA and sham groups. G) TNFα mRNA levels remain similar between MIA and sham groups (A–B: for mRNA expression normalized to sham controls a Kruskall–Wallis test was used, C–G: for mRNA expression expressed as 2-ΔCT values an independent samples t -test was used. * P

    Techniques Used: Expressing

    8) Product Images from "The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein"

    Article Title: The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006937

    OASL and ORF20 are expressed in reactivated HuARLT2-rKSHV.219 cells. Uninfected HuARLT2 and latently infected HuARLT2-rKSHV.219 cells were seeded in 6-well plates. HuARLT2-rKSHV.219 cells were untreated (-) or reactivated by addition of sodium butyrate and RTA-expressing baculovirus, then incubated for the indicated times. RNA was prepared and (A) OASL (B) ORF20 (C) ORF16 (D) ORF46 or (E) K8.1 mRNA levels were determined by q-RT-PCR. The averages + SD of duplicates from 1 representative of 3 total experiments are shown. nd, not detected, ns, not significant, ** P
    Figure Legend Snippet: OASL and ORF20 are expressed in reactivated HuARLT2-rKSHV.219 cells. Uninfected HuARLT2 and latently infected HuARLT2-rKSHV.219 cells were seeded in 6-well plates. HuARLT2-rKSHV.219 cells were untreated (-) or reactivated by addition of sodium butyrate and RTA-expressing baculovirus, then incubated for the indicated times. RNA was prepared and (A) OASL (B) ORF20 (C) ORF16 (D) ORF46 or (E) K8.1 mRNA levels were determined by q-RT-PCR. The averages + SD of duplicates from 1 representative of 3 total experiments are shown. nd, not detected, ns, not significant, ** P

    Techniques Used: Infection, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

    9) Product Images from "The somatic piRNA pathway controls germline transposition over generations"

    Article Title: The somatic piRNA pathway controls germline transposition over generations

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky761

    Somatic piwi depletion may lead to de novo ERV insertions in the offspring genome, with a transposition rate stable over generations. ( A ) Bar plot displaying the ratio of de novo insertions per ERV family (n = 62) in embryos of the F2 generation after piwi-sKD versus no-KD embryos. ( B ) PCR-validation of a de novo ZAM insertion detected by DNA-seq at position chrX:10.241.013. gDNA from flies, of the F2 generation after piwi-sKD, in which the de novo ZAM insertion was detected and, as control, their no-KD ancestors was used. The upper panel shows a schematic representation of the genomic location of the de novo ZAM insertion with the two primer pairs spanning the break point between the 5′ end of the insertion and the genome. The lower panel shows the PCR products on agarose gel. Primer pair 1 and 2 detect the de novo ZAM insertion at position chrX:10.241.013 and a control primer pair C detects a fixed ZAM insertion (detected in all our libraries) at position chr2L:19.841.922. ( C ) Schema depicting the experimental set up of the single and successive piwi-sKD. (D) Bar plot displaying the TE load for the depicted ERVs in no-KD and after 30, 41 and 72 generations of successive piwi-sKD (G30, G41 and G72). The F2 generation after the last piwi-sKD was analyzed by qPCR. The mean and standard deviation from three biological replicates is shown. The P -values where calculated with a two-tailed t -test (* P
    Figure Legend Snippet: Somatic piwi depletion may lead to de novo ERV insertions in the offspring genome, with a transposition rate stable over generations. ( A ) Bar plot displaying the ratio of de novo insertions per ERV family (n = 62) in embryos of the F2 generation after piwi-sKD versus no-KD embryos. ( B ) PCR-validation of a de novo ZAM insertion detected by DNA-seq at position chrX:10.241.013. gDNA from flies, of the F2 generation after piwi-sKD, in which the de novo ZAM insertion was detected and, as control, their no-KD ancestors was used. The upper panel shows a schematic representation of the genomic location of the de novo ZAM insertion with the two primer pairs spanning the break point between the 5′ end of the insertion and the genome. The lower panel shows the PCR products on agarose gel. Primer pair 1 and 2 detect the de novo ZAM insertion at position chrX:10.241.013 and a control primer pair C detects a fixed ZAM insertion (detected in all our libraries) at position chr2L:19.841.922. ( C ) Schema depicting the experimental set up of the single and successive piwi-sKD. (D) Bar plot displaying the TE load for the depicted ERVs in no-KD and after 30, 41 and 72 generations of successive piwi-sKD (G30, G41 and G72). The F2 generation after the last piwi-sKD was analyzed by qPCR. The mean and standard deviation from three biological replicates is shown. The P -values where calculated with a two-tailed t -test (* P

    Techniques Used: Polymerase Chain Reaction, DNA Sequencing, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

    10) Product Images from "A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications"

    Article Title: A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0132441

    (A) Gel electrophoresis of PCR amplified 16S rDNA for the DNA extracted using the powdered glass method M6. Samples were analyzed on 1% agarose gel in 0.5X TBE buffer. Lane M: 1 Kb DNA ladder (Merck, India); Lane 1: Garden soil; Lane 2: Sewage sludge; Lane 3: Lake soil; Lane 4: Compost; Lane 5: E . coli MTCC 40; Lane 6: Bacillus subtilis NRRL-B-201. Lane 7: Negative control. (B). Gel electrophoresis of PCR amplified ITS -2 for the DNA extracted using the powdered glass method M6. Lane M: 100bp DNA ladder (Merck, India); Lane 1: Negative control; Lane 2: PCR amplification of ITS -2 region of Chlorella sorokiniana UTEX# 1666.
    Figure Legend Snippet: (A) Gel electrophoresis of PCR amplified 16S rDNA for the DNA extracted using the powdered glass method M6. Samples were analyzed on 1% agarose gel in 0.5X TBE buffer. Lane M: 1 Kb DNA ladder (Merck, India); Lane 1: Garden soil; Lane 2: Sewage sludge; Lane 3: Lake soil; Lane 4: Compost; Lane 5: E . coli MTCC 40; Lane 6: Bacillus subtilis NRRL-B-201. Lane 7: Negative control. (B). Gel electrophoresis of PCR amplified ITS -2 for the DNA extracted using the powdered glass method M6. Lane M: 100bp DNA ladder (Merck, India); Lane 1: Negative control; Lane 2: PCR amplification of ITS -2 region of Chlorella sorokiniana UTEX# 1666.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Negative Control

    11) Product Images from "Micro(mi) RNA-34a targets protein phosphatase (PP)1γ to regulate DNA damage tolerance"

    Article Title: Micro(mi) RNA-34a targets protein phosphatase (PP)1γ to regulate DNA damage tolerance

    Journal: Cell Cycle

    doi: 10.1080/15384101.2015.1064202

    Dose-dependent miR-34a induction after IR is accompanied by down-regulation of PP1γ mRNA and protein. ( A ) Cal51 cells were irradiated with 1, 3 and 9 Gy and were harvested every 24 h up to 96 h. The 0 h sample not exposed to IR was deemed as a negative control. miR-34a expression was studied by qRT-PCR (n = 3; ±SEM). ( B ) PP1γ and CDK6 mRNA expression was studied by qRT-PCR (n = 3; ±SEM). ( C ) Western blot analysis. Numbers shown below PP1γ, PP1α and PP1β blots are densitometry values representing relative protein expression.
    Figure Legend Snippet: Dose-dependent miR-34a induction after IR is accompanied by down-regulation of PP1γ mRNA and protein. ( A ) Cal51 cells were irradiated with 1, 3 and 9 Gy and were harvested every 24 h up to 96 h. The 0 h sample not exposed to IR was deemed as a negative control. miR-34a expression was studied by qRT-PCR (n = 3; ±SEM). ( B ) PP1γ and CDK6 mRNA expression was studied by qRT-PCR (n = 3; ±SEM). ( C ) Western blot analysis. Numbers shown below PP1γ, PP1α and PP1β blots are densitometry values representing relative protein expression.

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

    PP1γ is a target of miR-34a. ( A ) Cal51 cells were transfected with 50 nM miR-34a mimic or Luc siRNA and harvested 72 h post-transfection for analysis. qRT-PCR of PP1γ and CDK6 was performed (one-way ANOVA with Dunett's post-test; * P
    Figure Legend Snippet: PP1γ is a target of miR-34a. ( A ) Cal51 cells were transfected with 50 nM miR-34a mimic or Luc siRNA and harvested 72 h post-transfection for analysis. qRT-PCR of PP1γ and CDK6 was performed (one-way ANOVA with Dunett's post-test; * P

    Techniques Used: Transfection, Quantitative RT-PCR

    12) Product Images from "TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends"

    Article Title: TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends

    Journal: Nature Communications

    doi: 10.1038/ncomms6467

    TRF2 and lamin A/C are enriched at ITSs. ( a ) The localization of BACs used for mitotic and interphase FISH analysis and primers used for ChIP–qPCR analysis are indicated. ( b ) Quantification of the co-localization between BACs on HSA1 and telomere signal in IMR90 interphase nuclei. ( c ) Representative image of 3D FISH analysis quantified in b showing the H17 BAC (magenta), the H22 BAC (red), telomere FISH signal (green) and DNA stained with DAPI (blue). One of the alleles (white box) is enlarged in the bottom right depicting co-localization of H17, but not H22, with the telomere. ( d , e ) TRF2 and lamin A/C ChIP–qPCR analysis at four ITSs (ITS1-1, ITS18-1, ITS19-1 and ITS19-2), a sequence immediately adjacent to the telomere (Tel-Adj), and sites without an ITS (HSA1-2 and RPLP0). ( f ) TRF2 ChIP–qPCR analysis at sites identified as positive in d , performed with cells treated with shLMNA or shSCR. Sites to the left of the dotted line show lamin A/C association in e , and sites to the right of the dotted line do not. Values are normalized to those of shSCR and data are presented as mean±s.e.m. ( n =3). * P
    Figure Legend Snippet: TRF2 and lamin A/C are enriched at ITSs. ( a ) The localization of BACs used for mitotic and interphase FISH analysis and primers used for ChIP–qPCR analysis are indicated. ( b ) Quantification of the co-localization between BACs on HSA1 and telomere signal in IMR90 interphase nuclei. ( c ) Representative image of 3D FISH analysis quantified in b showing the H17 BAC (magenta), the H22 BAC (red), telomere FISH signal (green) and DNA stained with DAPI (blue). One of the alleles (white box) is enlarged in the bottom right depicting co-localization of H17, but not H22, with the telomere. ( d , e ) TRF2 and lamin A/C ChIP–qPCR analysis at four ITSs (ITS1-1, ITS18-1, ITS19-1 and ITS19-2), a sequence immediately adjacent to the telomere (Tel-Adj), and sites without an ITS (HSA1-2 and RPLP0). ( f ) TRF2 ChIP–qPCR analysis at sites identified as positive in d , performed with cells treated with shLMNA or shSCR. Sites to the left of the dotted line show lamin A/C association in e , and sites to the right of the dotted line do not. Values are normalized to those of shSCR and data are presented as mean±s.e.m. ( n =3). * P

    Techniques Used: Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, BAC Assay, Staining, Sequencing

    13) Product Images from "The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein"

    Article Title: The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006937

    OASL expression is beneficial for MHV68 and KSHV replication. (A-F, H) 293T OASL -/- cells were reconstituted with empty vector (EV), mOASL1, or hOASL as indicated. (A-B) 24 h later, cells were infected with VSV-GFP for 16 h. The GFP intensity in fixed cells was determined by flow cytometry. (A) Histograms of the VSV-GFP signal for duplicates from one representative experiment are shown. (B) The number of highly-GFP positive cells was determined and scaled to EV. Means + SD of duplicates from two independent experiments are shown. (C-D) 24 h post transfection, cells were infected with MHV68-GFP at an MOI of 0.05 for 2 h at 37°C. Supernatant was harvested at the indicated times and the viral titer was determined by TCID 50 on M2-10B4 cells. Means ± SD of triplicates from two independent experiments are shown. (E, F, H) 24 h post transfection, cells were infected with (E) MHV68-GFP, (F) KSHV LYT wildtype, or (H) KSHV LYT ORF20stop. 20–24 h later, cells were detached, fixed, and the number of GFP-positive cells was determined by flow cytometry. Means + SD of 4–6 replicates total from two independent experiments are shown. (G) In KSHV LYT ORF20stop, the ORF20 GAG codon for E69 is replaced with the stop codon TAG. For all subfigures: ns, not significant, * P
    Figure Legend Snippet: OASL expression is beneficial for MHV68 and KSHV replication. (A-F, H) 293T OASL -/- cells were reconstituted with empty vector (EV), mOASL1, or hOASL as indicated. (A-B) 24 h later, cells were infected with VSV-GFP for 16 h. The GFP intensity in fixed cells was determined by flow cytometry. (A) Histograms of the VSV-GFP signal for duplicates from one representative experiment are shown. (B) The number of highly-GFP positive cells was determined and scaled to EV. Means + SD of duplicates from two independent experiments are shown. (C-D) 24 h post transfection, cells were infected with MHV68-GFP at an MOI of 0.05 for 2 h at 37°C. Supernatant was harvested at the indicated times and the viral titer was determined by TCID 50 on M2-10B4 cells. Means ± SD of triplicates from two independent experiments are shown. (E, F, H) 24 h post transfection, cells were infected with (E) MHV68-GFP, (F) KSHV LYT wildtype, or (H) KSHV LYT ORF20stop. 20–24 h later, cells were detached, fixed, and the number of GFP-positive cells was determined by flow cytometry. Means + SD of 4–6 replicates total from two independent experiments are shown. (G) In KSHV LYT ORF20stop, the ORF20 GAG codon for E69 is replaced with the stop codon TAG. For all subfigures: ns, not significant, * P

    Techniques Used: Expressing, Plasmid Preparation, Infection, Flow Cytometry, Cytometry, Transfection

