gfp  (Roche)


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    Structured Review

    Roche gfp
    Colocalization and effects of direct NPFFR2 signalling on <t>NPY</t> neurons. a Representative image of <t>GFP</t> expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p
    Gfp, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 1118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 1118 article reviews
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    gfp - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Diet-induced adaptive thermogenesis requires neuropeptide FF receptor-2 signalling"

    Article Title: Diet-induced adaptive thermogenesis requires neuropeptide FF receptor-2 signalling

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06462-0

    Colocalization and effects of direct NPFFR2 signalling on NPY neurons. a Representative image of GFP expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p
    Figure Legend Snippet: Colocalization and effects of direct NPFFR2 signalling on NPY neurons. a Representative image of GFP expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p

    Techniques Used: Expressing, Immunoprecipitation, Isolation, Mouse Assay

    2) Product Images from "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 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

    Journal: Cancer Science

    doi: 10.1111/cas.13754

    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P
    Figure Legend Snippet: Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase, Construct, Chromatin Immunoprecipitation, Over Expression, Activity Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

    3) Product Images from "Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits"

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

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01259

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

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

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

    Techniques Used: Expressing, Quantitative RT-PCR

    4) Product Images from "Angiogenesis‐related gene expression profile in clinical cases of canine cancer"

    Article Title: Angiogenesis‐related gene expression profile in clinical cases of canine cancer

    Journal: Veterinary Medicine and Science

    doi: 10.1002/vms3.127

    The expression of angiogenesis‐related genes in canine benign tumour tissues and the normal tissues surrounding each tumours. (a) The expression of the CD 34 gene. (b) The expression of the VEGF ‐A gene. (c) The expression of the VEGFR ‐1 gene. (d) The expression of the VEGFR ‐2 gene. (e) The expression of the Ang‐1 gene. (f) The expression of the Ang‐2 gene. (g) The expression of the Tie1 gene. (h) The expression of the Tie2 gene. The expression of each gene was compared between tumour tissues and the normal tissues surrounding the tumours using the Wilcoxon rank‐sum test. n. s. = not significant.
    Figure Legend Snippet: The expression of angiogenesis‐related genes in canine benign tumour tissues and the normal tissues surrounding each tumours. (a) The expression of the CD 34 gene. (b) The expression of the VEGF ‐A gene. (c) The expression of the VEGFR ‐1 gene. (d) The expression of the VEGFR ‐2 gene. (e) The expression of the Ang‐1 gene. (f) The expression of the Ang‐2 gene. (g) The expression of the Tie1 gene. (h) The expression of the Tie2 gene. The expression of each gene was compared between tumour tissues and the normal tissues surrounding the tumours using the Wilcoxon rank‐sum test. n. s. = not significant.

    Techniques Used: Expressing

    The expression of angiogenesis‐related genes in canine malignant tumour tissues and the normal tissues surrounding each tumours. (a) The mRNA expression of the CD 34 gene. (b) The expression of the VEGF ‐A gene. (c) The expression of the VEGFR ‐1 gene. (d) The expression of the VEGFR ‐2 gene. (e) The expression of the Ang‐1 gene. (f) The expression of the Ang‐2 gene. (g) The expression of the Tie1 gene. (h) The expression of the Tie2 gene. The expression of each gene was compared between tumour tissues and the normal tissues surrounding the tumours using the Wilcoxon rank‐sum test. n. s. = not significant.
    Figure Legend Snippet: The expression of angiogenesis‐related genes in canine malignant tumour tissues and the normal tissues surrounding each tumours. (a) The mRNA expression of the CD 34 gene. (b) The expression of the VEGF ‐A gene. (c) The expression of the VEGFR ‐1 gene. (d) The expression of the VEGFR ‐2 gene. (e) The expression of the Ang‐1 gene. (f) The expression of the Ang‐2 gene. (g) The expression of the Tie1 gene. (h) The expression of the Tie2 gene. The expression of each gene was compared between tumour tissues and the normal tissues surrounding the tumours using the Wilcoxon rank‐sum test. n. s. = not significant.

    Techniques Used: Expressing

    5) Product Images from "Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro"

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand HCV RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time RT-PCR quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.
    Figure Legend Snippet: Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand HCV RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time RT-PCR quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.

    Techniques Used: Infection, In Vitro, Quantitative RT-PCR, Concentration Assay, Inhibition

    Qualitative assay detection of positive- and negative-strand HCV RNA in a primary culture of healthy human hepatocytes infected in vitro with an HCV-positive serum and effect of IFN-α. The hepatocyte culture FT147, infected 3 days after plating by HCV-positive serum S26, is shown. Positive-strand (+) RNA but not negative-strand (-) RNA was present in the inoculum. (a) Primary hepatocyte culture in the absence of IFN-α. Positive-strand HCV RNA was detected with the qualitative strand-specific r Tth PCR assay from day 1 to the end of the culture (day 12), whereas negative-strand RNA was detected from days 2 to 10. (b) Culture in the presence of 5,000 U of IFN-α per ml. Positive-strand HCV RNA was detected from days 1 to 10, whereas negative-strand RNA was never detected. (c) Culture treated on day 3 with 5,000 U of IFN-α per ml. Positive-strand RNA was detected throughout the culture period, whereas negative-strand RNA was no longer detected after day 5. Similar patterns (not shown) were observed with the following cultures infected with the corresponding sera: FT141 and S23, FT143 and S34, FT144 and S27, FT154 and S23, FT155 and S20, and FT156 and S17. MK, molecular size standards.
    Figure Legend Snippet: Qualitative assay detection of positive- and negative-strand HCV RNA in a primary culture of healthy human hepatocytes infected in vitro with an HCV-positive serum and effect of IFN-α. The hepatocyte culture FT147, infected 3 days after plating by HCV-positive serum S26, is shown. Positive-strand (+) RNA but not negative-strand (-) RNA was present in the inoculum. (a) Primary hepatocyte culture in the absence of IFN-α. Positive-strand HCV RNA was detected with the qualitative strand-specific r Tth PCR assay from day 1 to the end of the culture (day 12), whereas negative-strand RNA was detected from days 2 to 10. (b) Culture in the presence of 5,000 U of IFN-α per ml. Positive-strand HCV RNA was detected from days 1 to 10, whereas negative-strand RNA was never detected. (c) Culture treated on day 3 with 5,000 U of IFN-α per ml. Positive-strand RNA was detected throughout the culture period, whereas negative-strand RNA was no longer detected after day 5. Similar patterns (not shown) were observed with the following cultures infected with the corresponding sera: FT141 and S23, FT143 and S34, FT144 and S27, FT154 and S23, FT155 and S20, and FT156 and S17. MK, molecular size standards.

    Techniques Used: Infection, In Vitro, Polymerase Chain Reaction

    Characteristics of the strand-specific HCV RNA assays used in this study. (a) Strand specificity of the positive-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of positive-strand (+) HCV RNA (100, 10, 1, and 0.1 fg) and of negative-strand (-) HCV RNA (10, 1, and 0.1 pg) synthesized from an appropriate plasmid were subjected to the r Tth RT-PCR assay. The products were analyzed by agarose gel electrophoresis. (b) Strand specificity of the negative-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of negative-strand HCV RNA (100, 10, 1, and 0.1 fg) and positive-strand HCV RNA (10, 1, and 0.1 pg) synthesized from the same plasmid as for panel a were analyzed by the same procedure. (c) Range of linear quantification of the quantitative assay based on real-time PCR using the LightCycler technology and SYBR green I dye for detection. The range of linear quantification of the assay was studied by testing 10-fold serial dilutions of synthetic positive- and negative-sense HCV RNA strands after RT at 70°C with the r Tth polymerase. Each point is the mean of three experimental values for each dilution. y is the slope of the linear plots.
    Figure Legend Snippet: Characteristics of the strand-specific HCV RNA assays used in this study. (a) Strand specificity of the positive-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of positive-strand (+) HCV RNA (100, 10, 1, and 0.1 fg) and of negative-strand (-) HCV RNA (10, 1, and 0.1 pg) synthesized from an appropriate plasmid were subjected to the r Tth RT-PCR assay. The products were analyzed by agarose gel electrophoresis. (b) Strand specificity of the negative-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of negative-strand HCV RNA (100, 10, 1, and 0.1 fg) and positive-strand HCV RNA (10, 1, and 0.1 pg) synthesized from the same plasmid as for panel a were analyzed by the same procedure. (c) Range of linear quantification of the quantitative assay based on real-time PCR using the LightCycler technology and SYBR green I dye for detection. The range of linear quantification of the assay was studied by testing 10-fold serial dilutions of synthetic positive- and negative-sense HCV RNA strands after RT at 70°C with the r Tth polymerase. Each point is the mean of three experimental values for each dilution. y is the slope of the linear plots.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Synthesized, Plasmid Preparation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, SYBR Green Assay

    Accumulation of positive- and negative-strand HCV RNA in hepatocyte cultures FT172 (a), FT189 (b), and FT195 (c), infected with sera S42, S155, and S155, respectively, as measured by the quantitative LightCycler real-time RT-PCR assay. The hepatocyte cultures were infected 3 days after plating. The cells were harvested 30 min and 1, 3, and 5 days after infection for positive-strand (gray) and negative-strand (black) HCV RNA quantification. The amounts of HCV RNA strands are shown as means ± SEMs of three determinations, expressed in numbers of HCV RNA copies per 2 × 10 6 cells, normalized to GAPDH mRNA. Similar results (not shown) were obtained with culture FT168 infected with serum S34.
    Figure Legend Snippet: Accumulation of positive- and negative-strand HCV RNA in hepatocyte cultures FT172 (a), FT189 (b), and FT195 (c), infected with sera S42, S155, and S155, respectively, as measured by the quantitative LightCycler real-time RT-PCR assay. The hepatocyte cultures were infected 3 days after plating. The cells were harvested 30 min and 1, 3, and 5 days after infection for positive-strand (gray) and negative-strand (black) HCV RNA quantification. The amounts of HCV RNA strands are shown as means ± SEMs of three determinations, expressed in numbers of HCV RNA copies per 2 × 10 6 cells, normalized to GAPDH mRNA. Similar results (not shown) were obtained with culture FT168 infected with serum S34.

    Techniques Used: Infection, Quantitative RT-PCR

    6) Product Images from "The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth"

    Article Title: The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-11-35

    Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and qRT-PCR experiments followed using primers for DUSP1 and DUSP5.
    Figure Legend Snippet: Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and qRT-PCR experiments followed using primers for DUSP1 and DUSP5.

    Techniques Used: Lysis, Derivative Assay, Isolation, Quantitative RT-PCR

    7) Product Images from "Efficacy of prosultiamine treatment in patients with human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis: results from an open-label clinical trial"

    Article Title: Efficacy of prosultiamine treatment in patients with human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis: results from an open-label clinical trial

    Journal: BMC Medicine

    doi: 10.1186/1741-7015-11-182

    Change in human T lymphotropic virus type I (HTLV-I) proviral copy numbers in peripheral blood mononuclear cells (PBMCs). (a) HTLV-I proviral copy numbers from 10 4 PBMCs decreased gradually until 12 weeks after prosultiamine treatment. The level of HTLV-I proviral copy numbers 12 weeks after prosultiamine treatment decreased by 15.4% compared with the time at pretreatment. (b) Changes in HTLV-I proviral copy numbers in each case between pretreatment and 12 weeks after prosultiamine treatment. Statistical significance was determined by the Wilcoxon signed-rank test.
    Figure Legend Snippet: Change in human T lymphotropic virus type I (HTLV-I) proviral copy numbers in peripheral blood mononuclear cells (PBMCs). (a) HTLV-I proviral copy numbers from 10 4 PBMCs decreased gradually until 12 weeks after prosultiamine treatment. The level of HTLV-I proviral copy numbers 12 weeks after prosultiamine treatment decreased by 15.4% compared with the time at pretreatment. (b) Changes in HTLV-I proviral copy numbers in each case between pretreatment and 12 weeks after prosultiamine treatment. Statistical significance was determined by the Wilcoxon signed-rank test.

    Techniques Used:

    8) Product Images from "BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells"

    Article Title: BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt163

    Microarray analysis and quantitative real-time polymerase chain reaction (QPCR) of genes upregulated by BAY11 . (a) Microarray data show four putative gene targets that were identified by over 3 × fold changes in human fibroblast (HUF) cells treated with BAY11compared with untreated HUF cells over the course of 3 days of daily synthetic mRNA transfections. (b) QPCR analysis confirmed microarray results for the four genes and demonstrated that relative expression of the four genes becomes more similar to human embryonic stem cell (hESC) expression in the presence of BAY11. Asterisks indicate statistically significant increases over control cells not receiving BAY11 where P value was less than 0.05. BAY11, BAY 11-7082; DUSP4 , dual-specificity phosphatase 4; SLC16A9 , solute carrier family 16, member 9.
    Figure Legend Snippet: Microarray analysis and quantitative real-time polymerase chain reaction (QPCR) of genes upregulated by BAY11 . (a) Microarray data show four putative gene targets that were identified by over 3 × fold changes in human fibroblast (HUF) cells treated with BAY11compared with untreated HUF cells over the course of 3 days of daily synthetic mRNA transfections. (b) QPCR analysis confirmed microarray results for the four genes and demonstrated that relative expression of the four genes becomes more similar to human embryonic stem cell (hESC) expression in the presence of BAY11. Asterisks indicate statistically significant increases over control cells not receiving BAY11 where P value was less than 0.05. BAY11, BAY 11-7082; DUSP4 , dual-specificity phosphatase 4; SLC16A9 , solute carrier family 16, member 9.

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Transfection, Expressing

    9) Product Images from "LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis"

    Article Title: LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1349

    LORD-Q allows the sequence-specific detection and accurate quantification of lesions in long DNA probes of mitochondrial and nuclear genomes. ( A ) Schematic illustration of the LORD-Q assay. Whole-cell DNA, either damaged or intact, is isolated (left). Undamaged template sequences of 3–4 kb-length are successfully amplified, whereas several types of DNA lesions or modifications impair or abrogate the PCR elongation reaction, as detected by reduced fluorescence (FL). Short fragments of 40–70 bp serve as reference templates (middle). Increasing DNA lesion/modification rates lead to elevated Δ C p values (right). ( B ) DNA damage induced by different stimuli can be quantified by LORD-Q. Jurkat cells were incubated with the indicated concentrations of bleomycin (20 min), cisplatin (16 h), etoposide (16 h) and different doses of UVC light. Both mtDNA and nDNA ( Col1a1 locus ) DNA lesions were quantified by LORD-Q ( n = 3, mean ± SD). ( C ) Effect of bleomycin on cell death and mtDNA damage. Jurkat cells were stimulated with the indicated concentrations of bleomycin. After 20 min, half of the cells were harvested and analyzed by LORD-Q, while the remaining half was washed and further incubated under standard culture conditions. After 6 h cell viability was determined by flow cytometry and annexin V-FITC/propidium iodide staining ( n = 3, mean ± SD). ( D ) Effect of the caspase inhibitor Q-VD-OPH on mtDNA and nDNA lesion rates. Jurkat cells were incubated with 100 µM cisplatin for 16 h (left) or irradiated with UVC (20 mJ/cm 2 , right) in the presence or absence of 10 µM Q-VD-OPH.
    Figure Legend Snippet: LORD-Q allows the sequence-specific detection and accurate quantification of lesions in long DNA probes of mitochondrial and nuclear genomes. ( A ) Schematic illustration of the LORD-Q assay. Whole-cell DNA, either damaged or intact, is isolated (left). Undamaged template sequences of 3–4 kb-length are successfully amplified, whereas several types of DNA lesions or modifications impair or abrogate the PCR elongation reaction, as detected by reduced fluorescence (FL). Short fragments of 40–70 bp serve as reference templates (middle). Increasing DNA lesion/modification rates lead to elevated Δ C p values (right). ( B ) DNA damage induced by different stimuli can be quantified by LORD-Q. Jurkat cells were incubated with the indicated concentrations of bleomycin (20 min), cisplatin (16 h), etoposide (16 h) and different doses of UVC light. Both mtDNA and nDNA ( Col1a1 locus ) DNA lesions were quantified by LORD-Q ( n = 3, mean ± SD). ( C ) Effect of bleomycin on cell death and mtDNA damage. Jurkat cells were stimulated with the indicated concentrations of bleomycin. After 20 min, half of the cells were harvested and analyzed by LORD-Q, while the remaining half was washed and further incubated under standard culture conditions. After 6 h cell viability was determined by flow cytometry and annexin V-FITC/propidium iodide staining ( n = 3, mean ± SD). ( D ) Effect of the caspase inhibitor Q-VD-OPH on mtDNA and nDNA lesion rates. Jurkat cells were incubated with 100 µM cisplatin for 16 h (left) or irradiated with UVC (20 mJ/cm 2 , right) in the presence or absence of 10 µM Q-VD-OPH.