    The interaction of ORF20 with OASL is conserved among the members of the UL24 family. (A) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, coverslips were fixed in PFA and processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (B) 293T cells were transfected with myc-tagged UL24, UL76, M76, or ORF20WT. Lysates were prepared 24 h later, separated by SDS-PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Data are representative of four independent experiments. (C) 293T cells were transfected with the indicated myc-tagged KSHV ORF20 form or MHV68 ORF20. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with an anti-myc antibody. Data are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged UL24 homologs MCMV M76, HCMV UL76, or HSV-1 UL24, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of two independent experiments.
    Figure Legend Snippet: The interaction of ORF20 with OASL is conserved among the members of the UL24 family. (A) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, coverslips were fixed in PFA and processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (B) 293T cells were transfected with myc-tagged UL24, UL76, M76, or ORF20WT. Lysates were prepared 24 h later, separated by SDS-PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Data are representative of four independent experiments. (C) 293T cells were transfected with the indicated myc-tagged KSHV ORF20 form or MHV68 ORF20. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with an anti-myc antibody. Data are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged UL24 homologs MCMV M76, HCMV UL76, or HSV-1 UL24, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of two independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Immunofluorescence, SDS Page, Immunoprecipitation

    ORF20 does not affect translation rates, but enhances expression of endogenous OASL. (A) 293T cells were co-transfected with EV, myc-tagged ORF20WT, ORF20FL, or ORF20B, and either EV or RIG-I N. 24 h post transfection, cells were treated for 15 minutes with 5μg/ml puromycin, then lysed and analyzed by immunoblotting. Anti-puromycin, anti-OASL, anti-myc, and anti-actin immunoblotting was performed sequentially. Data are representative of two independent experiments. *: Nonspecific background band. (B) 293T cells were co-transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (C) HEK293 cells were transfected with either EV or ORF20WT-myc. 24h later, cells were transfected with 5’pppRNA complexed with Lipofectamine 2000 for approximately 24h. (D) 293T cells were reverse transfected with control, IRF3, IFNAR, or STAT1 siRNAs as indicated. 48h later, cells were transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (B, C, D) Cells were lysed, RNA was isolated, mRNA-specific cDNA was reverse transcribed, and the amount of OASL was determined using relative quantification to GAPDH levels and the 2 -ΔΔ C T method. Data shown are means + SD of combined duplicates from at least two experiments. ns, not significant, * P
    Figure Legend Snippet: ORF20 does not affect translation rates, but enhances expression of endogenous OASL. (A) 293T cells were co-transfected with EV, myc-tagged ORF20WT, ORF20FL, or ORF20B, and either EV or RIG-I N. 24 h post transfection, cells were treated for 15 minutes with 5μg/ml puromycin, then lysed and analyzed by immunoblotting. Anti-puromycin, anti-OASL, anti-myc, and anti-actin immunoblotting was performed sequentially. Data are representative of two independent experiments. *: Nonspecific background band. (B) 293T cells were co-transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (C) HEK293 cells were transfected with either EV or ORF20WT-myc. 24h later, cells were transfected with 5’pppRNA complexed with Lipofectamine 2000 for approximately 24h. (D) 293T cells were reverse transfected with control, IRF3, IFNAR, or STAT1 siRNAs as indicated. 48h later, cells were transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (B, C, D) Cells were lysed, RNA was isolated, mRNA-specific cDNA was reverse transcribed, and the amount of OASL was determined using relative quantification to GAPDH levels and the 2 -ΔΔ C T method. Data shown are means + SD of combined duplicates from at least two experiments. ns, not significant, * P

    Techniques Used: Expressing, Transfection, Isolation

    KSHV ORF20 isoforms localize predominantly to the nuclei and nucleoli. (A-B) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, (A) coverslips were fixed directly in 4% paraformaldehyde in PBS (PFA) for whole cell immunofluorescence. (B) For nuclear immunofluorescence, coverslips were incubated in cold 1% NP-40 extraction buffer for 5 min to remove the cytoplasm before PFA fixation. (A-B) All samples were then processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm.
    Figure Legend Snippet: KSHV ORF20 isoforms localize predominantly to the nuclei and nucleoli. (A-B) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, (A) coverslips were fixed directly in 4% paraformaldehyde in PBS (PFA) for whole cell immunofluorescence. (B) For nuclear immunofluorescence, coverslips were incubated in cold 1% NP-40 extraction buffer for 5 min to remove the cytoplasm before PFA fixation. (A-B) All samples were then processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm.

    Techniques Used: Transfection, Plasmid Preparation, Immunofluorescence, Incubation

    14) Product Images from "Identification of Polycystic Ovary Syndrome (PCOS) Specific Genes in Cumulus and Mural Granulosa Cells"

    Article Title: Identification of Polycystic Ovary Syndrome (PCOS) Specific Genes in Cumulus and Mural Granulosa Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168875

    Relative expression values and heatmap figure of MAPK signaling pathway-related genes. (A) Relative expression values obtained from qRT-PCR analysis for each gene are shown on the y-axis in arbitrary units. The data were obtained from 12 independent PCOS patients. Each patient provided both cell types, CC and MGC. The data are presented as mean ± SEM. Asterisk (*) indicates a significant difference (p
    Figure Legend Snippet: Relative expression values and heatmap figure of MAPK signaling pathway-related genes. (A) Relative expression values obtained from qRT-PCR analysis for each gene are shown on the y-axis in arbitrary units. The data were obtained from 12 independent PCOS patients. Each patient provided both cell types, CC and MGC. The data are presented as mean ± SEM. Asterisk (*) indicates a significant difference (p

    Techniques Used: Expressing, Quantitative RT-PCR

    Relative expression of candidate genes (FZD3, INSR, PTPRC, RUNX2 and JUNB) obtained from qRT-PCR analysis. The validation study was performed with 12 independent patients who provided two somatic cell types: CC and MGC. Relative expression values for each gene is shown on the y-axis in arbitrary units. The data are presented as mean ± SEM. Asterisk (*) indicates a significant difference (p
    Figure Legend Snippet: Relative expression of candidate genes (FZD3, INSR, PTPRC, RUNX2 and JUNB) obtained from qRT-PCR analysis. The validation study was performed with 12 independent patients who provided two somatic cell types: CC and MGC. Relative expression values for each gene is shown on the y-axis in arbitrary units. The data are presented as mean ± SEM. Asterisk (*) indicates a significant difference (p

    Techniques Used: Expressing, Quantitative RT-PCR

    15) Product Images from "TRIM5α and TRIM22 Are Differentially Regulated According to HIV-1 Infection Phase and Compartment"

    Article Title: TRIM5α and TRIM22 Are Differentially Regulated According to HIV-1 Infection Phase and Compartment

    Journal: Journal of Virology

    doi: 10.1128/JVI.03603-13

    TRIM5α and TRIM22 expression in different immune cells. (A) Representative flow cytometry plots showing the gating strategy employed to define different cell populations. (Top) Cells were first gated for singlets (forward scatter height [FSC-H]
    Figure Legend Snippet: TRIM5α and TRIM22 expression in different immune cells. (A) Representative flow cytometry plots showing the gating strategy employed to define different cell populations. (Top) Cells were first gated for singlets (forward scatter height [FSC-H]

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Expression of antiviral factors according to HIV-1 infection status and compartment. (A to C) Expression of the IFN-I-responsive gene (MxA) (A), TRIM5α (B), and TRIM22 (C) in PBMCs from HIV-1-uninfected versus -infected subjects. (D to F) Expression
    Figure Legend Snippet: Expression of antiviral factors according to HIV-1 infection status and compartment. (A to C) Expression of the IFN-I-responsive gene (MxA) (A), TRIM5α (B), and TRIM22 (C) in PBMCs from HIV-1-uninfected versus -infected subjects. (D to F) Expression

    Techniques Used: Expressing, Infection

    Regulation of TRIM5α and TRIM22 by IFN-α and select proinflammatory cytokines in immune cells. Shown is the effect of IFN-α stimulation and proinflammatory cytokine stimulation of CD4 cells and monocytes on antiviral gene expression.
    Figure Legend Snippet: Regulation of TRIM5α and TRIM22 by IFN-α and select proinflammatory cytokines in immune cells. Shown is the effect of IFN-α stimulation and proinflammatory cytokine stimulation of CD4 cells and monocytes on antiviral gene expression.

    Techniques Used: Expressing

    siRNA-mediated silencing of TRIM5α or TRIM22 in CD4 cells. To determine the functional impact of TRIM5α and TRIM22 on HIV-1 replication in CD4 cells, gene knockdown experiments were performed by transducing the cells with siRNA against
    Figure Legend Snippet: siRNA-mediated silencing of TRIM5α or TRIM22 in CD4 cells. To determine the functional impact of TRIM5α and TRIM22 on HIV-1 replication in CD4 cells, gene knockdown experiments were performed by transducing the cells with siRNA against

    Techniques Used: Functional Assay

    siRNA-mediated silencing of TRIM5α or TRIM22 in neuroblasts. To determine the functional impacts of TRIM5α and TRIM22 on HIV-1 infection in neuroblasts, gene knockdown experiments were performed by transducing the cells with siRNA against
    Figure Legend Snippet: siRNA-mediated silencing of TRIM5α or TRIM22 in neuroblasts. To determine the functional impacts of TRIM5α and TRIM22 on HIV-1 infection in neuroblasts, gene knockdown experiments were performed by transducing the cells with siRNA against

    Techniques Used: Functional Assay, Infection

    16) Product Images from "Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis"

    Article Title: Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis

    Journal: Nature

    doi: 10.1038/nature13322

    A20 inhibits Nlrp3 inflammasome priming a, b, Nlrp3 (a) and proIL-1β (b) mRNA levels of LPS-treated BMDMs. c, Expression of the indicated proteins in BMDMs 6 h after LPS treatment. d, e, Rapid Nlrp3 inflammasome activation as described in Methods . Expression of the indicated proteins (d), and secreted cytokines (e) were determined. f-i, A20 myel-KO BMDMs were treated as indicated. Nlrp3 mRNA levels (f), caspase-1 expression (g), secreted IL-1β (h) and LDH activity (i) were determined. Black arrow, procaspase-1; white arrow, p20. Data represent mean ± s.d. of 1 out of 3 biological replicates, with 3 technical replicates each (* P
    Figure Legend Snippet: A20 inhibits Nlrp3 inflammasome priming a, b, Nlrp3 (a) and proIL-1β (b) mRNA levels of LPS-treated BMDMs. c, Expression of the indicated proteins in BMDMs 6 h after LPS treatment. d, e, Rapid Nlrp3 inflammasome activation as described in Methods . Expression of the indicated proteins (d), and secreted cytokines (e) were determined. f-i, A20 myel-KO BMDMs were treated as indicated. Nlrp3 mRNA levels (f), caspase-1 expression (g), secreted IL-1β (h) and LDH activity (i) were determined. Black arrow, procaspase-1; white arrow, p20. Data represent mean ± s.d. of 1 out of 3 biological replicates, with 3 technical replicates each (* P

    Techniques Used: Expressing, Activation Assay, Activity Assay

    17) Product Images from "High Levels of SOX5 Decrease Proliferative Capacity of Human B Cells, but Permit Plasmablast Differentiation"

    Article Title: High Levels of SOX5 Decrease Proliferative Capacity of Human B Cells, but Permit Plasmablast Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100328

    Human B cells express at least three different transcript variants of SOX5 . (A) Schematic representation of sequence verified human SOX5 transcript variants in B lymphocytes. Non-coding exons are depicted as open rectangles, partial coding exons - as half open rectangles and coding exons - as filled rectangles. Cloning primer locations are indicated with appropriate arrows. Exons and coding exons are numbered according to their location along the genomic sequence, which are drawn as black lines. (B) PCR analysis of SOX5 transcript expression in B cells and in single clones picked for sequence analysis. B lymphocytes express at least three different SOX5 transcript splice variants as evidenced by representative single TOPO clones 1, 2 and 3.
    Figure Legend Snippet: Human B cells express at least three different transcript variants of SOX5 . (A) Schematic representation of sequence verified human SOX5 transcript variants in B lymphocytes. Non-coding exons are depicted as open rectangles, partial coding exons - as half open rectangles and coding exons - as filled rectangles. Cloning primer locations are indicated with appropriate arrows. Exons and coding exons are numbered according to their location along the genomic sequence, which are drawn as black lines. (B) PCR analysis of SOX5 transcript expression in B cells and in single clones picked for sequence analysis. B lymphocytes express at least three different SOX5 transcript splice variants as evidenced by representative single TOPO clones 1, 2 and 3.

    Techniques Used: Sequencing, Clone Assay, Polymerase Chain Reaction, Expressing

    Expression of SOX5 transcript variants in human B cells. (A) Schematic representation of human SOX5 transcript variants. Non-coding exons are depicted as open rectangles, partial coding exons - as half open rectangles and coding exons - as filled rectangles. Primer regions are indicated with appropriate arrows. Exons and coding exons are numbered according to their location along the genomic sequence, which are drawn as black lines. (B) RT-PCR analysis for the expression of β-actin , CD19 genes and SOX5 transcript variants. HD PBMCs were separated into: A – PBMCs; B – B cells and C – non-B lymphocytes. Except for SOX5 transcript variant 3 ( SOX5 -var 3) in which human testis RNA sample served as a control, human costal cartilage cells used as a positive control in all RT-PCR reactions. In agarose gel pictures DNA markers were cut out, since they were loaded between the tested samples and the control sample. (C) RT-PCR assay performed to discriminate between SOX5 transcript variant 2 and variant 4 in samples of peripheral blood lymphocytes: A – PBMCs; B – B cells and C – non-B lymphocytes.
    Figure Legend Snippet: Expression of SOX5 transcript variants in human B cells. (A) Schematic representation of human SOX5 transcript variants. Non-coding exons are depicted as open rectangles, partial coding exons - as half open rectangles and coding exons - as filled rectangles. Primer regions are indicated with appropriate arrows. Exons and coding exons are numbered according to their location along the genomic sequence, which are drawn as black lines. (B) RT-PCR analysis for the expression of β-actin , CD19 genes and SOX5 transcript variants. HD PBMCs were separated into: A – PBMCs; B – B cells and C – non-B lymphocytes. Except for SOX5 transcript variant 3 ( SOX5 -var 3) in which human testis RNA sample served as a control, human costal cartilage cells used as a positive control in all RT-PCR reactions. In agarose gel pictures DNA markers were cut out, since they were loaded between the tested samples and the control sample. (C) RT-PCR assay performed to discriminate between SOX5 transcript variant 2 and variant 4 in samples of peripheral blood lymphocytes: A – PBMCs; B – B cells and C – non-B lymphocytes.