    Techniques Used: Sequencing, Isolation, Amplification, Polymerase Chain Reaction, Fluorescence, Modification, Incubation, Flow Cytometry, Cytometry, Staining, Irradiation

    Exemplary applications of the LORD-Q method. ( A ) Comparative analysis of genotoxic vulnerability. Human primary fibroblasts (hFib) and corresponding isogenic hiPS cells were treated in serum-free medium with 10 µM bleomycin (20 min) or 10 mJ/cm 2 UVC radiation before mitochondrial and genomic lesion rates were determined by LORD-Q analysis ( n = 3, mean ± SD). ( B ) Investigation of mtDNA repair. Human primary fibroblasts and corresponding isogenic hiPS cells were treated with low and high doses of bleomycin (see Materials and methods section for details) or UV radiation in serum-free medium, and then harvested either directly after treatment (indicated ‘initial’) or after 2 h of recovery under cell culture conditions ( n = 3, mean ± SD). ( C ) Comparison of DNA damage in different genes. QRT-PCR expression analysis of Oct4 and Col1a1 in fibroblasts and corresponding hiPS cells ( left , n = 3, mean ± SD). Corresponding qPCR analysis of histone H3 acetylation in ChIP-enriched Oct4 and Col1a1 loci (middle). Fibroblasts and corresponding hiPS cell clones were stimulated with 10 µM bleomycin (20 min) and the DNA lesion rates in both Oct4 and Col1a1 gene loci were determined (right, n = 6, mean ± SD). The ratio of detected lesions per 10 kb in both genes is shown. * P
    Figure Legend Snippet: Exemplary applications of the LORD-Q method. ( A ) Comparative analysis of genotoxic vulnerability. Human primary fibroblasts (hFib) and corresponding isogenic hiPS cells were treated in serum-free medium with 10 µM bleomycin (20 min) or 10 mJ/cm 2 UVC radiation before mitochondrial and genomic lesion rates were determined by LORD-Q analysis ( n = 3, mean ± SD). ( B ) Investigation of mtDNA repair. Human primary fibroblasts and corresponding isogenic hiPS cells were treated with low and high doses of bleomycin (see Materials and methods section for details) or UV radiation in serum-free medium, and then harvested either directly after treatment (indicated ‘initial’) or after 2 h of recovery under cell culture conditions ( n = 3, mean ± SD). ( C ) Comparison of DNA damage in different genes. QRT-PCR expression analysis of Oct4 and Col1a1 in fibroblasts and corresponding hiPS cells ( left , n = 3, mean ± SD). Corresponding qPCR analysis of histone H3 acetylation in ChIP-enriched Oct4 and Col1a1 loci (middle). Fibroblasts and corresponding hiPS cell clones were stimulated with 10 µM bleomycin (20 min) and the DNA lesion rates in both Oct4 and Col1a1 gene loci were determined (right, n = 6, mean ± SD). The ratio of detected lesions per 10 kb in both genes is shown. * P

    Techniques Used: Cell Culture, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Clone Assay

    Comparison of sensitivity of the semi-long-run rtPCR method and the LORD-Q assay by ROC analysis. Jurkat cells were treated with the indicated concentrations of bleomycin for 20 min in serum-free medium. Real-time PCR experiments were carried out in triplicate ( n = 15 per concentration and experiment). ( A ) Quantification of detectable DNA lesions per 10 kb by the LORD-Q method. ( B ) Representative ROC analyses for LORD-Q after treatment of Jurkat cells with the indicated concentrations of bleomycin (Bleo, left panel), and averaged ROC AUC values for the semi-long-run rtPCR method and LORD-Q (right panel). ( C ) Effect of different DNA lesions and nucleotide modifications on relative template amplification rates. Synthetic DNA oligonucleotides with inserted single-base modifications were amplified applying LORD-Q ( n = 4, mean ± SD). * P
    Figure Legend Snippet: Comparison of sensitivity of the semi-long-run rtPCR method and the LORD-Q assay by ROC analysis. Jurkat cells were treated with the indicated concentrations of bleomycin for 20 min in serum-free medium. Real-time PCR experiments were carried out in triplicate ( n = 15 per concentration and experiment). ( A ) Quantification of detectable DNA lesions per 10 kb by the LORD-Q method. ( B ) Representative ROC analyses for LORD-Q after treatment of Jurkat cells with the indicated concentrations of bleomycin (Bleo, left panel), and averaged ROC AUC values for the semi-long-run rtPCR method and LORD-Q (right panel). ( C ) Effect of different DNA lesions and nucleotide modifications on relative template amplification rates. Synthetic DNA oligonucleotides with inserted single-base modifications were amplified applying LORD-Q ( n = 4, mean ± SD). * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Concentration Assay, Amplification

    10) Product Images from "Prospective Evaluation of Whole Genome MicroRNA Expression Profiling in Childhood Acute Lymphoblastic Leukemia"

    Article Title: Prospective Evaluation of Whole Genome MicroRNA Expression Profiling in Childhood Acute Lymphoblastic Leukemia

    Journal: BioMed Research International

    doi: 10.1155/2014/967585

    Diagram of significantly changed miRNAs in ALL patients after 6 months of treatment. The figure shows the change in expression levels of 4 significantly dysregulated miRNAs after 6 months of treatment. The change in miRNA levels over time was determined by comparing the ratio at day 0 with the ratio of those measured at 6 months by using quantitative real time RT-PCR.
    Figure Legend Snippet: Diagram of significantly changed miRNAs in ALL patients after 6 months of treatment. The figure shows the change in expression levels of 4 significantly dysregulated miRNAs after 6 months of treatment. The change in miRNA levels over time was determined by comparing the ratio at day 0 with the ratio of those measured at 6 months by using quantitative real time RT-PCR.

    Techniques Used: Expressing, Quantitative RT-PCR

    11) Product Images from "Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression"

    Article Title: Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095180

    LPA-induced angiogenic factor expression was mediated through Gi/NF-κB signaling in chondrocytes. (A) Expression of LPA receptors in CHON001 chondrocytes and HC were detected by RT-PCR. The gel shown was representative of three independent experiments. (B) CHON001 chondrocytes were pretreated with PTX (100 ng/mL) or PDTC (100 nM) for 1 h prior to the LPA treatment. The protein levels of angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF expressions were determined after 24 h by EIA. Data were compared with the vehicle-treated group. n = 5. (C) Cells were pretreated with PTX (100 ng/mL) or PDTC (100 nM) for 1 h prior to LPA treatment. The protein levels of NF-kB p50 and p65 in nuclear or cytosolic fractions were determined by Western blot analysis. The quantitative results are presented as the ratio of nuclear-to-cytosolic p50 or p65. Data were compared with the LPA-treated group. n = 3 (D) Cells were transfected with an NF-κB–driven luciferase reporter and then pretreated with indicated inhibitors for 1 h prior to LPA treatment. After 4 h, NF-κB promoter activities were determined. Data are compared between indicated groups. n = 5; * p
    Figure Legend Snippet: LPA-induced angiogenic factor expression was mediated through Gi/NF-κB signaling in chondrocytes. (A) Expression of LPA receptors in CHON001 chondrocytes and HC were detected by RT-PCR. The gel shown was representative of three independent experiments. (B) CHON001 chondrocytes were pretreated with PTX (100 ng/mL) or PDTC (100 nM) for 1 h prior to the LPA treatment. The protein levels of angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF expressions were determined after 24 h by EIA. Data were compared with the vehicle-treated group. n = 5. (C) Cells were pretreated with PTX (100 ng/mL) or PDTC (100 nM) for 1 h prior to LPA treatment. The protein levels of NF-kB p50 and p65 in nuclear or cytosolic fractions were determined by Western blot analysis. The quantitative results are presented as the ratio of nuclear-to-cytosolic p50 or p65. Data were compared with the LPA-treated group. n = 3 (D) Cells were transfected with an NF-κB–driven luciferase reporter and then pretreated with indicated inhibitors for 1 h prior to LPA treatment. After 4 h, NF-κB promoter activities were determined. Data are compared between indicated groups. n = 5; * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Luciferase

    LPA significantly induced angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF expression in CHON001and HC. Cells were treated with 5 µM LPA or vehicle (1% BSA). (A) After 24 h, the cell culture supernatant from CHON001 was used to detect angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF proteins by EIA. Data were compared between indicated groups. (B) After 24 h, the cell culture supernatant from HC was assayed as described in (A). (C) CHON001chondrocytes were treated with 5 µM LPA or vehicle (1% BSA). After 6 h, mRNA levels of angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF were determined by real-time quantitative RT-PCR and compared. (D) HC was treated with 5 µM LPA or vehicle (1% BSA). After 6 h, mRNA was assayed as described in (C). Data were compared between indicated groups. n = 3 for each assay. * p
    Figure Legend Snippet: LPA significantly induced angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF expression in CHON001and HC. Cells were treated with 5 µM LPA or vehicle (1% BSA). (A) After 24 h, the cell culture supernatant from CHON001 was used to detect angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF proteins by EIA. Data were compared between indicated groups. (B) After 24 h, the cell culture supernatant from HC was assayed as described in (A). (C) CHON001chondrocytes were treated with 5 µM LPA or vehicle (1% BSA). After 6 h, mRNA levels of angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF were determined by real-time quantitative RT-PCR and compared. (D) HC was treated with 5 µM LPA or vehicle (1% BSA). After 6 h, mRNA was assayed as described in (C). Data were compared between indicated groups. n = 3 for each assay. * p

    Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    12) Product Images from "Inhibition of hepatitis C virus by an M1GS ribozyme derived from the catalytic RNA subunit of Escherichia coli RNase P"

    Article Title: Inhibition of hepatitis C virus by an M1GS ribozyme derived from the catalytic RNA subunit of Escherichia coli RNase P

    Journal: Virology Journal

    doi: 10.1186/1743-422X-11-86

    In vitro cleavage of the targeted RNA by M1GS ribozymes. No ribozyme was added to the reaction mixture in lane 1; 10 nM of M1GS-HCV/C 67 (lanes 2), M1GS-HCV/C 67 * (lanes 3) and Chol-M1GS/C 67 (lane 4) were incubated with 32 P-labeled substrate RNA (S 1–584 , 10 nM) at 37°C in a volume of 10 μl for 30 min in cleavage buffer. As a control, equal volumes of M1GS-HCV/C 67 and Chol-M1GS-HCV/C 67 were separately incubated with another 32 P-labeled substrate RNA (HCMV UL97 RNA) in the same condition (lanes 5 and 6) Cleavage products were separated on 15% polyacrylamide gels containing 8 M urea. The RNA makers were transcribed in vitro by T7 RNA polymerase with DNA templates linearized by different restriction enzymes (lane M).
    Figure Legend Snippet: In vitro cleavage of the targeted RNA by M1GS ribozymes. No ribozyme was added to the reaction mixture in lane 1; 10 nM of M1GS-HCV/C 67 (lanes 2), M1GS-HCV/C 67 * (lanes 3) and Chol-M1GS/C 67 (lane 4) were incubated with 32 P-labeled substrate RNA (S 1–584 , 10 nM) at 37°C in a volume of 10 μl for 30 min in cleavage buffer. As a control, equal volumes of M1GS-HCV/C 67 and Chol-M1GS-HCV/C 67 were separately incubated with another 32 P-labeled substrate RNA (HCMV UL97 RNA) in the same condition (lanes 5 and 6) Cleavage products were separated on 15% polyacrylamide gels containing 8 M urea. The RNA makers were transcribed in vitro by T7 RNA polymerase with DNA templates linearized by different restriction enzymes (lane M).

    Techniques Used: In Vitro, Incubation, Labeling

    Viral titers in supernatant of cultured cells that transfected with M1GS ribozyme. Huh7.5.1 cells were infected with JFH1 at a MOI of 1. The culture supernatants were then harvested at the indicated times. Quantitation of HCV titer was determined from viral RNA copies by real-time PCR method. Data shown were the means from triplicate experiments.
    Figure Legend Snippet: Viral titers in supernatant of cultured cells that transfected with M1GS ribozyme. Huh7.5.1 cells were infected with JFH1 at a MOI of 1. The culture supernatants were then harvested at the indicated times. Quantitation of HCV titer was determined from viral RNA copies by real-time PCR method. Data shown were the means from triplicate experiments.

    Techniques Used: Cell Culture, Transfection, Infection, Quantitation Assay, Real-time Polymerase Chain Reaction

    Secondary structure of a portion of HCV 5′ UTR predicted by the RNA Structure software. The three sites indicated by triangular arrows were potential cleavage sites of M1GS ribozyme. The site indicated by scissors was an optimal cleavage site, with the sequence marked by a yellow line capable of forming a long single-strand structure and facilitating complementary binding with the GS sequence of M1GS ribozyme.
    Figure Legend Snippet: Secondary structure of a portion of HCV 5′ UTR predicted by the RNA Structure software. The three sites indicated by triangular arrows were potential cleavage sites of M1GS ribozyme. The site indicated by scissors was an optimal cleavage site, with the sequence marked by a yellow line capable of forming a long single-strand structure and facilitating complementary binding with the GS sequence of M1GS ribozyme.

    Techniques Used: Software, Sequencing, Binding Assay

    Levels of HCV RNA as determined by Northern analysis. Cells (n = 1 × 10 6 ) were either mock-infected (lanes 1, 6 and 11) or infected with JFH1 (MOI = 5) and harvested at 48 h after infection. Northern analysis was carried out with RNA isolated from Huh7.5.1 cells transfected with M1GS-HCV/C 67 * (lanes 3, 8 and 13), M1GS-HCV/C 67 (lanes 4, 9 and 14), and Chol-M1GS-HCV/C 67 (lanes 5, 10 and 15). Equal amount of each RNA sample was separated on agarose gels containing formaldehyde, transferred to a nitrocellulose membrane, and hybridized to a 32 P-radiolabeled probe containing the cDNA sequences of HCV core coding region (A) , M1 RNA gene (B) , and human β-actin gene (C) , respectively.
    Figure Legend Snippet: Levels of HCV RNA as determined by Northern analysis. Cells (n = 1 × 10 6 ) were either mock-infected (lanes 1, 6 and 11) or infected with JFH1 (MOI = 5) and harvested at 48 h after infection. Northern analysis was carried out with RNA isolated from Huh7.5.1 cells transfected with M1GS-HCV/C 67 * (lanes 3, 8 and 13), M1GS-HCV/C 67 (lanes 4, 9 and 14), and Chol-M1GS-HCV/C 67 (lanes 5, 10 and 15). Equal amount of each RNA sample was separated on agarose gels containing formaldehyde, transferred to a nitrocellulose membrane, and hybridized to a 32 P-radiolabeled probe containing the cDNA sequences of HCV core coding region (A) , M1 RNA gene (B) , and human β-actin gene (C) , respectively.

    Techniques Used: Northern Blot, Infection, Isolation, Transfection

    13) Product Images from "Interferon-alpha Induces High Expression of APOBEC3G and STAT-1 in Vitro and in Vivo"

    Article Title: Interferon-alpha Induces High Expression of APOBEC3G and STAT-1 in Vitro and in Vivo

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms11093501

    STAT-1 expression in HepG2.2.15 cells after IFN-α stimulation. ( A ) Detection of STAT-1 mRNA by real-time fluorescent quantitative RT-PCR after HepG2.2.15 cells were treated with various concentrations of IFN-α; ( B ) Detection of STAT-1 protein by Western-blot after HepG2.2.15 cells were treated with various concentrations of IFN-α for 8 hours.
    Figure Legend Snippet: STAT-1 expression in HepG2.2.15 cells after IFN-α stimulation. ( A ) Detection of STAT-1 mRNA by real-time fluorescent quantitative RT-PCR after HepG2.2.15 cells were treated with various concentrations of IFN-α; ( B ) Detection of STAT-1 protein by Western-blot after HepG2.2.15 cells were treated with various concentrations of IFN-α for 8 hours.