    Techniques Used: Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Positive Control, Agarose Gel Electrophoresis

    18) Product Images from "Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease"

    Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

    Journal: Journal of Cardiovascular Development and Disease

    doi: 10.3390/jcdd6040039

    Abnormal aortic valve morphology in Krox20+/−;Nos3+/− mice. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Nos3+/− , Krox20+/− , Nos3+/− ; Krox20+/− and Nos3−/− mice. ( B – D ) H E images showing representative Nos3+/− and Krox20+/− with tri-leaflets aortic valve, and an example of Nos3+/− ; Krox20+/− aortic valve with 2 leaflets. Aortic valve leaflets of Nos3+/− ; Krox20+/− mice appear equal in size. ( E ) Real-time qPCR demonstrates a reduction of Nos3 at a transcriptional level in Nos3+/− ; Krox20+/− and Krox20−/− compared to wild-type embryos ( n = 5 for each genotype). qPCR experiments were performed in triplicate and expressed as mean ± SEM (* p
    Figure Legend Snippet: Abnormal aortic valve morphology in Krox20+/−;Nos3+/− mice. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Nos3+/− , Krox20+/− , Nos3+/− ; Krox20+/− and Nos3−/− mice. ( B – D ) H E images showing representative Nos3+/− and Krox20+/− with tri-leaflets aortic valve, and an example of Nos3+/− ; Krox20+/− aortic valve with 2 leaflets. Aortic valve leaflets of Nos3+/− ; Krox20+/− mice appear equal in size. ( E ) Real-time qPCR demonstrates a reduction of Nos3 at a transcriptional level in Nos3+/− ; Krox20+/− and Krox20−/− compared to wild-type embryos ( n = 5 for each genotype). qPCR experiments were performed in triplicate and expressed as mean ± SEM (* p

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction

    Endothelial nitric oxide synthase (Nos3) expression is altered in Krox20 deficient mice. ( A – F ) Immunohistochemistry showing Nos3 protein (green) in the aortic valve of wild-type (WT, A ), Nos3−/− ( B ), Krox20−/− ( C ), Nos3+/− ( D ), Krox20+/− ( E ), and Nos3+/− ; Krox20+/− ( F ) embryos at E18.5. Immunohistochemistry showing abundant expression of Nos3 in the valve endothelial cells (arrows). Note the reduction of Nos3 expression in the mesenchyme of Krox20−/− ( C ) aortic valve leaflets (arrowhead; compared C with A ). Scale bars: 100 μm.
    Figure Legend Snippet: Endothelial nitric oxide synthase (Nos3) expression is altered in Krox20 deficient mice. ( A – F ) Immunohistochemistry showing Nos3 protein (green) in the aortic valve of wild-type (WT, A ), Nos3−/− ( B ), Krox20−/− ( C ), Nos3+/− ( D ), Krox20+/− ( E ), and Nos3+/− ; Krox20+/− ( F ) embryos at E18.5. Immunohistochemistry showing abundant expression of Nos3 in the valve endothelial cells (arrows). Note the reduction of Nos3 expression in the mesenchyme of Krox20−/− ( C ) aortic valve leaflets (arrowhead; compared C with A ). Scale bars: 100 μm.

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry

    Modulation of gene expression in Krox20−/− embryos. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Krox20−/− , Tie2-cre;Krox20f/f , and Wnt1-cre;Krox20f/f mice. ( B , C ) Cross-sectional H E images through the aortic valve of Krox20+/+ ( B ) and Krox20−/− ( C ) littermate embryos. At E18.5, left and right-coronary leaflets are observed in BAV of Krox20−/− ( C ) embryos. ( D ) Real-time qPCR analyses were performed from isolated aortic valve of Krox20+/+ ( n = 5) and Krox20−/− ( n = 5) embryos at E18.5. qPCR showing normal levels of Alk2 , Gata5 , and Hey1 , and altered expression of Nos3 , Notch1 , and Hey2 in the aortic valve of Krox20−/− embryos at E18.5. qRT-PCR experiments were performed in triplicate and expressed as mean ±SEM (* p
    Figure Legend Snippet: Modulation of gene expression in Krox20−/− embryos. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Krox20−/− , Tie2-cre;Krox20f/f , and Wnt1-cre;Krox20f/f mice. ( B , C ) Cross-sectional H E images through the aortic valve of Krox20+/+ ( B ) and Krox20−/− ( C ) littermate embryos. At E18.5, left and right-coronary leaflets are observed in BAV of Krox20−/− ( C ) embryos. ( D ) Real-time qPCR analyses were performed from isolated aortic valve of Krox20+/+ ( n = 5) and Krox20−/− ( n = 5) embryos at E18.5. qPCR showing normal levels of Alk2 , Gata5 , and Hey1 , and altered expression of Nos3 , Notch1 , and Hey2 in the aortic valve of Krox20−/− embryos at E18.5. qRT-PCR experiments were performed in triplicate and expressed as mean ±SEM (* p

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

    Krox20 promotes the transcriptional activity of the Nos3 proximal promoter. ( A ) Schematic representation of the 1.5-kb Nos3 proximal promoters. The putative Krox20 binding sites located around −513 bp and −136 bp on Nos3 promoters are represented by black squares. Asterisk (*) indicates conservation of Krox20-binding sites with human and rat sequences. Numbers indicate position of the sequences from the ATG. ( B ) EMSA showing binding of Krox20 to the wild-type K1 (GTGTGGGAC) and K2 (GTGTGGGTT) motifs. Mutation of the K1 and K2 motif impairs Krox20 binding. ( C ) Relative luciferase activity in Cos7 cells transiently co-transfected with reporter constructs containing a 1544 bp and 265 bp regions of Nos3 promoter cloned upstream of the luciferase gene, 10 ng of control pGL4.74( hRluc /TK) vector and 50, 150, and 300 ng of CMV-Krox20 or CMV control expressing vectors. Transfection of CMV- Krox20 has a trans-activating effect on −1544 bp reporter. Mutation of the K2 motif abolishes the trans-activation of Krox20 on the −1544 bp reporter. Data is represented as a fold change in luciferase activity normalized to Renilla . ( D ) qPCR showing significant enrichment of DNA/Krox20 complexes on Krox20-binding sites within the Nos3 (regions A) proximal promoters following chromatin immunoprecipitation using dissected outflow tract (OFT) and left ventricle (LV) from E13.5 hearts. Anti-Histone H3 was used as positive control of immunoprecipitation. Primers used for qPCR correspond to Nos3 proximal promoter region ( Nos3-A , Nos3-B ) as indicated in ( A ). Primers in region without Krox20 binding sites (no binding site) were used as a negative control. Relative quantities of each chromatin bound fragment were normalized relative to the amount of input DNA. Note significant enrichment in region A within the Nos3 promoters. ( E ) Nos3 transcriptional level was quantified by qRT-PCR in rat AVICs, 24 h, 48 h, and 72 h after Krox20 transfection. Experiments were performed in triplicate and expressed as means ± SEM.
    Figure Legend Snippet: Krox20 promotes the transcriptional activity of the Nos3 proximal promoter. ( A ) Schematic representation of the 1.5-kb Nos3 proximal promoters. The putative Krox20 binding sites located around −513 bp and −136 bp on Nos3 promoters are represented by black squares. Asterisk (*) indicates conservation of Krox20-binding sites with human and rat sequences. Numbers indicate position of the sequences from the ATG. ( B ) EMSA showing binding of Krox20 to the wild-type K1 (GTGTGGGAC) and K2 (GTGTGGGTT) motifs. Mutation of the K1 and K2 motif impairs Krox20 binding. ( C ) Relative luciferase activity in Cos7 cells transiently co-transfected with reporter constructs containing a 1544 bp and 265 bp regions of Nos3 promoter cloned upstream of the luciferase gene, 10 ng of control pGL4.74( hRluc /TK) vector and 50, 150, and 300 ng of CMV-Krox20 or CMV control expressing vectors. Transfection of CMV- Krox20 has a trans-activating effect on −1544 bp reporter. Mutation of the K2 motif abolishes the trans-activation of Krox20 on the −1544 bp reporter. Data is represented as a fold change in luciferase activity normalized to Renilla . ( D ) qPCR showing significant enrichment of DNA/Krox20 complexes on Krox20-binding sites within the Nos3 (regions A) proximal promoters following chromatin immunoprecipitation using dissected outflow tract (OFT) and left ventricle (LV) from E13.5 hearts. Anti-Histone H3 was used as positive control of immunoprecipitation. Primers used for qPCR correspond to Nos3 proximal promoter region ( Nos3-A , Nos3-B ) as indicated in ( A ). Primers in region without Krox20 binding sites (no binding site) were used as a negative control. Relative quantities of each chromatin bound fragment were normalized relative to the amount of input DNA. Note significant enrichment in region A within the Nos3 promoters. ( E ) Nos3 transcriptional level was quantified by qRT-PCR in rat AVICs, 24 h, 48 h, and 72 h after Krox20 transfection. Experiments were performed in triplicate and expressed as means ± SEM.

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis, Luciferase, Transfection, Construct, Clone Assay, Plasmid Preparation, Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control, Immunoprecipitation, Negative Control, Quantitative RT-PCR

    Absence of Nos3 does not affect Krox20 contribution. ( A , B ) Embryos were harvested from Krox20 Cre/+ ;Rosa tdTomato mice at E18.5 and immunohistochemistry is performed to detect Tomato expression (red) as an indicator of recombination, while Pecam (green) identifies endothelial cells in the aortic valve. No major difference is observed between Nos3−/− and control littermate embryos. ( C – F ) X-gal staining was performed on −31/−23.5 Krox20/LacZ transgenic mice to follow the Krox20 -expressing cells at E9.5 ( C , D ), and E13.5 ( E,F ) stages. ( C , D ) At E9.5, β-galactosidase (β-gal) activity is detected in migratory neural crest cells. No defect is observed in Nos3−/− compared with control embryos (compared D with C ). ( E , F ) Transverse section through the outflow tract cushions at E13.5 showing β-gal-positive cells in the arterial valve leaflets. AoV, aortic valve; ba1, branchial arch 1; PV, pulmonary valve; NCC, neural crest cells. Scale bars: 100 μm ( A , B ); 50 μm ( C – F ).
    Figure Legend Snippet: Absence of Nos3 does not affect Krox20 contribution. ( A , B ) Embryos were harvested from Krox20 Cre/+ ;Rosa tdTomato mice at E18.5 and immunohistochemistry is performed to detect Tomato expression (red) as an indicator of recombination, while Pecam (green) identifies endothelial cells in the aortic valve. No major difference is observed between Nos3−/− and control littermate embryos. ( C – F ) X-gal staining was performed on −31/−23.5 Krox20/LacZ transgenic mice to follow the Krox20 -expressing cells at E9.5 ( C , D ), and E13.5 ( E,F ) stages. ( C , D ) At E9.5, β-galactosidase (β-gal) activity is detected in migratory neural crest cells. No defect is observed in Nos3−/− compared with control embryos (compared D with C ). ( E , F ) Transverse section through the outflow tract cushions at E13.5 showing β-gal-positive cells in the arterial valve leaflets. AoV, aortic valve; ba1, branchial arch 1; PV, pulmonary valve; NCC, neural crest cells. Scale bars: 100 μm ( A , B ); 50 μm ( C – F ).

    Techniques Used: Mouse Assay, Immunohistochemistry, Expressing, Staining, Transgenic Assay, Activity Assay

    19) Product Images from "Partial depletion of yolk during zebrafish embryogenesis changes the dynamics of methionine cycle and metabolic genes"

    Article Title: Partial depletion of yolk during zebrafish embryogenesis changes the dynamics of methionine cycle and metabolic genes

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1654-6

    Study design, developmental outcome and differential expression datasets of YD vs. SP embryos. a Study design. Transcriptome profiles were generated at 4 time-points until 48 hpf. The images represent YD and SP embryos at the given time points. YD embryos show a reduced yolk volume (stained by ORO and marked within dashed line) and size. b Survival of YD (25 % yolk reduction) and SP embryos during one week post-fertilization in YD (treated) and SP (control-punctured) embryos during 7 days after treatment is > 90 % in each group. c Average somite number from 11 hpf to 15 hpf in YD (24 % yolk reduction) and SP embryos. d Gender of adult fish, number of fish per treatment noted inside bars. A paired T -test for the three batches shows a significant increase in females. e Box-plot of differential expression (DE) levels at each time-point for genes that were significantly differentially expressed (DE genes). f Number of DE genes obtained at each time-point. SP: down-regulated in YD; YD: up-regulated in YD; ALL: all genes which passes expression level threshold and were included in this study (see methods for details); Ens: genes from microarray data with Ensembl identifier. Union indicates union of genes with Ensembl identifier
    Figure Legend Snippet: Study design, developmental outcome and differential expression datasets of YD vs. SP embryos. a Study design. Transcriptome profiles were generated at 4 time-points until 48 hpf. The images represent YD and SP embryos at the given time points. YD embryos show a reduced yolk volume (stained by ORO and marked within dashed line) and size. b Survival of YD (25 % yolk reduction) and SP embryos during one week post-fertilization in YD (treated) and SP (control-punctured) embryos during 7 days after treatment is > 90 % in each group. c Average somite number from 11 hpf to 15 hpf in YD (24 % yolk reduction) and SP embryos. d Gender of adult fish, number of fish per treatment noted inside bars. A paired T -test for the three batches shows a significant increase in females. e Box-plot of differential expression (DE) levels at each time-point for genes that were significantly differentially expressed (DE genes). f Number of DE genes obtained at each time-point. SP: down-regulated in YD; YD: up-regulated in YD; ALL: all genes which passes expression level threshold and were included in this study (see methods for details); Ens: genes from microarray data with Ensembl identifier. Union indicates union of genes with Ensembl identifier

    Techniques Used: Expressing, Generated, Staining, Fluorescence In Situ Hybridization, Microarray

    20) Product Images from "Nucleic acid polymer REP 2139 and nucleos(T)ide analogues act synergistically against chronic hepadnaviral infection in vivo in Pekin ducks"

    Article Title: Nucleic acid polymer REP 2139 and nucleos(T)ide analogues act synergistically against chronic hepadnaviral infection in vivo in Pekin ducks

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.29737

    Changes in serum DHBsAg and DHBV DNA during treatment and follow‐up. Mean ± SD of duplicate measurements are presented for serum DHBsAg (left column) and DHBV DNA (right column) taken at the start of treatment (4 weeks postinoculation), mid‐treatment (6 weeks postinoculation), end of treatment (8 weeks postinoculation), 4 weeks after discontinuing treatment (12 weeks postinoculation), and 8 weeks after discontinuing treatment (16 weeks postinoculation). Serum DHBsAg values have had background reactivity (from uninfected duck sera) removed. Lower limit of detection of DHBV DNA (3.30 × 10 3 vge/mL) is indicated by horizontal dashed lines. Individual sampling points where no detectable DHBV DNA was present were set to 3.30 × 10 3 vge/mL for analysis purposes. Individual ducks either establishing functional control in the NS group or rebounding in REP 2139 treatment groups are identified.
    Figure Legend Snippet: Changes in serum DHBsAg and DHBV DNA during treatment and follow‐up. Mean ± SD of duplicate measurements are presented for serum DHBsAg (left column) and DHBV DNA (right column) taken at the start of treatment (4 weeks postinoculation), mid‐treatment (6 weeks postinoculation), end of treatment (8 weeks postinoculation), 4 weeks after discontinuing treatment (12 weeks postinoculation), and 8 weeks after discontinuing treatment (16 weeks postinoculation). Serum DHBsAg values have had background reactivity (from uninfected duck sera) removed. Lower limit of detection of DHBV DNA (3.30 × 10 3 vge/mL) is indicated by horizontal dashed lines. Individual sampling points where no detectable DHBV DNA was present were set to 3.30 × 10 3 vge/mL for analysis purposes. Individual ducks either establishing functional control in the NS group or rebounding in REP 2139 treatment groups are identified.

    Techniques Used: Sampling, Functional Assay

    Liver DHBV DNA and cccDNA at the end of follow‐up. (A) Individual measurements for each animal are presented. Mean ± SD of duplicate measurements for each individual animal are presented for liver DHBV DNA (left column) and liver cccDNA (right column) taken from liver tissue harvested 8 weeks after discontinuing treatment. Lower limit of detection of 2.1 × 10 −5 vge/cell (for liver DHBV DNA) and 2.1 × 10 −5 copies/cell (for liver cccDNA) are indicated in (A) by horizontal dashed lines. (B) Group mean values are presented ± SD and P values for the differences between the means of the NS and treatment groups. Reductions in liver DHBV DNA (left) and cccDNA (right) relative to the NS group were significant ( P
    Figure Legend Snippet: Liver DHBV DNA and cccDNA at the end of follow‐up. (A) Individual measurements for each animal are presented. Mean ± SD of duplicate measurements for each individual animal are presented for liver DHBV DNA (left column) and liver cccDNA (right column) taken from liver tissue harvested 8 weeks after discontinuing treatment. Lower limit of detection of 2.1 × 10 −5 vge/cell (for liver DHBV DNA) and 2.1 × 10 −5 copies/cell (for liver cccDNA) are indicated in (A) by horizontal dashed lines. (B) Group mean values are presented ± SD and P values for the differences between the means of the NS and treatment groups. Reductions in liver DHBV DNA (left) and cccDNA (right) relative to the NS group were significant ( P

    Techniques Used:

    Detection of DHBV cccDNA by Southern blotting assay. DNA was isolated from approximately 300 mg of liver tissue using the Hirt extraction technique. DHBV DNA was detected using a radiolabeled RNA probe specific for the detection of the plus strand. Signals are from approximately 4.20E+06 liver cells. Controls of cloned linear (L) DHBV DNA, corresponding to 1.62E+07, 3.25E+07, 6.50E+07, and 1.30E+08 vge, were used for quantification by Phosphorimager. The cccDNA copy numbers for samples 936, 965, 940, 949, 959, 962, 964, and 981 were estimated at 1.6, 1.8, 1.8, 1.2, 1.1, 0.03, 1.0, and 0.01 vge/cell, respectively. To validate the authenticity of cccDNA, extract from sample 936 was digested with single cutter EcoR1 restriction enzyme resulting in conversion to linear (L) DHBV DNA. Abbreviations: CCC, covalently closed circular DNA; RC, relaxed circular DNA; L, linear DNA; MW, molecular weight.
    Figure Legend Snippet: Detection of DHBV cccDNA by Southern blotting assay. DNA was isolated from approximately 300 mg of liver tissue using the Hirt extraction technique. DHBV DNA was detected using a radiolabeled RNA probe specific for the detection of the plus strand. Signals are from approximately 4.20E+06 liver cells. Controls of cloned linear (L) DHBV DNA, corresponding to 1.62E+07, 3.25E+07, 6.50E+07, and 1.30E+08 vge, were used for quantification by Phosphorimager. The cccDNA copy numbers for samples 936, 965, 940, 949, 959, 962, 964, and 981 were estimated at 1.6, 1.8, 1.8, 1.2, 1.1, 0.03, 1.0, and 0.01 vge/cell, respectively. To validate the authenticity of cccDNA, extract from sample 936 was digested with single cutter EcoR1 restriction enzyme resulting in conversion to linear (L) DHBV DNA. Abbreviations: CCC, covalently closed circular DNA; RC, relaxed circular DNA; L, linear DNA; MW, molecular weight.

    Techniques Used: Southern Blot, Isolation, Clone Assay, Countercurrent Chromatography, Molecular Weight

    21) Product Images from "Autologous Cell Therapy Approach for Duchenne Muscular Dystrophy using PiggyBac Transposons and Mesoangioblasts"

    Article Title: Autologous Cell Therapy Approach for Duchenne Muscular Dystrophy using PiggyBac Transposons and Mesoangioblasts

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2018.01.021

    Characterization of mdx MABs Carrying Dystrophin- and GFP-Expressing PB Transposons (A) Phase contrast (left) and fluorescence microscopy (right) analysis of mdx (DYS GFP PB) MABs transfected with PB transposon harboring dystrophin and GFP expression cassettes and selected for puromycin resistance. Scale bar, 100 μm. (B) In vitro differentiation of the mdx (DYS GFP PB) MABs by co-culture in differentiation medium with a GFP-negative mouse myoblast (C2C12) cell line. GFP fluorescence (green), with myosin heavy-chain immunofluorescence of myotubes (red) and DAPI nuclear staining (blue, right), was performed after 5 days of culture in differentiation medium showing a multinucleated GFP- and MyHC-positive myotube. Scale bar, 50 μm. (C) Flow cytometry analysis for the presence of MAB cell surface markers on untransfected mdx MABs or transfected mdx (DYS PB) MABs populations, as indicated (n = 4). (D) Reverse transcriptase and quantitative real-time PCR (real-time qPCR) analysis of relative transcript levels for different MAB markers in mdx MABs and mdx (DYS PB) MABs. Data are represented as the mean ± SD of three independent experiments, each performed in triplicate (E). In vitro transmigration of MABs performed on endothelial cells (H5V cell line) cultured on a gelatin-coated filter. GFP-positive MABs were added on the upper surface of the filters, and after 11 hr, the MABs that had migrated on the other side of the filter were quantified. The number of mdx (DYS GFP PB) MABs and mdx (GFP PB) MABs normalized to the area is represented as the mean ± SD from three experiments, each performed in triplicate (p
    Figure Legend Snippet: Characterization of mdx MABs Carrying Dystrophin- and GFP-Expressing PB Transposons (A) Phase contrast (left) and fluorescence microscopy (right) analysis of mdx (DYS GFP PB) MABs transfected with PB transposon harboring dystrophin and GFP expression cassettes and selected for puromycin resistance. Scale bar, 100 μm. (B) In vitro differentiation of the mdx (DYS GFP PB) MABs by co-culture in differentiation medium with a GFP-negative mouse myoblast (C2C12) cell line. GFP fluorescence (green), with myosin heavy-chain immunofluorescence of myotubes (red) and DAPI nuclear staining (blue, right), was performed after 5 days of culture in differentiation medium showing a multinucleated GFP- and MyHC-positive myotube. Scale bar, 50 μm. (C) Flow cytometry analysis for the presence of MAB cell surface markers on untransfected mdx MABs or transfected mdx (DYS PB) MABs populations, as indicated (n = 4). (D) Reverse transcriptase and quantitative real-time PCR (real-time qPCR) analysis of relative transcript levels for different MAB markers in mdx MABs and mdx (DYS PB) MABs. Data are represented as the mean ± SD of three independent experiments, each performed in triplicate (E). In vitro transmigration of MABs performed on endothelial cells (H5V cell line) cultured on a gelatin-coated filter. GFP-positive MABs were added on the upper surface of the filters, and after 11 hr, the MABs that had migrated on the other side of the filter were quantified. The number of mdx (DYS GFP PB) MABs and mdx (GFP PB) MABs normalized to the area is represented as the mean ± SD from three experiments, each performed in triplicate (p

    Techniques Used: Expressing, Fluorescence, Microscopy, Transfection, In Vitro, Co-Culture Assay, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Transmigration Assay, Cell Culture

    22) Product Images from "CDNF rescues motor neurons in three animal models of ALS by targeting ER stress"

    Article Title: CDNF rescues motor neurons in three animal models of ALS by targeting ER stress

    Journal: bioRxiv

    doi: 10.1101/2020.05.05.078618

    CDNF protein administration increases the survival of SOD1-G93A mouse-derived embryonic MNs and reduces the expression of UPR markers after toxin treatment. ( A ) Survival of WT and SOD1-G93A embryonic MNs upon CDNF or CNTF and BDNF treatment. ( B ) Effect of CDNF administration on WT and SOD1 MNs survival upon 5 nM thapsigargin (TP) treatment or no treatment. ( C ) Dose-response curve of CDNF administration upon TP treatment on WT and SOD1-G93A MNs survival. ( D) Effect of CDNF on ATF6 translocation from cytoplasm to nucleus in MNs treated with TP or tunicamycin (TM). ( E ) Quantification of D . ( F ) mRNA expression of UPR markers Xbp1s in the MNs treated as in Figure B . ( G-I ) Protein expression of UPR markers CHOP and phosphorylation of eIF2α ( I ) in the MNs treated as in Figure D . Quantification of the blot ( G , H ). Mean ± SEM of 3 independent experiments in A, B and F , 4 experiments in C and 7 experiments in G-I *p
    Figure Legend Snippet: CDNF protein administration increases the survival of SOD1-G93A mouse-derived embryonic MNs and reduces the expression of UPR markers after toxin treatment. ( A ) Survival of WT and SOD1-G93A embryonic MNs upon CDNF or CNTF and BDNF treatment. ( B ) Effect of CDNF administration on WT and SOD1 MNs survival upon 5 nM thapsigargin (TP) treatment or no treatment. ( C ) Dose-response curve of CDNF administration upon TP treatment on WT and SOD1-G93A MNs survival. ( D) Effect of CDNF on ATF6 translocation from cytoplasm to nucleus in MNs treated with TP or tunicamycin (TM). ( E ) Quantification of D . ( F ) mRNA expression of UPR markers Xbp1s in the MNs treated as in Figure B . ( G-I ) Protein expression of UPR markers CHOP and phosphorylation of eIF2α ( I ) in the MNs treated as in Figure D . Quantification of the blot ( G , H ). Mean ± SEM of 3 independent experiments in A, B and F , 4 experiments in C and 7 experiments in G-I *p

    Techniques Used: Derivative Assay, Expressing, Translocation Assay

    CDNF treatment decreases UPR markers expression in TDP43-M337V expressing MNs. ( A-B ) Protein expression of UPR markers phosphorylated eIF2α and CHOP in the MNs expressing TDP43 WT and TDP43-M337V, which were previously stressed with thapsigargin (TP) and tunicamycin (TM). ( C-D ) Representative blots of p-eIF2 α and CHOP. Mean ± SEM of 5 different experiments in A-B . *p
    Figure Legend Snippet: CDNF treatment decreases UPR markers expression in TDP43-M337V expressing MNs. ( A-B ) Protein expression of UPR markers phosphorylated eIF2α and CHOP in the MNs expressing TDP43 WT and TDP43-M337V, which were previously stressed with thapsigargin (TP) and tunicamycin (TM). ( C-D ) Representative blots of p-eIF2 α and CHOP. Mean ± SEM of 5 different experiments in A-B . *p

    Techniques Used: Expressing

    CDNF treatment decreases UPR markers expression in a novel TDP43-M337V mouse model with ER stress pathology. ( A ) Schematic representation of the appearance of ER stress and MNs loss in the novel TDP43-M337V mouse model. ( B ) TDP43-M337V mice were injected at 6 weeks of age and UPR markers were analyzed by qPCR at 6 months. ( C-H ) mRNA expression of UPR markers Atf6, Atf4, Chop, Xbp1t, Xbp1s and Grp78 in microdissected lumbar MNs from 6 months TDP43-M337V mice treated with CDNF or vehicle at 6 weeks of age. ( I ) mRNA expression of UPR markers Atf6, Chop, Xbp1s and Grp78 in total lysates of motor cortex from 6 months TDP43-M337V mice treated with CDNF or vehicle at 6 weeks of age; results are presented as fold change increase compared to naïve control. Mean ± SEM, n=5/6/group in C-H , n=5/group in I . *p
    Figure Legend Snippet: CDNF treatment decreases UPR markers expression in a novel TDP43-M337V mouse model with ER stress pathology. ( A ) Schematic representation of the appearance of ER stress and MNs loss in the novel TDP43-M337V mouse model. ( B ) TDP43-M337V mice were injected at 6 weeks of age and UPR markers were analyzed by qPCR at 6 months. ( C-H ) mRNA expression of UPR markers Atf6, Atf4, Chop, Xbp1t, Xbp1s and Grp78 in microdissected lumbar MNs from 6 months TDP43-M337V mice treated with CDNF or vehicle at 6 weeks of age. ( I ) mRNA expression of UPR markers Atf6, Chop, Xbp1s and Grp78 in total lysates of motor cortex from 6 months TDP43-M337V mice treated with CDNF or vehicle at 6 weeks of age; results are presented as fold change increase compared to naïve control. Mean ± SEM, n=5/6/group in C-H , n=5/group in I . *p