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

    APOBEC3G expression in HepG2.2.15 cells in response to IFN-α stimulation. ( A ) RT-PCR electrophotogram of APOBEC3G mRNA after HepG2.2.15 cells were treated with various concentrations and times of IFN-α; ( B ) Detection of APOBEC3G mRNA by real-time fluorescent quantitative RT-PCR after HepG2.2.15 cells were treated with various concentrations of IFN-α (0 U/mL, 1 U/mL, 10 1 U/mL, 10 2 U/mL, 10 3 U/mL, 10 4 U/mL) for 8 hours; ( C ) Detection of APOBEC3G mRNA by real-time fluorescent quantitative RT-PCR after HepG2.2.15 cells were treated with IFN-α of 10 3 U/mL for 2, 4, 6, 8, 10, 12 hours; ( D ) Detection of APOBEC3G protein by Western-blot after HepG2.2.15 cells were treated with various concentrations of IFN-α for 8 hours.
    Figure Legend Snippet: APOBEC3G expression in HepG2.2.15 cells in response to IFN-α stimulation. ( A ) RT-PCR electrophotogram of APOBEC3G mRNA after HepG2.2.15 cells were treated with various concentrations and times of IFN-α; ( B ) Detection of APOBEC3G mRNA by real-time fluorescent quantitative RT-PCR after HepG2.2.15 cells were treated with various concentrations of IFN-α (0 U/mL, 1 U/mL, 10 1 U/mL, 10 2 U/mL, 10 3 U/mL, 10 4 U/mL) for 8 hours; ( C ) Detection of APOBEC3G mRNA by real-time fluorescent quantitative RT-PCR after HepG2.2.15 cells were treated with IFN-α of 10 3 U/mL for 2, 4, 6, 8, 10, 12 hours; ( D ) Detection of APOBEC3G protein by Western-blot after HepG2.2.15 cells were treated with various concentrations of IFN-α for 8 hours.

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

    14) Product Images from "NUCKS overexpression in breast cancer"

    Article Title: NUCKS overexpression in breast cancer

    Journal: Cancer Cell International

    doi: 10.1186/1475-2867-9-19

    Quantitative mRNA expression of NUCKS in primary cultures from different biopsies . (A) Graphical presentation of the ratio of NUCKS to PBGD mRNA expression quantitated by qRT PCR as median values of 3 independent experiments (p = 0.05) per culture. The most representative cases are illustrated. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from grade II breast cancer biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1 . MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (B) Median values of the ratio of NUCKS to PBGD mRNA expression in the studied groups (p = 0.05).
    Figure Legend Snippet: Quantitative mRNA expression of NUCKS in primary cultures from different biopsies . (A) Graphical presentation of the ratio of NUCKS to PBGD mRNA expression quantitated by qRT PCR as median values of 3 independent experiments (p = 0.05) per culture. The most representative cases are illustrated. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from grade II breast cancer biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1 . MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (B) Median values of the ratio of NUCKS to PBGD mRNA expression in the studied groups (p = 0.05).

    Techniques Used: Expressing, Quantitative RT-PCR, Derivative Assay, Multiple Displacement Amplification, Positive Control

    Semi-quantitative mRNA expression of NUCKS in primary cultures from different biopsies . (A) The RT-PCR products, generated with NUCKS and GAPDH gene specific primers, were electrophorized in a 2% agarose gel. GAPDH mRNA was used as an internal control. The most representative cases are illustrated. (B) Graphical presentation of the ratio of NUCKS to GAPDH mRNA levels corresponding to the samples illustrated in (A), as median values of 3 independent experiments (p = 0.05). The mRNA levels were quantitated semiquantitatively as described in the Methods section. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from IDC, grade II biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1 . MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (C) Median values of the ratio of NUCKS to GAPDH mRNA levels in the studied groups (p = 0.05).
    Figure Legend Snippet: Semi-quantitative mRNA expression of NUCKS in primary cultures from different biopsies . (A) The RT-PCR products, generated with NUCKS and GAPDH gene specific primers, were electrophorized in a 2% agarose gel. GAPDH mRNA was used as an internal control. The most representative cases are illustrated. (B) Graphical presentation of the ratio of NUCKS to GAPDH mRNA levels corresponding to the samples illustrated in (A), as median values of 3 independent experiments (p = 0.05). The mRNA levels were quantitated semiquantitatively as described in the Methods section. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from IDC, grade II biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1 . MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (C) Median values of the ratio of NUCKS to GAPDH mRNA levels in the studied groups (p = 0.05).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Agarose Gel Electrophoresis, Derivative Assay, Multiple Displacement Amplification, Positive Control

    15) Product Images from "Comparative Transcriptome and iTRAQ Proteome Analyses of Citrus Root Responses to Candidatus Liberibacter asiaticus Infection"

    Article Title: Comparative Transcriptome and iTRAQ Proteome Analyses of Citrus Root Responses to Candidatus Liberibacter asiaticus Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126973

    Expression of 16 differentially expressed genes at 20 dpi (A) and 50 dpi (B) as determined by quantitative real time PCR. C indicates the expression level determined by RNA-seq. RIN4 : RPM1 interacting protein 4 ; RPS2 : disease resistant protein ribosomal protein S2 ; NPR1 : regulatory protein nonexpresser of PR genes 1 ; DRP : disease resistant protein (TIR-NBS-LRR class) ; PP2-B15 : Phloem protein 2-B15 ; BAM : β-amylase ; PMEI : pectin methylesterase inhibitor ; TPP : trehalose-6-phosphate phosphatase ; XTR6 : Xyloglucan endotransglycosylase 6 ; BZIP : bZIP transcription factor ; CRPK : cysteine-rich protein kinase ; ACR4 : Act repeat 4 ; BRI1 : BRI1 kinase inhibitor 1 ; KCS6 : 3-ketoacyl-CoA synthase 6 .
    Figure Legend Snippet: Expression of 16 differentially expressed genes at 20 dpi (A) and 50 dpi (B) as determined by quantitative real time PCR. C indicates the expression level determined by RNA-seq. RIN4 : RPM1 interacting protein 4 ; RPS2 : disease resistant protein ribosomal protein S2 ; NPR1 : regulatory protein nonexpresser of PR genes 1 ; DRP : disease resistant protein (TIR-NBS-LRR class) ; PP2-B15 : Phloem protein 2-B15 ; BAM : β-amylase ; PMEI : pectin methylesterase inhibitor ; TPP : trehalose-6-phosphate phosphatase ; XTR6 : Xyloglucan endotransglycosylase 6 ; BZIP : bZIP transcription factor ; CRPK : cysteine-rich protein kinase ; ACR4 : Act repeat 4 ; BRI1 : BRI1 kinase inhibitor 1 ; KCS6 : 3-ketoacyl-CoA synthase 6 .

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Activated Clotting Time Assay

    16) Product Images from "The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells"

    Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-015-0099-5

    The transcription factor MITF is expressed in progenitor cells, leucocytes and primary moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or IL-10 (10 ng/mL). For the analysis of CD34 + progenitor and blood cells, cell-type specific total RNA was used. qRT-PCR analysis: (A, B) relative level of MITF and (C, D) GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (E) MITF protein level and phosphorylation status was analyzed by western blotting in two different donors (lanes 1–4 and lanes 5–7, respectively). Phosphorylated MITF was detected by mobility shift (slower migrating band at 70 kDa). Western blotting revealed an additional, slower migrating band that supposedly represents the phosphorylated protein of higher molecular weight of approximately 70 kDa. “+ phosphatase”: cell lysates were incubated with phosphatase. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.
    Figure Legend Snippet: The transcription factor MITF is expressed in progenitor cells, leucocytes and primary moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or IL-10 (10 ng/mL). For the analysis of CD34 + progenitor and blood cells, cell-type specific total RNA was used. qRT-PCR analysis: (A, B) relative level of MITF and (C, D) GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (E) MITF protein level and phosphorylation status was analyzed by western blotting in two different donors (lanes 1–4 and lanes 5–7, respectively). Phosphorylated MITF was detected by mobility shift (slower migrating band at 70 kDa). Western blotting revealed an additional, slower migrating band that supposedly represents the phosphorylated protein of higher molecular weight of approximately 70 kDa. “+ phosphatase”: cell lysates were incubated with phosphatase. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Techniques Used: Generated, In Vitro, Quantitative RT-PCR, Western Blot, Mobility Shift, Molecular Weight, Incubation