    Techniques Used: Expressing, Mouse Assay, Injection, Real-time Polymerase Chain Reaction

    CDNF administration attenuates the expression of UPR markers in the spinal cord of ChAT-tTA/TRE-TDP43-M337V rats at 21 days after transgene activation. ( A ) Representative fluorescence images of ChAT (Green) and GRP78 (Red) protein expression in PBS/CDNF-treated ChAT-tTA/TRE-TDP43-M337V compared to WT controls. Scale bar 50 µm. ( B ) Quantification of the pixel value of GRP78 in the lumbar ventral horn area. ( C ) Quantification of the pixel value of GRP78 in the ChAT + MNs. ( D ) Representative fluorescence images of ChAT (Green) and p-PERK (Red) protein expression in PBS/CDNF-treated ChAT-tTA/TRE-TDP43-M337V compared to WT controls. Scale bar 50 µm. ( E ) Quantification the of pixel value of p-PERK in the lumbar ventral horn area. ( F ) Quantification of pixel value of GRP78 within ChAT + MNs. Mean ± SEM, n=5-8/group. *p
    Figure Legend Snippet: CDNF administration attenuates the expression of UPR markers in the spinal cord of ChAT-tTA/TRE-TDP43-M337V rats at 21 days after transgene activation. ( A ) Representative fluorescence images of ChAT (Green) and GRP78 (Red) protein expression in PBS/CDNF-treated ChAT-tTA/TRE-TDP43-M337V compared to WT controls. Scale bar 50 µm. ( B ) Quantification of the pixel value of GRP78 in the lumbar ventral horn area. ( C ) Quantification of the pixel value of GRP78 in the ChAT + MNs. ( D ) Representative fluorescence images of ChAT (Green) and p-PERK (Red) protein expression in PBS/CDNF-treated ChAT-tTA/TRE-TDP43-M337V compared to WT controls. Scale bar 50 µm. ( E ) Quantification the of pixel value of p-PERK in the lumbar ventral horn area. ( F ) Quantification of pixel value of GRP78 within ChAT + MNs. Mean ± SEM, n=5-8/group. *p

    Techniques Used: Expressing, Activation Assay, Fluorescence

    A single i.c.v. injection of CDNF decreases the expression of UPR markers in the lumbar spinal cord of SOD1-G93A animals at 17 weeks. ( A ) Representative pictures of a lumbar spinal cord section before and after the MNs dissection and mRNA level of ChAT , marker of spinal cord MNs, in the putative MNs area and in a control region where no MNs should be present (marked with *). ( B-F ) mRNA expression of UPR markers Atf6, Atf4, Chop, Xbp1t and Xbp1s in microdissected lumbar MNs after CDNF injection in 13 and 17 weeks SOD1-G93A mice and WT littermates. ( G ) Expression of GRP78 protein in lumbar MNs after CDNF treatment in 17 weeks SOD1-G93A mice and WT littermates. ( H ) Quantification of G . ( I ) Expression of p-eIF2α protein in lumbar MNs after CDNF treatment in 17 weeks SOD1-G93A mice. ( J ) Quantification of I . Mean ± SEM, n=6-9/group in B-F , n=3-5/group in F-J . *p
    Figure Legend Snippet: A single i.c.v. injection of CDNF decreases the expression of UPR markers in the lumbar spinal cord of SOD1-G93A animals at 17 weeks. ( A ) Representative pictures of a lumbar spinal cord section before and after the MNs dissection and mRNA level of ChAT , marker of spinal cord MNs, in the putative MNs area and in a control region where no MNs should be present (marked with *). ( B-F ) mRNA expression of UPR markers Atf6, Atf4, Chop, Xbp1t and Xbp1s in microdissected lumbar MNs after CDNF injection in 13 and 17 weeks SOD1-G93A mice and WT littermates. ( G ) Expression of GRP78 protein in lumbar MNs after CDNF treatment in 17 weeks SOD1-G93A mice and WT littermates. ( H ) Quantification of G . ( I ) Expression of p-eIF2α protein in lumbar MNs after CDNF treatment in 17 weeks SOD1-G93A mice. ( J ) Quantification of I . Mean ± SEM, n=6-9/group in B-F , n=3-5/group in F-J . *p

    Techniques Used: Injection, Expressing, Dissection, Marker, Mouse Assay

    23) Product Images from "Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells"

    Article Title: Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20100977

    DMF-induced HO-1 selectively prevents IL-23 induction. (A) DCs were treated with DMSO or 70 µM DMF, and HO-1 mRNA expression was determined by quantitative RT-PCR. HO-1 data were normalized to β-actin, and HO-1 level in control siRNA–transfected DMF–treated DCs was set as 1.0. The results are representative of three independent experiments. Error bars represent SEM. (B) HO-1 was knocked down, and levels of IL-12/IL-23p40, IL-23p19, or IL-12p35 mRNA were determined by RT-PCR. Data (mean ± SEM) were normalized to β-actin, and message levels in control siRNA–transfected DMF-treated DCs were set as 1.0. (C) DCs were treated as in A and lysed, and nuclear or cytoplasmic cell extracts were analyzed by Western blotting using antibodies directed against C- or N-terminal HO-1 protein. (D and E) DCs treated as in A were activated with LPS, cross-linked, and immunoprecipitated with anti–HO-1 (D) or anti-H3Ac (E). Bound DNA was amplified by quantitative PCR for primer sites P1 (AP-1; position 412–422 bp), P2 (c-Rel; position 560–584 bp), and P3 (RelA/c-Rel; position 394–406 bp). Data were pooled from four separate experiments and represent mean ± SEM (*, P
    Figure Legend Snippet: DMF-induced HO-1 selectively prevents IL-23 induction. (A) DCs were treated with DMSO or 70 µM DMF, and HO-1 mRNA expression was determined by quantitative RT-PCR. HO-1 data were normalized to β-actin, and HO-1 level in control siRNA–transfected DMF–treated DCs was set as 1.0. The results are representative of three independent experiments. Error bars represent SEM. (B) HO-1 was knocked down, and levels of IL-12/IL-23p40, IL-23p19, or IL-12p35 mRNA were determined by RT-PCR. Data (mean ± SEM) were normalized to β-actin, and message levels in control siRNA–transfected DMF-treated DCs were set as 1.0. (C) DCs were treated as in A and lysed, and nuclear or cytoplasmic cell extracts were analyzed by Western blotting using antibodies directed against C- or N-terminal HO-1 protein. (D and E) DCs treated as in A were activated with LPS, cross-linked, and immunoprecipitated with anti–HO-1 (D) or anti-H3Ac (E). Bound DNA was amplified by quantitative PCR for primer sites P1 (AP-1; position 412–422 bp), P2 (c-Rel; position 560–584 bp), and P3 (RelA/c-Rel; position 394–406 bp). Data were pooled from four separate experiments and represent mean ± SEM (*, P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    24) Product Images from "FOXM1 Upregulation Is an Early Event in Human Squamous Cell Carcinoma and it Is Enhanced by Nicotine during Malignant Transformation"

    Article Title: FOXM1 Upregulation Is an Early Event in Human Squamous Cell Carcinoma and it Is Enhanced by Nicotine during Malignant Transformation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004849

    Upregulation of FOXM1 in both human oral premalignant and HNSCC tissues. (A) Semi-quantitative RT-PCR showing the relative expression levels of total FOXM1 mRNA in normal oral mucosa (NOM; #1–2), moderate dysplasia (MD; #3–4), severe dysplasia (SD; #5–6), primary HNSCC (#7–8), premalignant oral keratinocytes SVpgC2a, HNSCC cell lines SqCC/Y1 and SCC25. POLR2A was used as an endogenous reference gene. (B) Bioinformatics analysis of microarray data performed on primary cells extracted from normal oral mucosa (NOM), leukoplakia, erythroplakia and primary HNSCC with sample number as indicated. Statistically significant (***P
    Figure Legend Snippet: Upregulation of FOXM1 in both human oral premalignant and HNSCC tissues. (A) Semi-quantitative RT-PCR showing the relative expression levels of total FOXM1 mRNA in normal oral mucosa (NOM; #1–2), moderate dysplasia (MD; #3–4), severe dysplasia (SD; #5–6), primary HNSCC (#7–8), premalignant oral keratinocytes SVpgC2a, HNSCC cell lines SqCC/Y1 and SCC25. POLR2A was used as an endogenous reference gene. (B) Bioinformatics analysis of microarray data performed on primary cells extracted from normal oral mucosa (NOM), leukoplakia, erythroplakia and primary HNSCC with sample number as indicated. Statistically significant (***P

    Techniques Used: Quantitative RT-PCR, Expressing, Microarray

    25) Product Images from "Leprosy at the edge of Europe—Biomolecular, isotopic and osteoarchaeological findings from medieval Ireland"

    Article Title: Leprosy at the edge of Europe—Biomolecular, isotopic and osteoarchaeological findings from medieval Ireland

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209495

    Amplification plots of the multi-copy PCR target RLEP reported with a specific FAM-labelled fluorogenic probe. Products were generated from extracts prepared from SkCXCV (left fibula, blue traces) and SkCCXXX from two extracts (palate, green traces) and first phalanx (fuchsia pink trace). SkCXLVIII was negative using this PCR method, as were the two template blanks run in parallel (black traces).
    Figure Legend Snippet: Amplification plots of the multi-copy PCR target RLEP reported with a specific FAM-labelled fluorogenic probe. Products were generated from extracts prepared from SkCXCV (left fibula, blue traces) and SkCCXXX from two extracts (palate, green traces) and first phalanx (fuchsia pink trace). SkCXLVIII was negative using this PCR method, as were the two template blanks run in parallel (black traces).

    Techniques Used: Amplification, Polymerase Chain Reaction, Generated

    26) Product Images from "Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae, et al. Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae"

    Article Title: Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae, et al. Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae

    Journal: Evolutionary Applications

    doi: 10.1111/eva.12619

    Duplication structure and primer positions of the internal deletion test ( ID test). (a) Amplicon structure. The whole amplicon is represented by the box. The predicted genes are represented by gray dots, except for ace‐1 , which is indicated by the black line (see Assogba et al., 2016 for details). The white box represents the area deleted in some amplicons, that is, the internal deletion ( ID ). The blue arrows represent the ID test primers positions. (b) PCR results of the ID test for different genotypes ( NB : This image has been produced by merging two parts of a single photograph). Only those containing an R x * copy are amplified ([ ID +]). M is the size marker
    Figure Legend Snippet: Duplication structure and primer positions of the internal deletion test ( ID test). (a) Amplicon structure. The whole amplicon is represented by the box. The predicted genes are represented by gray dots, except for ace‐1 , which is indicated by the black line (see Assogba et al., 2016 for details). The white box represents the area deleted in some amplicons, that is, the internal deletion ( ID ). The blue arrows represent the ID test primers positions. (b) PCR results of the ID test for different genotypes ( NB : This image has been produced by merging two parts of a single photograph). Only those containing an R x * copy are amplified ([ ID +]). M is the size marker

    Techniques Used: Amplification, Polymerase Chain Reaction, Produced, Marker

    27) Product Images from "The Effect of Deoxycholic Acid on Secretion and Motility in the Rat and Guinea Pig Large Intestine"

    Article Title: The Effect of Deoxycholic Acid on Secretion and Motility in the Rat and Guinea Pig Large Intestine

    Journal: Journal of Neurogastroenterology and Motility

    doi: 10.5056/jnm16201

    Expression levels of G protein-coupled bile acid receptor 1 (GpBAR1) between proximal and distal colon. The distribution of GpBAR1 was similar in both colon segments (n = 5).
    Figure Legend Snippet: Expression levels of G protein-coupled bile acid receptor 1 (GpBAR1) between proximal and distal colon. The distribution of GpBAR1 was similar in both colon segments (n = 5).

    Techniques Used: Expressing

    28) Product Images from "The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein"

    Article Title: The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006937

    OASL RNA binding and ubiquitin-like domains are not required for interaction with ORF20. (A) Multiple OASL constructs were utilized, including WT OASL and OASL mutants, including ΔUBL lacking the ubiquitin-like domain, the P-loop mutants V67G and N72K, the RNA binding mutants R45E/K66E/R196E/K200E (RKRK), K63E, and K66E, and the catalytic triad mutants, E81A, E83A, and T152A. (B) 293T cells were transfected with myc-tagged ORF20WT or LacZ, V5-tagged OAS1, WT or mutant OASL, and/or empty vector (EV) as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of at least three independent experiments. Similar results were obtained with simultaneous and sequential antibody exposures. (C) The amino acid sequence for ORF20B is shown, with the predicted nuclear localization sequence indicated in red, the predicted nucleolar localization sequence indicated in green, and predicted disordered regions underlined. The predictions formed the basis for three ORF20B C-terminal truncation mutants: ORF20B 1–186, 1–220, and 1–235. (D) 293T cells were transfected with myc-tagged ORF20 isoforms, ORF20B mutants, or LacZ, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed. Input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of three independent experiments.
    Figure Legend Snippet: OASL RNA binding and ubiquitin-like domains are not required for interaction with ORF20. (A) Multiple OASL constructs were utilized, including WT OASL and OASL mutants, including ΔUBL lacking the ubiquitin-like domain, the P-loop mutants V67G and N72K, the RNA binding mutants R45E/K66E/R196E/K200E (RKRK), K63E, and K66E, and the catalytic triad mutants, E81A, E83A, and T152A. (B) 293T cells were transfected with myc-tagged ORF20WT or LacZ, V5-tagged OAS1, WT or mutant OASL, and/or empty vector (EV) as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of at least three independent experiments. Similar results were obtained with simultaneous and sequential antibody exposures. (C) The amino acid sequence for ORF20B is shown, with the predicted nuclear localization sequence indicated in red, the predicted nucleolar localization sequence indicated in green, and predicted disordered regions underlined. The predictions formed the basis for three ORF20B C-terminal truncation mutants: ORF20B 1–186, 1–220, and 1–235. (D) 293T cells were transfected with myc-tagged ORF20 isoforms, ORF20B mutants, or LacZ, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed. Input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of three independent experiments.

    Techniques Used: RNA Binding Assay, Construct, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Western Blot, Sequencing

    ORF20 does not affect translation rates, but enhances expression of endogenous OASL. (A) 293T cells were co-transfected with EV, myc-tagged ORF20WT, ORF20FL, or ORF20B, and either EV or RIG-I N. 24 h post transfection, cells were treated for 15 minutes with 5μg/ml puromycin, then lysed and analyzed by immunoblotting. Anti-puromycin, anti-OASL, anti-myc, and anti-actin immunoblotting was performed sequentially. Data are representative of two independent experiments. *: Nonspecific background band. (B) 293T cells were co-transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (C) HEK293 cells were transfected with either EV or ORF20WT-myc. 24h later, cells were transfected with 5’pppRNA complexed with Lipofectamine 2000 for approximately 24h. (D) 293T cells were reverse transfected with control, IRF3, IFNAR, or STAT1 siRNAs as indicated. 48h later, cells were transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (B, C, D) Cells were lysed, RNA was isolated, mRNA-specific cDNA was reverse transcribed, and the amount of OASL was determined using relative quantification to GAPDH levels and the 2 -ΔΔ C T method. Data shown are means + SD of combined duplicates from at least two experiments. ns, not significant, * P
    Figure Legend Snippet: ORF20 does not affect translation rates, but enhances expression of endogenous OASL. (A) 293T cells were co-transfected with EV, myc-tagged ORF20WT, ORF20FL, or ORF20B, and either EV or RIG-I N. 24 h post transfection, cells were treated for 15 minutes with 5μg/ml puromycin, then lysed and analyzed by immunoblotting. Anti-puromycin, anti-OASL, anti-myc, and anti-actin immunoblotting was performed sequentially. Data are representative of two independent experiments. *: Nonspecific background band. (B) 293T cells were co-transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (C) HEK293 cells were transfected with either EV or ORF20WT-myc. 24h later, cells were transfected with 5’pppRNA complexed with Lipofectamine 2000 for approximately 24h. (D) 293T cells were reverse transfected with control, IRF3, IFNAR, or STAT1 siRNAs as indicated. 48h later, cells were transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (B, C, D) Cells were lysed, RNA was isolated, mRNA-specific cDNA was reverse transcribed, and the amount of OASL was determined using relative quantification to GAPDH levels and the 2 -ΔΔ C T method. Data shown are means + SD of combined duplicates from at least two experiments. ns, not significant, * P

    Techniques Used: Expressing, Transfection, Isolation

    29) Product Images from "Correlation between ERK1 and STAT3 expression and chemoresistance in patients with conventional osteosarcoma"

    Article Title: Correlation between ERK1 and STAT3 expression and chemoresistance in patients with conventional osteosarcoma

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-606

    RTQ-PCR analysis of STAT3 and ERK1 genes. A : Quantification of STAT3 mRNA with 18S rRNA reference gene transcript confirmed higher STAT3 mRNA levels in poor responder (PR) samples compared with good responder (GR) samples (p = 0.019). B : Quantification of MAPK3 (ERK1) mRNA with 18S rRNA reference gene transcript confirmed higher MAPK3 (ERK1) mRNA levels in PR samples compared with GR samples (p = 0.046).
    Figure Legend Snippet: RTQ-PCR analysis of STAT3 and ERK1 genes. A : Quantification of STAT3 mRNA with 18S rRNA reference gene transcript confirmed higher STAT3 mRNA levels in poor responder (PR) samples compared with good responder (GR) samples (p = 0.019). B : Quantification of MAPK3 (ERK1) mRNA with 18S rRNA reference gene transcript confirmed higher MAPK3 (ERK1) mRNA levels in PR samples compared with GR samples (p = 0.046).

    Techniques Used: Polymerase Chain Reaction

    30) Product Images from "Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis"

    Article Title: Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00940-17

    PZA susceptibility testing of strains overexpressing Rv0191 , Rv3756c , Rv3008 , and Rv1667c . (A) pOLYG vector control. (B) pOLYG- Rv0191 . (C) pOLYG- Rv3756c . (D) pOLYG- Rv3008 . (E) pOLYG- Rv1667c . (F) pOLYG- Rv2535c .
    Figure Legend Snippet: PZA susceptibility testing of strains overexpressing Rv0191 , Rv3756c , Rv3008 , and Rv1667c . (A) pOLYG vector control. (B) pOLYG- Rv0191 . (C) pOLYG- Rv3756c . (D) pOLYG- Rv3008 . (E) pOLYG- Rv1667c . (F) pOLYG- Rv2535c .

    Techniques Used: Plasmid Preparation

    POA susceptibility testing of strains overexpressing Rv0191 , Rv3756c , Rv3008 , and Rv1667c . (A) pOLYG vector control. (B) pOLYG- Rv0191 . (C) pOLYG- Rv3756c . (D) pOLYG- Rv3008 . (E) pOLYG- Rv1667c .
    Figure Legend Snippet: POA susceptibility testing of strains overexpressing Rv0191 , Rv3756c , Rv3008 , and Rv1667c . (A) pOLYG vector control. (B) pOLYG- Rv0191 . (C) pOLYG- Rv3756c . (D) pOLYG- Rv3008 . (E) pOLYG- Rv1667c .

    Techniques Used: Plasmid Preparation

    Efflux inhibitors decreased PZA resistance caused by overexpression of Rv0191 , Rv3756c , Rv3008 , and Rv1667c . (A) PZA plus resperine. (B) PZA plus piperine. (C) PZA plus verapamil.
    Figure Legend Snippet: Efflux inhibitors decreased PZA resistance caused by overexpression of Rv0191 , Rv3756c , Rv3008 , and Rv1667c . (A) PZA plus resperine. (B) PZA plus piperine. (C) PZA plus verapamil.

    Techniques Used: Over Expression

    31) Product Images from "Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease"

    Article Title: Krox20 Regulates Endothelial Nitric Oxide Signaling in Aortic Valve Development and Disease

    Journal: Journal of Cardiovascular Development and Disease

    doi: 10.3390/jcdd6040039

    Abnormal aortic valve morphology in Krox20+/−;Nos3+/− mice. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Nos3+/− , Krox20+/− , Nos3+/− ; Krox20+/− and Nos3−/− mice. ( B – D ) H E images showing representative Nos3+/− and Krox20+/− with tri-leaflets aortic valve, and an example of Nos3+/− ; Krox20+/− aortic valve with 2 leaflets. Aortic valve leaflets of Nos3+/− ; Krox20+/− mice appear equal in size. ( E ) Real-time qPCR demonstrates a reduction of Nos3 at a transcriptional level in Nos3+/− ; Krox20+/− and Krox20−/− compared to wild-type embryos ( n = 5 for each genotype). qPCR experiments were performed in triplicate and expressed as mean ± SEM (* p
    Figure Legend Snippet: Abnormal aortic valve morphology in Krox20+/−;Nos3+/− mice. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Nos3+/− , Krox20+/− , Nos3+/− ; Krox20+/− and Nos3−/− mice. ( B – D ) H E images showing representative Nos3+/− and Krox20+/− with tri-leaflets aortic valve, and an example of Nos3+/− ; Krox20+/− aortic valve with 2 leaflets. Aortic valve leaflets of Nos3+/− ; Krox20+/− mice appear equal in size. ( E ) Real-time qPCR demonstrates a reduction of Nos3 at a transcriptional level in Nos3+/− ; Krox20+/− and Krox20−/− compared to wild-type embryos ( n = 5 for each genotype). qPCR experiments were performed in triplicate and expressed as mean ± SEM (* p

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction

    Endothelial nitric oxide synthase (Nos3) expression is altered in Krox20 deficient mice. ( A – F ) Immunohistochemistry showing Nos3 protein (green) in the aortic valve of wild-type (WT, A ), Nos3−/− ( B ), Krox20−/− ( C ), Nos3+/− ( D ), Krox20+/− ( E ), and Nos3+/− ; Krox20+/− ( F ) embryos at E18.5. Immunohistochemistry showing abundant expression of Nos3 in the valve endothelial cells (arrows). Note the reduction of Nos3 expression in the mesenchyme of Krox20−/− ( C ) aortic valve leaflets (arrowhead; compared C with A ). Scale bars: 100 μm.
    Figure Legend Snippet: Endothelial nitric oxide synthase (Nos3) expression is altered in Krox20 deficient mice. ( A – F ) Immunohistochemistry showing Nos3 protein (green) in the aortic valve of wild-type (WT, A ), Nos3−/− ( B ), Krox20−/− ( C ), Nos3+/− ( D ), Krox20+/− ( E ), and Nos3+/− ; Krox20+/− ( F ) embryos at E18.5. Immunohistochemistry showing abundant expression of Nos3 in the valve endothelial cells (arrows). Note the reduction of Nos3 expression in the mesenchyme of Krox20−/− ( C ) aortic valve leaflets (arrowhead; compared C with A ). Scale bars: 100 μm.

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry

    Modulation of gene expression in Krox20−/− embryos. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Krox20−/− , Tie2-cre;Krox20f/f , and Wnt1-cre;Krox20f/f mice. ( B , C ) Cross-sectional H E images through the aortic valve of Krox20+/+ ( B ) and Krox20−/− ( C ) littermate embryos. At E18.5, left and right-coronary leaflets are observed in BAV of Krox20−/− ( C ) embryos. ( D ) Real-time qPCR analyses were performed from isolated aortic valve of Krox20+/+ ( n = 5) and Krox20−/− ( n = 5) embryos at E18.5. qPCR showing normal levels of Alk2 , Gata5 , and Hey1 , and altered expression of Nos3 , Notch1 , and Hey2 in the aortic valve of Krox20−/− embryos at E18.5. qRT-PCR experiments were performed in triplicate and expressed as mean ±SEM (* p
    Figure Legend Snippet: Modulation of gene expression in Krox20−/− embryos. ( A ) Table depicting penetrance of bicuspid aortic valve (BAV) in Krox20−/− , Tie2-cre;Krox20f/f , and Wnt1-cre;Krox20f/f mice. ( B , C ) Cross-sectional H E images through the aortic valve of Krox20+/+ ( B ) and Krox20−/− ( C ) littermate embryos. At E18.5, left and right-coronary leaflets are observed in BAV of Krox20−/− ( C ) embryos. ( D ) Real-time qPCR analyses were performed from isolated aortic valve of Krox20+/+ ( n = 5) and Krox20−/− ( n = 5) embryos at E18.5. qPCR showing normal levels of Alk2 , Gata5 , and Hey1 , and altered expression of Nos3 , Notch1 , and Hey2 in the aortic valve of Krox20−/− embryos at E18.5. qRT-PCR experiments were performed in triplicate and expressed as mean ±SEM (* p

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

    Krox20 promotes the transcriptional activity of the Nos3 proximal promoter. ( A ) Schematic representation of the 1.5-kb Nos3 proximal promoters. The putative Krox20 binding sites located around −513 bp and −136 bp on Nos3 promoters are represented by black squares. Asterisk (*) indicates conservation of Krox20-binding sites with human and rat sequences. Numbers indicate position of the sequences from the ATG. ( B ) EMSA showing binding of Krox20 to the wild-type K1 (GTGTGGGAC) and K2 (GTGTGGGTT) motifs. Mutation of the K1 and K2 motif impairs Krox20 binding. ( C ) Relative luciferase activity in Cos7 cells transiently co-transfected with reporter constructs containing a 1544 bp and 265 bp regions of Nos3 promoter cloned upstream of the luciferase gene, 10 ng of control pGL4.74( hRluc /TK) vector and 50, 150, and 300 ng of CMV-Krox20 or CMV control expressing vectors. Transfection of CMV- Krox20 has a trans-activating effect on −1544 bp reporter. Mutation of the K2 motif abolishes the trans-activation of Krox20 on the −1544 bp reporter. Data is represented as a fold change in luciferase activity normalized to Renilla . ( D ) qPCR showing significant enrichment of DNA/Krox20 complexes on Krox20-binding sites within the Nos3 (regions A) proximal promoters following chromatin immunoprecipitation using dissected outflow tract (OFT) and left ventricle (LV) from E13.5 hearts. Anti-Histone H3 was used as positive control of immunoprecipitation. Primers used for qPCR correspond to Nos3 proximal promoter region ( Nos3-A , Nos3-B ) as indicated in ( A ). Primers in region without Krox20 binding sites (no binding site) were used as a negative control. Relative quantities of each chromatin bound fragment were normalized relative to the amount of input DNA. Note significant enrichment in region A within the Nos3 promoters. ( E ) Nos3 transcriptional level was quantified by qRT-PCR in rat AVICs, 24 h, 48 h, and 72 h after Krox20 transfection. Experiments were performed in triplicate and expressed as means ± SEM.
    Figure Legend Snippet: Krox20 promotes the transcriptional activity of the Nos3 proximal promoter. ( A ) Schematic representation of the 1.5-kb Nos3 proximal promoters. The putative Krox20 binding sites located around −513 bp and −136 bp on Nos3 promoters are represented by black squares. Asterisk (*) indicates conservation of Krox20-binding sites with human and rat sequences. Numbers indicate position of the sequences from the ATG. ( B ) EMSA showing binding of Krox20 to the wild-type K1 (GTGTGGGAC) and K2 (GTGTGGGTT) motifs. Mutation of the K1 and K2 motif impairs Krox20 binding. ( C ) Relative luciferase activity in Cos7 cells transiently co-transfected with reporter constructs containing a 1544 bp and 265 bp regions of Nos3 promoter cloned upstream of the luciferase gene, 10 ng of control pGL4.74( hRluc /TK) vector and 50, 150, and 300 ng of CMV-Krox20 or CMV control expressing vectors. Transfection of CMV- Krox20 has a trans-activating effect on −1544 bp reporter. Mutation of the K2 motif abolishes the trans-activation of Krox20 on the −1544 bp reporter. Data is represented as a fold change in luciferase activity normalized to Renilla . ( D ) qPCR showing significant enrichment of DNA/Krox20 complexes on Krox20-binding sites within the Nos3 (regions A) proximal promoters following chromatin immunoprecipitation using dissected outflow tract (OFT) and left ventricle (LV) from E13.5 hearts. Anti-Histone H3 was used as positive control of immunoprecipitation. Primers used for qPCR correspond to Nos3 proximal promoter region ( Nos3-A , Nos3-B ) as indicated in ( A ). Primers in region without Krox20 binding sites (no binding site) were used as a negative control. Relative quantities of each chromatin bound fragment were normalized relative to the amount of input DNA. Note significant enrichment in region A within the Nos3 promoters. ( E ) Nos3 transcriptional level was quantified by qRT-PCR in rat AVICs, 24 h, 48 h, and 72 h after Krox20 transfection. Experiments were performed in triplicate and expressed as means ± SEM.