    PI3K/Akt-inhibition upregulates GPNMB gene expression in human moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or inhibitors of signal transduction (300 nM Akt inhibitor MK2206 (Akt-inh.), 300 nM Erk inhibitor FR180204 (Erk-inh.), 100 nM PI3K inhibitor LY294002 (PI3K-inh.), 20 nM c-Raf inhibitor 553003 (c-Raf-inh.)) and analyzed for GPNMB expression. Exemplary results from at least three independent experiments using different donors are presented. (A) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (B, C) GPNMB protein level of CD209 + moDC (of three different donors) was analyzed by flow cytometry. Where indicated, maturation of moDC was induced by LPS. Data were analyzed using FlowJo software and Difference in Median Fluorescence Intensity (DMFI) of CD209 + cells is shown in the upper right quadrants. (D) Phenotypic changes of immature moDC in the absence (4/GM) or presence of nilotinib or Akt inhibitor were analyzed by flow cytometry. Double stainings were performed with monoclonal antibodies recognizing CD209, CD1a or CD14. DMFI of CD209 + cells is shown in the upper right quadrants.
    Figure Legend Snippet: PI3K/Akt-inhibition upregulates GPNMB gene expression in human moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or inhibitors of signal transduction (300 nM Akt inhibitor MK2206 (Akt-inh.), 300 nM Erk inhibitor FR180204 (Erk-inh.), 100 nM PI3K inhibitor LY294002 (PI3K-inh.), 20 nM c-Raf inhibitor 553003 (c-Raf-inh.)) and analyzed for GPNMB expression. Exemplary results from at least three independent experiments using different donors are presented. (A) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (B, C) GPNMB protein level of CD209 + moDC (of three different donors) was analyzed by flow cytometry. Where indicated, maturation of moDC was induced by LPS. Data were analyzed using FlowJo software and Difference in Median Fluorescence Intensity (DMFI) of CD209 + cells is shown in the upper right quadrants. (D) Phenotypic changes of immature moDC in the absence (4/GM) or presence of nilotinib or Akt inhibitor were analyzed by flow cytometry. Double stainings were performed with monoclonal antibodies recognizing CD209, CD1a or CD14. DMFI of CD209 + cells is shown in the upper right quadrants.

    Techniques Used: Inhibition, Expressing, Generated, In Vitro, Transduction, Quantitative RT-PCR, Flow Cytometry, Cytometry, Software, Fluorescence

    MITF-Inhibition decreases GPNMB gene expression in moDC. moDC were generated in vitro with GM-CSF, IL-4 and DMSO alone (4/GM) with or without IL-10 and additional MITF inhibitor ML329 (MITF-inh.; 600 nM - 2000 nM) or KLF5 expression inhibitor CID (2000 nM) as control and analyzed for GPNMB expression. (A, B) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (C) GPNMB protein levels were analyzed by western blotting. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.
    Figure Legend Snippet: MITF-Inhibition decreases GPNMB gene expression in moDC. moDC were generated in vitro with GM-CSF, IL-4 and DMSO alone (4/GM) with or without IL-10 and additional MITF inhibitor ML329 (MITF-inh.; 600 nM - 2000 nM) or KLF5 expression inhibitor CID (2000 nM) as control and analyzed for GPNMB expression. (A, B) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (C) GPNMB protein levels were analyzed by western blotting. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Techniques Used: Inhibition, Expressing, Generated, In Vitro, Quantitative RT-PCR, Western Blot

    17) Product Images from "Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells"

    Article Title: Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00942.x

    High ALDH activity and Aldh1a1 expression are restricted to human myoblasts. (A) Primary cultures of mouse myoblasts (upper panels) and cornea cells (lower panels) were stained with ALDEFLUOR in the presence (left panels) or not (right panels) of DEAB and analysed by FACS. (B) Protein extracts from human myoblasts (human), mouse myoblasts (mouse), mouse cornea cells (cornea (m)), rat myoblasts (rat), rabbit myoblasts (rabbit) and non-human primate myoblasts (n.h. primate) were analysed for Aldh1a1 expression. Loading control was assessed with α tubulin (αTub) expression. (C) Quantitative reverse transcriptase-polymerase chain reaction analysis (RT-PCR) of relative levels of Aldh1a1 mRNA in mouse myoblasts, fractionated ALDH high and ALDH low human myoblasts and in mouse cornea cells. Expression was normalized against 28s rRNA and the lowest level of Aldh1a1 expression was set at 1. Means were given ±S.E.M.; n = 3.
    Figure Legend Snippet: High ALDH activity and Aldh1a1 expression are restricted to human myoblasts. (A) Primary cultures of mouse myoblasts (upper panels) and cornea cells (lower panels) were stained with ALDEFLUOR in the presence (left panels) or not (right panels) of DEAB and analysed by FACS. (B) Protein extracts from human myoblasts (human), mouse myoblasts (mouse), mouse cornea cells (cornea (m)), rat myoblasts (rat), rabbit myoblasts (rabbit) and non-human primate myoblasts (n.h. primate) were analysed for Aldh1a1 expression. Loading control was assessed with α tubulin (αTub) expression. (C) Quantitative reverse transcriptase-polymerase chain reaction analysis (RT-PCR) of relative levels of Aldh1a1 mRNA in mouse myoblasts, fractionated ALDH high and ALDH low human myoblasts and in mouse cornea cells. Expression was normalized against 28s rRNA and the lowest level of Aldh1a1 expression was set at 1. Means were given ±S.E.M.; n = 3.

    Techniques Used: Activity Assay, Expressing, Staining, FACS, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Aldh1a1 expression is associated with ALDH activity. (A) Proteins extracts from CD56 + myoblasts and CD56 − cells were analysed for Aldh1a1 expression. (B) Fractionated ALDH high and ALDH low myoblasts (clone hm1 myoblasts) were analysed for Aldh1a1 expression. Loading control was assessed with a tubulin (αTub) expression. (C) Quantitative reverse transcriptase-polymerase chain reaction analysis (RT-PCR) of relative levels of Aldh1a1 mRNA in human myoblasts (myoblasts) and in fractionated ALDH high and ALDH low human myoblasts (clone hm1 myoblasts). Expression was normalized against 28s rRNA and the lowest level of Aldh1a1 expression was set at 1. Means were given ±S.E.M.; n = 3.
    Figure Legend Snippet: Aldh1a1 expression is associated with ALDH activity. (A) Proteins extracts from CD56 + myoblasts and CD56 − cells were analysed for Aldh1a1 expression. (B) Fractionated ALDH high and ALDH low myoblasts (clone hm1 myoblasts) were analysed for Aldh1a1 expression. Loading control was assessed with a tubulin (αTub) expression. (C) Quantitative reverse transcriptase-polymerase chain reaction analysis (RT-PCR) of relative levels of Aldh1a1 mRNA in human myoblasts (myoblasts) and in fractionated ALDH high and ALDH low human myoblasts (clone hm1 myoblasts). Expression was normalized against 28s rRNA and the lowest level of Aldh1a1 expression was set at 1. Means were given ±S.E.M.; n = 3.

    Techniques Used: Expressing, Activity Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Aldh1a1 contributes to most if not all ALDH activity in human myoblasts. (A, B) Human myoblasts, transduced or not (not-inf.) with lentiviral mediated expression of control shRNA (shc) or Aldh1a1 shRNA (sh98, sh82, sh02) were analysed for Aldh1a1 expression by Western blot (A) and ALDH activity in the presence or not of DEAB by FACS (B).
    Figure Legend Snippet: Aldh1a1 contributes to most if not all ALDH activity in human myoblasts. (A, B) Human myoblasts, transduced or not (not-inf.) with lentiviral mediated expression of control shRNA (shc) or Aldh1a1 shRNA (sh98, sh82, sh02) were analysed for Aldh1a1 expression by Western blot (A) and ALDH activity in the presence or not of DEAB by FACS (B).

    Techniques Used: Activity Assay, Expressing, shRNA, Western Blot, FACS

    18) Product Images from "Cybrid studies establish the causal link between the mtDNA m.3890G > A/MT-ND1 mutation and optic atrophy with bilateral brainstem lesions"

    Article Title: Cybrid studies establish the causal link between the mtDNA m.3890G > A/MT-ND1 mutation and optic atrophy with bilateral brainstem lesions

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbadis.2012.12.002

    Quantification of the m.3890G > A/MT-ND1 mutation load by ARMS qPCR (A) in proband's skeletal muscle, blood cells and urinary epithelium, and in family (B) members' urinary epithelium, expressed as % of mutant 3890A compared to wild-type (wt) 3890G. The arrow indicates the proband, while asterisks indicate family members who were investigated.
    Figure Legend Snippet: Quantification of the m.3890G > A/MT-ND1 mutation load by ARMS qPCR (A) in proband's skeletal muscle, blood cells and urinary epithelium, and in family (B) members' urinary epithelium, expressed as % of mutant 3890A compared to wild-type (wt) 3890G. The arrow indicates the proband, while asterisks indicate family members who were investigated.