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis, Luciferase, Transfection, Construct, Clone Assay, Plasmid Preparation, Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control, Immunoprecipitation, Negative Control, Quantitative RT-PCR

    Absence of Nos3 does not affect Krox20 contribution. ( A , B ) Embryos were harvested from Krox20 Cre/+ ;Rosa tdTomato mice at E18.5 and immunohistochemistry is performed to detect Tomato expression (red) as an indicator of recombination, while Pecam (green) identifies endothelial cells in the aortic valve. No major difference is observed between Nos3−/− and control littermate embryos. ( C – F ) X-gal staining was performed on −31/−23.5 Krox20/LacZ transgenic mice to follow the Krox20 -expressing cells at E9.5 ( C , D ), and E13.5 ( E,F ) stages. ( C , D ) At E9.5, β-galactosidase (β-gal) activity is detected in migratory neural crest cells. No defect is observed in Nos3−/− compared with control embryos (compared D with C ). ( E , F ) Transverse section through the outflow tract cushions at E13.5 showing β-gal-positive cells in the arterial valve leaflets. AoV, aortic valve; ba1, branchial arch 1; PV, pulmonary valve; NCC, neural crest cells. Scale bars: 100 μm ( A , B ); 50 μm ( C – F ).
    Figure Legend Snippet: Absence of Nos3 does not affect Krox20 contribution. ( A , B ) Embryos were harvested from Krox20 Cre/+ ;Rosa tdTomato mice at E18.5 and immunohistochemistry is performed to detect Tomato expression (red) as an indicator of recombination, while Pecam (green) identifies endothelial cells in the aortic valve. No major difference is observed between Nos3−/− and control littermate embryos. ( C – F ) X-gal staining was performed on −31/−23.5 Krox20/LacZ transgenic mice to follow the Krox20 -expressing cells at E9.5 ( C , D ), and E13.5 ( E,F ) stages. ( C , D ) At E9.5, β-galactosidase (β-gal) activity is detected in migratory neural crest cells. No defect is observed in Nos3−/− compared with control embryos (compared D with C ). ( E , F ) Transverse section through the outflow tract cushions at E13.5 showing β-gal-positive cells in the arterial valve leaflets. AoV, aortic valve; ba1, branchial arch 1; PV, pulmonary valve; NCC, neural crest cells. Scale bars: 100 μm ( A , B ); 50 μm ( C – F ).

    Techniques Used: Mouse Assay, Immunohistochemistry, Expressing, Staining, Transgenic Assay, Activity Assay

    32) Product Images from "Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis"

    Article Title: Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis

    Journal: Nature

    doi: 10.1038/nature13322

    A20 inhibits Nlrp3 inflammasome priming a, b, Nlrp3 (a) and proIL-1β (b) mRNA levels of LPS-treated BMDMs. c, Expression of the indicated proteins in BMDMs 6 h after LPS treatment. d, e, Rapid Nlrp3 inflammasome activation as described in Methods . Expression of the indicated proteins (d), and secreted cytokines (e) were determined. f-i, A20 myel-KO BMDMs were treated as indicated. Nlrp3 mRNA levels (f), caspase-1 expression (g), secreted IL-1β (h) and LDH activity (i) were determined. Black arrow, procaspase-1; white arrow, p20. Data represent mean ± s.d. of 1 out of 3 biological replicates, with 3 technical replicates each (* P
    Figure Legend Snippet: A20 inhibits Nlrp3 inflammasome priming a, b, Nlrp3 (a) and proIL-1β (b) mRNA levels of LPS-treated BMDMs. c, Expression of the indicated proteins in BMDMs 6 h after LPS treatment. d, e, Rapid Nlrp3 inflammasome activation as described in Methods . Expression of the indicated proteins (d), and secreted cytokines (e) were determined. f-i, A20 myel-KO BMDMs were treated as indicated. Nlrp3 mRNA levels (f), caspase-1 expression (g), secreted IL-1β (h) and LDH activity (i) were determined. Black arrow, procaspase-1; white arrow, p20. Data represent mean ± s.d. of 1 out of 3 biological replicates, with 3 technical replicates each (* P

    Techniques Used: Expressing, Activation Assay, Activity Assay

    33) Product Images from "Cytidine-phosphate-guanosine oligodeoxynucleotides in combination with CD40 ligand decrease periodontal inflammation and alveolar bone loss in a TLR9-independent manner"

    Article Title: Cytidine-phosphate-guanosine oligodeoxynucleotides in combination with CD40 ligand decrease periodontal inflammation and alveolar bone loss in a TLR9-independent manner

    Journal: Journal of Applied Oral Science

    doi: 10.1590/1678-7757-2017-0451

    CpG+CD40L increased gingival mRNA levels of IL-10 and decreased gingival mRNA levels of RANKL and IFN-γ in ligature-induced experimental periodontitis of WT and TLR9 KO mice. Silk ligatures were tied around the maxillary secondary molars on day 0 and injection of CpG (1 μM)+CD40L (0.1 μg/ml) or PBS was performed in palatal gingiva on days 3, 6 and 9 in WT mice and TLR9 KO mice. The levels of mRNA expression for IL-10 (A), OPG (B), RANKL (C), IL-1β (D), TNF-α (E), IFN-γ (F) in gingival tissues were detected by qRT-PCR and their relative levels of expression were normalized to the level of GAPDH expression (means±SD, n=4 WT or TLR9 KO mice per group, *p
    Figure Legend Snippet: CpG+CD40L increased gingival mRNA levels of IL-10 and decreased gingival mRNA levels of RANKL and IFN-γ in ligature-induced experimental periodontitis of WT and TLR9 KO mice. Silk ligatures were tied around the maxillary secondary molars on day 0 and injection of CpG (1 μM)+CD40L (0.1 μg/ml) or PBS was performed in palatal gingiva on days 3, 6 and 9 in WT mice and TLR9 KO mice. The levels of mRNA expression for IL-10 (A), OPG (B), RANKL (C), IL-1β (D), TNF-α (E), IFN-γ (F) in gingival tissues were detected by qRT-PCR and their relative levels of expression were normalized to the level of GAPDH expression (means±SD, n=4 WT or TLR9 KO mice per group, *p

    Techniques Used: Mouse Assay, Injection, Expressing, Quantitative RT-PCR

    34) Product Images from "In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method"

    Article Title: In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118841

    Real-time quantitative PCR analysis of albumin and CYP1A1 gene expression in cultured hepatocytes. Panel A shows relative transcript level of albumin and, Panel B shows CYP1A1 relative transcript level expressed as fold change.
    Figure Legend Snippet: Real-time quantitative PCR analysis of albumin and CYP1A1 gene expression in cultured hepatocytes. Panel A shows relative transcript level of albumin and, Panel B shows CYP1A1 relative transcript level expressed as fold change.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Cell Culture

    Agarose gel electrophoresis of RT-PCR products of hepatocyte-specific marker genes expressed in 5 days old cultured hepatocytes. Panel A shows 293 bp amplicon of albumin; B—130 bp amplicon of hepatocyte nuclear factor 4α; C—240 bp amplicon of glucose-6-phosphatase; D—136 bp amplicon of CYP1A1; E—164 bp amplicon of CYP3A4; F—142 bp amplicon of tyrosine aminotransferase. Lane 1: 100 bp ladder; Lane 2: RT-PCR of liver tissue (positive control) by using the gene-specific primers; Lane 3: RT-PCR of respective genes from cultured buffalo hepatocytes; Lane 4: RT-PCR from skin fibroblasts (negative control). Amplification of Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) was used as housekeeping gene.
    Figure Legend Snippet: Agarose gel electrophoresis of RT-PCR products of hepatocyte-specific marker genes expressed in 5 days old cultured hepatocytes. Panel A shows 293 bp amplicon of albumin; B—130 bp amplicon of hepatocyte nuclear factor 4α; C—240 bp amplicon of glucose-6-phosphatase; D—136 bp amplicon of CYP1A1; E—164 bp amplicon of CYP3A4; F—142 bp amplicon of tyrosine aminotransferase. Lane 1: 100 bp ladder; Lane 2: RT-PCR of liver tissue (positive control) by using the gene-specific primers; Lane 3: RT-PCR of respective genes from cultured buffalo hepatocytes; Lane 4: RT-PCR from skin fibroblasts (negative control). Amplification of Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) was used as housekeeping gene.

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Marker, Cell Culture, Amplification, Positive Control, Negative Control

    35) Product Images from "Identification of Herpesvirus Proteins That Contribute to G1/S Arrest"

    Article Title: Identification of Herpesvirus Proteins That Contribute to G1/S Arrest

    Journal: Journal of Virology

    doi: 10.1128/JVI.00059-14

    BGLF2 induces p21 protein levels in a p53-independent manner. (A) Fucci cells were transfected with pMZS3F or pMZS3F-BGLF2 and, 24 h later, cell lysates were analyzed by Western blotting for p53, phosphorylated p53 [p-p53 (ser15)], and p21, using actin as a loading control. (B and C) H1299 p53-null cells were transfected as described for panel A and then lysates were Western blotted for p21 (B) or EAPP (C) in addition to FLAG-BGLF2 and actin. CNE2Z (D) and H1299 (E) cells were transfected as described above, and then p21 transcript levels were determined by quantitative PCR and normalized to GAPDH. Average values with standard deviations are shown from three independent experiments.
    Figure Legend Snippet: BGLF2 induces p21 protein levels in a p53-independent manner. (A) Fucci cells were transfected with pMZS3F or pMZS3F-BGLF2 and, 24 h later, cell lysates were analyzed by Western blotting for p53, phosphorylated p53 [p-p53 (ser15)], and p21, using actin as a loading control. (B and C) H1299 p53-null cells were transfected as described for panel A and then lysates were Western blotted for p21 (B) or EAPP (C) in addition to FLAG-BGLF2 and actin. CNE2Z (D) and H1299 (E) cells were transfected as described above, and then p21 transcript levels were determined by quantitative PCR and normalized to GAPDH. Average values with standard deviations are shown from three independent experiments.

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

    36) Product Images from "Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient"

    Article Title: Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2014.11

    Generation and characterization of iPSC-derived RPE. ( a ) Pigmented foci in confluent iPSC plates following bFGF depletion (arrowheads). ( b ) At confluence, passaged pigmented foci form a layer of polygonal pigmented cells. Bar = 100 µm. ( c ) Semi-thin section of iPSC-derived epithelium cultured on a porous filter and stained with toluidine blue demonstrates a regular monolayer (in blue) superposed on the filter (in white). Bar = 50 µm. ( d ) Transmission electron microscopy shows the iPSC-derived monolayer as a polarized epithelium with microvilli (m) on the apical side, desmosomes (arrowheads) at the apical junctions, melanosomes (asterisks) distributed throughout the cytosol, a nucleus (n) on the basal side, and a basal lamina (arrow) between the epithelium and the filter. Bar = 2 µm. ( e ) Expression of classic RPE genes, as determined by reverse transcriptase (RT)–polymerase chain reaction analysis in both wild-type (WT) and patient (CHM1) RPEs in the presence of RT (+RT). In the absence of RT (−RT) or complementary DNA (cDNA) (−), an amplicon was not detected. Immunofluorescence studies of the RPE monolayer, followed by confocal analysis, demonstrate the expression of ( f ) MERTK in the microvilli (in red), ( g ) CRALBP and ( h ) RPE65 in the cytoplasm (in green), and ( i ) ZO-1 at the apical junctions (in red). Bars = 100 µm (in  f ,  g ), 30 µm (in  h ), and 50 µm (in  i ). Apicobasal fluid transport causes the RPE to form fluid-filled domes, detaching it from the cell culture plate: ( j ) focus on the RPE adhered to the plate; ( k ) focus on the RPE at the top of the dome. Bars = 100 µm. ( l ) Confocal analysis of the RPE 6 hours postincubation with FluoSpheres showing the internalized beads (in green), nuclei (in blue), and F-actin (in red). Bar = 15 µm. ( m ) Flow cytometry analysis showing the percentage of RPE cells that internalized FluoSpheres over time. bFGF, basic fibroblast growth factor; BEST1, bestrophin 1; CRALPB, cellular retinaldehyde-binding protein; GAPDH, glycerglyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LRAT, lecithin retinol acyltransferase; MERTK , C-mer proto-oncogene tyrosine kinase; PAX6 , paired box 6; RDH, retinal dehydrogenase 5; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium; TYR, tyrosinase; ZO1, zona occludens protein 1.
    Figure Legend Snippet: Generation and characterization of iPSC-derived RPE. ( a ) Pigmented foci in confluent iPSC plates following bFGF depletion (arrowheads). ( b ) At confluence, passaged pigmented foci form a layer of polygonal pigmented cells. Bar = 100 µm. ( c ) Semi-thin section of iPSC-derived epithelium cultured on a porous filter and stained with toluidine blue demonstrates a regular monolayer (in blue) superposed on the filter (in white). Bar = 50 µm. ( d ) Transmission electron microscopy shows the iPSC-derived monolayer as a polarized epithelium with microvilli (m) on the apical side, desmosomes (arrowheads) at the apical junctions, melanosomes (asterisks) distributed throughout the cytosol, a nucleus (n) on the basal side, and a basal lamina (arrow) between the epithelium and the filter. Bar = 2 µm. ( e ) Expression of classic RPE genes, as determined by reverse transcriptase (RT)–polymerase chain reaction analysis in both wild-type (WT) and patient (CHM1) RPEs in the presence of RT (+RT). In the absence of RT (−RT) or complementary DNA (cDNA) (−), an amplicon was not detected. Immunofluorescence studies of the RPE monolayer, followed by confocal analysis, demonstrate the expression of ( f ) MERTK in the microvilli (in red), ( g ) CRALBP and ( h ) RPE65 in the cytoplasm (in green), and ( i ) ZO-1 at the apical junctions (in red). Bars = 100 µm (in f , g ), 30 µm (in h ), and 50 µm (in i ). Apicobasal fluid transport causes the RPE to form fluid-filled domes, detaching it from the cell culture plate: ( j ) focus on the RPE adhered to the plate; ( k ) focus on the RPE at the top of the dome. Bars = 100 µm. ( l ) Confocal analysis of the RPE 6 hours postincubation with FluoSpheres showing the internalized beads (in green), nuclei (in blue), and F-actin (in red). Bar = 15 µm. ( m ) Flow cytometry analysis showing the percentage of RPE cells that internalized FluoSpheres over time. bFGF, basic fibroblast growth factor; BEST1, bestrophin 1; CRALPB, cellular retinaldehyde-binding protein; GAPDH, glycerglyceraldehyde 3-phosphate dehydrogenase; iPSC, induced pluripotent stem cell; LRAT, lecithin retinol acyltransferase; MERTK , C-mer proto-oncogene tyrosine kinase; PAX6 , paired box 6; RDH, retinal dehydrogenase 5; RLBP1, retinaldehyde binding protein 1; RPE, retinal pigment epithelium; TYR, tyrosinase; ZO1, zona occludens protein 1.

    Techniques Used: Derivative Assay, Cell Culture, Staining, Transmission Assay, Electron Microscopy, Expressing, Polymerase Chain Reaction, Amplification, Immunofluorescence, Flow Cytometry, Cytometry, Binding Assay

    37) Product Images from "Comparative transcriptome analysis reveals host-associated differentiation in Chilo suppressalis (Lepidoptera: Crambidae)"

    Article Title: Comparative transcriptome analysis reveals host-associated differentiation in Chilo suppressalis (Lepidoptera: Crambidae)

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14137-x

    De novo assembly of the transcriptome
    Figure Legend Snippet: De novo assembly of the transcriptome

    Techniques Used:

    Transcriptome sequencing
    Figure Legend Snippet: Transcriptome sequencing

    Techniques Used: Sequencing

    38) Product Images from "Cytidine-phosphate-guanosine oligodeoxynucleotides in combination with CD40 ligand decrease periodontal inflammation and alveolar bone loss in a TLR9-independent manner"

    Article Title: Cytidine-phosphate-guanosine oligodeoxynucleotides in combination with CD40 ligand decrease periodontal inflammation and alveolar bone loss in a TLR9-independent manner

    Journal: Journal of Applied Oral Science

    doi: 10.1590/1678-7757-2017-0451

    CpG+CD40L increased gingival mRNA levels of IL-10 and decreased gingival mRNA levels of RANKL and IFN-γ in ligature-induced experimental periodontitis of WT and TLR9 KO mice. Silk ligatures were tied around the maxillary secondary molars on day 0 and injection of CpG (1 μM)+CD40L (0.1 μg/ml) or PBS was performed in palatal gingiva on days 3, 6 and 9 in WT mice and TLR9 KO mice. The levels of mRNA expression for IL-10 (A), OPG (B), RANKL (C), IL-1β (D), TNF-α (E), IFN-γ (F) in gingival tissues were detected by qRT-PCR and their relative levels of expression were normalized to the level of GAPDH expression (means±SD, n=4 WT or TLR9 KO mice per group, *p
    Figure Legend Snippet: CpG+CD40L increased gingival mRNA levels of IL-10 and decreased gingival mRNA levels of RANKL and IFN-γ in ligature-induced experimental periodontitis of WT and TLR9 KO mice. Silk ligatures were tied around the maxillary secondary molars on day 0 and injection of CpG (1 μM)+CD40L (0.1 μg/ml) or PBS was performed in palatal gingiva on days 3, 6 and 9 in WT mice and TLR9 KO mice. The levels of mRNA expression for IL-10 (A), OPG (B), RANKL (C), IL-1β (D), TNF-α (E), IFN-γ (F) in gingival tissues were detected by qRT-PCR and their relative levels of expression were normalized to the level of GAPDH expression (means±SD, n=4 WT or TLR9 KO mice per group, *p

    Techniques Used: Mouse Assay, Injection, Expressing, Quantitative RT-PCR

    39) Product Images from "Phytophthora palmivora establishes tissue-specific intracellular infection structures in the earliest divergent land plant lineage"

    Article Title: Phytophthora palmivora establishes tissue-specific intracellular infection structures in the earliest divergent land plant lineage

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

    doi: 10.1073/pnas.1717900115

    P. palmivora completes a full infection cycle that includes the intracellular colonization of living Marchantia cells. ( A ) Confocal fluorescence microscopy demonstrating key morphological transitions in P. palmivora lifestyle during the colonization of TAK1 plants from 1 to 3 dpi. Z-stack projections of pathogen fluorescence merged with plastid autofluorescence (turquoise). Intracellular infection structures are denoted by an asterisk. Sporangia are indicated by dashed arrows. (Scale bars, 10 μm.) ( B ) Quantification of P. palmivora lifestyle marker genes during the colonization of TAK1 thalli from 1 to 4 dpi via qRT-PCR analysis. Pathogen biomass ( PpEF1a ) was quantified relative to M. polymorpha biomass markers ( MpACT and MpEF1a ). Haustoria ( PpHmp1 ) and sporulation ( PpCdc14 ) marker genes were quantified relative to pathogen biomass controls ( PpEF1a and PpWS21 ). Different letters signify statistically significant differences in transcript abundance [ANOVA, Tukey’s honest significant difference (HSD), P
    Figure Legend Snippet: P. palmivora completes a full infection cycle that includes the intracellular colonization of living Marchantia cells. ( A ) Confocal fluorescence microscopy demonstrating key morphological transitions in P. palmivora lifestyle during the colonization of TAK1 plants from 1 to 3 dpi. Z-stack projections of pathogen fluorescence merged with plastid autofluorescence (turquoise). Intracellular infection structures are denoted by an asterisk. Sporangia are indicated by dashed arrows. (Scale bars, 10 μm.) ( B ) Quantification of P. palmivora lifestyle marker genes during the colonization of TAK1 thalli from 1 to 4 dpi via qRT-PCR analysis. Pathogen biomass ( PpEF1a ) was quantified relative to M. polymorpha biomass markers ( MpACT and MpEF1a ). Haustoria ( PpHmp1 ) and sporulation ( PpCdc14 ) marker genes were quantified relative to pathogen biomass controls ( PpEF1a and PpWS21 ). Different letters signify statistically significant differences in transcript abundance [ANOVA, Tukey’s honest significant difference (HSD), P

    Techniques Used: Infection, Fluorescence, Microscopy, Marker, Quantitative RT-PCR

    A colonization-induced host syntaxin accumulates at intracellular infection structures. ( A ) qRT-PCR analysis of MpSYP13A and MpSYP13B transcripts in mock-treated or P. palmivora -colonized (ARI-td) TAK1 plants from 1 to 4 dpi. Expression values are shown relative to internal MpACT and MpEF1a controls. Different letters signify statistically significant differences in transcript abundance (ANOVA, Tukey’s HSD, P
    Figure Legend Snippet: A colonization-induced host syntaxin accumulates at intracellular infection structures. ( A ) qRT-PCR analysis of MpSYP13A and MpSYP13B transcripts in mock-treated or P. palmivora -colonized (ARI-td) TAK1 plants from 1 to 4 dpi. Expression values are shown relative to internal MpACT and MpEF1a controls. Different letters signify statistically significant differences in transcript abundance (ANOVA, Tukey’s HSD, P

    Techniques Used: Infection, Quantitative RT-PCR, Expressing

    40) Product Images from "Induction of Hepatic Metabolic Functions by a Novel Variant of Hepatocyte Nuclear Factor 4γ"

    Article Title: Induction of Hepatic Metabolic Functions by a Novel Variant of Hepatocyte Nuclear Factor 4γ

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00213-18

    Hepatic expression of Hnf4a , Hnf4g1 , and Hnf4g2 mRNA in Hnf4a f/f and Hnf4a ΔH mice. (A to C) Real-time PCR for hepatic Hnf4a (A), Hnf4g1 (B), and Hnf4g2 (C) in Hnf4a f/f and Hnf4a ΔH mice ( n = 5 mice for each genotype). The normalized expression in Hnf4a ΔH mice is presented relative to that in Hnf4a f/f mice. (D and E) Expression of hepatic Hnf4a , Hnf4g1 , and Hnf4g2 mRNA in Hnf4a f/f (D) and Hnf4a ΔH mice (E) ( n = 5 mice for each genotype). The normalized expression of Hnf4g1 and Hnf4g2 is presented relative to Hnf4a expression. Gapdh mRNA expression was used as an internal control to normalize Hnf4 mRNA expression. Data are presented as the mean ± SD. *, P
    Figure Legend Snippet: Hepatic expression of Hnf4a , Hnf4g1 , and Hnf4g2 mRNA in Hnf4a f/f and Hnf4a ΔH mice. (A to C) Real-time PCR for hepatic Hnf4a (A), Hnf4g1 (B), and Hnf4g2 (C) in Hnf4a f/f and Hnf4a ΔH mice ( n = 5 mice for each genotype). The normalized expression in Hnf4a ΔH mice is presented relative to that in Hnf4a f/f mice. (D and E) Expression of hepatic Hnf4a , Hnf4g1 , and Hnf4g2 mRNA in Hnf4a f/f (D) and Hnf4a ΔH mice (E) ( n = 5 mice for each genotype). The normalized expression of Hnf4g1 and Hnf4g2 is presented relative to Hnf4a expression. Gapdh mRNA expression was used as an internal control to normalize Hnf4 mRNA expression. Data are presented as the mean ± SD. *, P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Hepatic expression of Hnf4g variants in Hnf4a ΔH mice. (A) Western blot analysis of the HNF4γ (top) and TUBG (bottom) proteins in the livers of Hnf4a f/f (lanes 1 to 3) and Hnf4a ΔH (lanes 4 to 6) mice and HeLa, H4IIE, Hepa1-6, and HepG2 cells (lanes 7 to 10) (top). (B) 5′ RACE analysis of the hepatic Hnf4g gene in Hnf4a ΔH mice. (Left) PCR products were separated by 2% agarose gel electrophoresis. From DNA sequencing analysis of the amplified bands, the upper and lower arrows indicate Hnf4g1 and Hnf4g2 cDNA, respectively. (Right) Schematic structure of the mouse Hnf4g1 and Hnf4g2 genes. The start codon is shown as ATG. (C) Exon 1B sequences of the mouse Hnf4g2 gene. The predicted start codon is shown in boxed letters. (D) 5′ RACE analysis of the human HNF4G gene in NCI-H28 cells. (Middle) PCR products were separated by 2% agarose gel electrophoresis. From DNA sequencing analysis of the amplified bands, the upper (lane 1) and lower (lane 2) arrows indicate HNF4G1 and HNF4G2 cDNA, respectively. (Left and right) Schematic structures of the human HNF4G1 (left) and HNF4G2 (right) genes. The start codon is shown as ATG. (E) Exon 1C sequences of the human HNF4G2 gene. The predicted start codon is shown in boxed letters. Lanes M, 100-bp DNA marker; NS, nonspecific band; nt, nucleotide.
    Figure Legend Snippet: Hepatic expression of Hnf4g variants in Hnf4a ΔH mice. (A) Western blot analysis of the HNF4γ (top) and TUBG (bottom) proteins in the livers of Hnf4a f/f (lanes 1 to 3) and Hnf4a ΔH (lanes 4 to 6) mice and HeLa, H4IIE, Hepa1-6, and HepG2 cells (lanes 7 to 10) (top). (B) 5′ RACE analysis of the hepatic Hnf4g gene in Hnf4a ΔH mice. (Left) PCR products were separated by 2% agarose gel electrophoresis. From DNA sequencing analysis of the amplified bands, the upper and lower arrows indicate Hnf4g1 and Hnf4g2 cDNA, respectively. (Right) Schematic structure of the mouse Hnf4g1 and Hnf4g2 genes. The start codon is shown as ATG. (C) Exon 1B sequences of the mouse Hnf4g2 gene. The predicted start codon is shown in boxed letters. (D) 5′ RACE analysis of the human HNF4G gene in NCI-H28 cells. (Middle) PCR products were separated by 2% agarose gel electrophoresis. From DNA sequencing analysis of the amplified bands, the upper (lane 1) and lower (lane 2) arrows indicate HNF4G1 and HNF4G2 cDNA, respectively. (Left and right) Schematic structures of the human HNF4G1 (left) and HNF4G2 (right) genes. The start codon is shown as ATG. (E) Exon 1C sequences of the human HNF4G2 gene. The predicted start codon is shown in boxed letters. Lanes M, 100-bp DNA marker; NS, nonspecific band; nt, nucleotide.

    Techniques Used: Expressing, Mouse Assay, Western Blot, Polymerase Chain Reaction, Agarose Gel Electrophoresis, DNA Sequencing, Amplification, Marker

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    Purification:

    Article Title: Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion
    Article Snippet: .. RNA purification and Real Time RT-PCR Entamoeba cells were harvested and suspended immediately in TriZol reagent (Invitrogen) and RNA was isolated and treated with DNase I (Roche) according to manufacturer's protocol. .. RNA was reverse transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen) and random hexamers used as a primer.

    SYBR Green Assay:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Article Title: Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases
    Article Snippet: .. RNA (1 μg) was used in each RT-PCR following the manufacturer's protocol (Titan One Tube RT-PCR System, Roche Diagnostics). ..

    Polymerase Chain Reaction:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    Chromatin Immunoprecipitation:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

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    Roche lightcyclertm 480 sybr green i master reaction mix
    Lightcyclertm 480 Sybr Green I Master Reaction Mix, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche lightcyclerã‚â 480 sybr green i master mix
    Lightcyclerã‚â 480 Sybr Green I Master Mix, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lightcyclerã‚â 480 sybr green i master mix/product/Roche
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    lightcyclerã‚â 480 sybr green i master mix - by Bioz Stars, 2020-08
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