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction

    19) Product Images from "Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems"

    Article Title: Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091156

    Phylogenetic trees of genome-length HCV RNA populations obtained in long-term cell culture. The phylogenetic trees are depicted on the basis of nucleotide sequences of all cDNA clones obtained by 0-year, 2-year, and 4-year cultures of OL, OL8, OL11, and OL14 cells. (A) The 5′-terminus-NS2 regions of genome-length HCV RNA. ON/C-2 indicates the original sequences of the 5′-terminus-NS2 regions of ON/C-5B/QR,KE,SR RNA [21] . (B) The NS3-NS5B regions of genome-length HCV RNA. O/3-5B/QR,KE,SR indicates the original sequences of the NS3-NS5B regions of ON/C-5B/QR,KE,SR RNA [21] .
    Figure Legend Snippet: Phylogenetic trees of genome-length HCV RNA populations obtained in long-term cell culture. The phylogenetic trees are depicted on the basis of nucleotide sequences of all cDNA clones obtained by 0-year, 2-year, and 4-year cultures of OL, OL8, OL11, and OL14 cells. (A) The 5′-terminus-NS2 regions of genome-length HCV RNA. ON/C-2 indicates the original sequences of the 5′-terminus-NS2 regions of ON/C-5B/QR,KE,SR RNA [21] . (B) The NS3-NS5B regions of genome-length HCV RNA. O/3-5B/QR,KE,SR indicates the original sequences of the NS3-NS5B regions of ON/C-5B/QR,KE,SR RNA [21] .

    Techniques Used: Cell Culture, Clone Assay

    Genetic deletions occurred in the first half of genome-length HCV RNAs during the long-term cell culture. The conservative deleted portions in the genome-length HCV RNAs derived from OL8(2Y), OL8(4Y), OL11(2Y), OL11(4Y), OL14(2Y), or OL14(4Y) cells were shown by boxes. The original sequence was from ON/C-5B/QR,KE,SR RNA [21] .
    Figure Legend Snippet: Genetic deletions occurred in the first half of genome-length HCV RNAs during the long-term cell culture. The conservative deleted portions in the genome-length HCV RNAs derived from OL8(2Y), OL8(4Y), OL11(2Y), OL11(4Y), OL14(2Y), or OL14(4Y) cells were shown by boxes. The original sequence was from ON/C-5B/QR,KE,SR RNA [21] .

    Techniques Used: Cell Culture, Derivative Assay, Sequencing

    Characterization of genome-length HCV RNA-replicating cells after 4 years in culture. (A) Quantitative analysis of intracellular genome-length HCV RNA. The total RNAs from OL(4Y), OL8(4Y), OL11(4Y), and OL14(4Y) cells used were analyzed. The levels of intracellular genome-length HCV RNA were quantified by LightCycler PCR. OL(0Y) and Li23 cells were used as a positive and a negative control, respectively. (B) Western blot analysis. The cellular lysates from the cells used for RT-PCR analysis were also used for comparison. NS4A and NS5B were detected by Western blot analysis. β-actin was used as a control for the amount of protein loaded per lane.
    Figure Legend Snippet: Characterization of genome-length HCV RNA-replicating cells after 4 years in culture. (A) Quantitative analysis of intracellular genome-length HCV RNA. The total RNAs from OL(4Y), OL8(4Y), OL11(4Y), and OL14(4Y) cells used were analyzed. The levels of intracellular genome-length HCV RNA were quantified by LightCycler PCR. OL(0Y) and Li23 cells were used as a positive and a negative control, respectively. (B) Western blot analysis. The cellular lysates from the cells used for RT-PCR analysis were also used for comparison. NS4A and NS5B were detected by Western blot analysis. β-actin was used as a control for the amount of protein loaded per lane.

    Techniques Used: Polymerase Chain Reaction, Negative Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Increased GC content of genome-length HCV RNAs occurring in long-term RNA replication. The GC content of cDNA clones obtained by 0-year, 2-year, and 4-year culture of OL, OL8, OL11, and OL14 cells was calculated. The values indicate the means of 10 clones (OL) or 3 clones (OL8, OL11, or OL14). (A) The 5′-terminus-NS2 regions. (B) The NS3-NS5B regions.
    Figure Legend Snippet: Increased GC content of genome-length HCV RNAs occurring in long-term RNA replication. The GC content of cDNA clones obtained by 0-year, 2-year, and 4-year culture of OL, OL8, OL11, and OL14 cells was calculated. The values indicate the means of 10 clones (OL) or 3 clones (OL8, OL11, or OL14). (A) The 5′-terminus-NS2 regions. (B) The NS3-NS5B regions.

    Techniques Used: Clone Assay

    Sensitivity to telaprevir of the 4-year cultured genome-length HCV RNA-replicating cells. (A) Telaprevir sensitivities on genome-length HCV RNA replication in OL(4Y), OL8(4Y), OL11(4Y), and OL14(4Y) cells. OL(0Y) cells were used as a control. The cells were treated with telaprevir for 72 h, and then the levels of intracellular genome-length HCV RNA were quantified by LightCycler PCR. (B) Telaprevir-treated OL(0Y) and OL(4Y) cells (designated as OL(0Y)T and OL(4Y)T, respectively) became telaprevir-resistant easily. Telaprevir treatment and quantitative RT-PCR were preformed as shown in (A).
    Figure Legend Snippet: Sensitivity to telaprevir of the 4-year cultured genome-length HCV RNA-replicating cells. (A) Telaprevir sensitivities on genome-length HCV RNA replication in OL(4Y), OL8(4Y), OL11(4Y), and OL14(4Y) cells. OL(0Y) cells were used as a control. The cells were treated with telaprevir for 72 h, and then the levels of intracellular genome-length HCV RNA were quantified by LightCycler PCR. (B) Telaprevir-treated OL(0Y) and OL(4Y) cells (designated as OL(0Y)T and OL(4Y)T, respectively) became telaprevir-resistant easily. Telaprevir treatment and quantitative RT-PCR were preformed as shown in (A).

    Techniques Used: Cell Culture, Polymerase Chain Reaction, Quantitative RT-PCR

    Mutation rates of genome-length HCV RNAs in long-term cell culture. The mutation rates of three regions (5′-terminus-EMCV-IRES, Core-NS2, and NS3-NS5B) of genome-length HCV RNAs (OL, OL8, OL11, and OL14) were calculated using the sequence data obtained from 2- or 4-year cell culture. The vertical line indicates the means of the mutation rates calculated using the nucleotide sequences of 10 clones (OL) or 3 clones (OL8, OL11, or OL14) of genome-length HCV RNAs, by comparison with the original sequence (ON/C-5B/QR,KE,SR RNA) [21] .
    Figure Legend Snippet: Mutation rates of genome-length HCV RNAs in long-term cell culture. The mutation rates of three regions (5′-terminus-EMCV-IRES, Core-NS2, and NS3-NS5B) of genome-length HCV RNAs (OL, OL8, OL11, and OL14) were calculated using the sequence data obtained from 2- or 4-year cell culture. The vertical line indicates the means of the mutation rates calculated using the nucleotide sequences of 10 clones (OL) or 3 clones (OL8, OL11, or OL14) of genome-length HCV RNAs, by comparison with the original sequence (ON/C-5B/QR,KE,SR RNA) [21] .

    Techniques Used: Mutagenesis, Cell Culture, Sequencing, Clone Assay

    Genetic variations occurring in long-term replication of genome-length HCV RNAs. (A) Genetic variations in the 5′-terminus-NS2 regions. The left vertical line indicates the mean numbers of base substitutions detected per cDNA clone, by comparison with ON/C-5B/QR,KE,SR RNA [21] . The right vertical line indicates the mean numbers of aa substitutions in the Core-NS2 regions deduced per cDNA clone, by comparison with the original aa sequences deduced from ON/C-5B/QR, KE, SR RNA [21] . (B) Genetic variations in the NS3-NS5B regions. The mean numbers of base substitutions and aa substitutions are indicated as shown in (A).
    Figure Legend Snippet: Genetic variations occurring in long-term replication of genome-length HCV RNAs. (A) Genetic variations in the 5′-terminus-NS2 regions. The left vertical line indicates the mean numbers of base substitutions detected per cDNA clone, by comparison with ON/C-5B/QR,KE,SR RNA [21] . The right vertical line indicates the mean numbers of aa substitutions in the Core-NS2 regions deduced per cDNA clone, by comparison with the original aa sequences deduced from ON/C-5B/QR, KE, SR RNA [21] . (B) Genetic variations in the NS3-NS5B regions. The mean numbers of base substitutions and aa substitutions are indicated as shown in (A).

    Techniques Used:

    20) Product Images from "Anti-Inflammatory Cytokine Interleukin-4 Inhibits Inducible Nitric Oxide Synthase Gene Expression in the Mouse Macrophage Cell Line RAW264.7 through the Repression of Octamer-Dependent Transcription"

    Article Title: Anti-Inflammatory Cytokine Interleukin-4 Inhibits Inducible Nitric Oxide Synthase Gene Expression in the Mouse Macrophage Cell Line RAW264.7 through the Repression of Octamer-Dependent Transcription

    Journal: Mediators of Inflammation

    doi: 10.1155/2013/369693

    Mutational analysis of the mouse Nos2 enhancer/promoter region by luciferase reporter assays in RAW264.7 cells. The diagram on the left shows the wild-type (pNOS-996 and pNOS-333) and mutant Nos2 luciferase reporter constructs. Mutations of the NF- κ B and/or OCT sites in the constructs are also indicated. RAW264.7 cells were transiently transfected with the wild-type or the mutant Nos2 luciferase reporter construct, as described above. The relative luciferase activities are shown as percentages of the activity in cells transfected with the wild-type construct (pNOS-996) and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P
    Figure Legend Snippet: Mutational analysis of the mouse Nos2 enhancer/promoter region by luciferase reporter assays in RAW264.7 cells. The diagram on the left shows the wild-type (pNOS-996 and pNOS-333) and mutant Nos2 luciferase reporter constructs. Mutations of the NF- κ B and/or OCT sites in the constructs are also indicated. RAW264.7 cells were transiently transfected with the wild-type or the mutant Nos2 luciferase reporter construct, as described above. The relative luciferase activities are shown as percentages of the activity in cells transfected with the wild-type construct (pNOS-996) and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P

    Techniques Used: Luciferase, Mutagenesis, Construct, Transfection, Activity Assay

    The OCT site in the Nos2 promoter region is required for IFN γ - and LPS-induced promoter activity. (a) The diagram shows the wild-type (wt) and mutant sequences of the OCT site (mOCT) in the Nos2 minimum promoter region. The numbers above the promoter region refer to the nucleotide positions relative to the transcription start site of the mouse Nos2 gene. (b) RAW264.7 cells were transiently transfected with the NOS-62 luciferase reporter construct or a mutant construct containing a mutated OCT site (mOCT), as described above. The relative luciferase activities are shown as percentages of the activity in cells transfected with the wild-type construct (pNOS-996) and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P
    Figure Legend Snippet: The OCT site in the Nos2 promoter region is required for IFN γ - and LPS-induced promoter activity. (a) The diagram shows the wild-type (wt) and mutant sequences of the OCT site (mOCT) in the Nos2 minimum promoter region. The numbers above the promoter region refer to the nucleotide positions relative to the transcription start site of the mouse Nos2 gene. (b) RAW264.7 cells were transiently transfected with the NOS-62 luciferase reporter construct or a mutant construct containing a mutated OCT site (mOCT), as described above. The relative luciferase activities are shown as percentages of the activity in cells transfected with the wild-type construct (pNOS-996) and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Luciferase, Construct

    Stat6 is required for the IL-4-mediated inhibition of Nos2 gene expression in RAW264.7 cells. (a) RAW264.7 cells were transfected with control siRNA (100 nM) or Stat6 siRNA (100 nM) for 36 hours; total RNA was then prepared for quantitative real-time RT-PCR. Each column and bar represents the mean ± SEM of three independent experiments. (b) RAW264.7 cells were transfected with siRNA, as described above, and then used to prepare total cellular lysates for a western blot analysis using an anti-Stat6 antibody. (c, d) RAW264.7 cells were transfected with control siRNA (100 nM) or Stat6 siRNA (100 nM). Thirty-six hours after transfection, the cells were either left untreated (UT) or treated with IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the preparation of total RNA for quantitative real-time RT-PCR. The relative Nos2 mRNA expression levels are shown as percentages of the activity of cells transfected with the control siRNA and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P
    Figure Legend Snippet: Stat6 is required for the IL-4-mediated inhibition of Nos2 gene expression in RAW264.7 cells. (a) RAW264.7 cells were transfected with control siRNA (100 nM) or Stat6 siRNA (100 nM) for 36 hours; total RNA was then prepared for quantitative real-time RT-PCR. Each column and bar represents the mean ± SEM of three independent experiments. (b) RAW264.7 cells were transfected with siRNA, as described above, and then used to prepare total cellular lysates for a western blot analysis using an anti-Stat6 antibody. (c, d) RAW264.7 cells were transfected with control siRNA (100 nM) or Stat6 siRNA (100 nM). Thirty-six hours after transfection, the cells were either left untreated (UT) or treated with IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the preparation of total RNA for quantitative real-time RT-PCR. The relative Nos2 mRNA expression levels are shown as percentages of the activity of cells transfected with the control siRNA and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P

    Techniques Used: Inhibition, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay

    Overexpression of the coactivator CBP partially attenuates the IL-4-mediated inhibition of Nos2 promoter activity in RAW264.7 cells. (a) Analysis of endogenous BOB.1 expression in RAW264.7 cells. The cells were treated with medium alone (UT) or IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the preparation of nuclear extracts. Twenty micrograms of nuclear extract was analyzed by western blotting using an antibody against BOB.1 or an antibody against TATA-binding protein (TBP), which was used as a loading control. Nuclear extracts from the mouse leukemia cell line BCL1-B20 (BCL) were used as a positive control for BOB.1 expression (lane 1). (b) RAW264.7 cells were transiently co-transfected with either the empty vector or wild-type CBP expression plasmid and the pNOS-62 luciferase reporter construct. Twenty-four hours after transfection, the cells were treated with medium alone (untreated: UT) or IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the measurement of luciferase activity. The relative luciferase activities are shown as the percentage of the activity of cells transfected with the empty vector and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P
    Figure Legend Snippet: Overexpression of the coactivator CBP partially attenuates the IL-4-mediated inhibition of Nos2 promoter activity in RAW264.7 cells. (a) Analysis of endogenous BOB.1 expression in RAW264.7 cells. The cells were treated with medium alone (UT) or IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the preparation of nuclear extracts. Twenty micrograms of nuclear extract was analyzed by western blotting using an antibody against BOB.1 or an antibody against TATA-binding protein (TBP), which was used as a loading control. Nuclear extracts from the mouse leukemia cell line BCL1-B20 (BCL) were used as a positive control for BOB.1 expression (lane 1). (b) RAW264.7 cells were transiently co-transfected with either the empty vector or wild-type CBP expression plasmid and the pNOS-62 luciferase reporter construct. Twenty-four hours after transfection, the cells were treated with medium alone (untreated: UT) or IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before the measurement of luciferase activity. The relative luciferase activities are shown as the percentage of the activity of cells transfected with the empty vector and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures treated with IL-4 ( P

    Techniques Used: Over Expression, Inhibition, Activity Assay, Expressing, Western Blot, Binding Assay, Positive Control, Transfection, Plasmid Preparation, Luciferase, Construct

    Deletion analysis of the mouse Nos2 enhancer/promoter region by luciferase reporter assays in RAW264.7 cells. The diagram on the left shows the wild-type (pNOS-996) and deletion constructs of the Nos2 luciferase reporter. The numbers above the enhancer/promoter region refer to the nucleotide positions relative to the transcriptional start site of the mouse Nos2 gene. NF- κ B, nuclear factor kappa B; GAS, gamma-IFN activation sequence; ISRE, interferon-stimulated responsive element; NF-IL-6, nuclear factor IL-6; OCT, octamer transcription factor; TATA, TATA-box. RAW264.7 cells were transiently transfected with wild-type or mutant Nos2 luciferase reporter constructs. Twenty-four hours after transfection, the cells were either left untreated (UT) or treated with IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before measurement of the luciferase activity. The relative luciferase activities are shown as percentages of the activity in cells transfected with the wild-type construct (pNOS-996) and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of five independent experiments. The asterisks denote a statistically significant difference compared to the cultures with IL-4 ( P
    Figure Legend Snippet: Deletion analysis of the mouse Nos2 enhancer/promoter region by luciferase reporter assays in RAW264.7 cells. The diagram on the left shows the wild-type (pNOS-996) and deletion constructs of the Nos2 luciferase reporter. The numbers above the enhancer/promoter region refer to the nucleotide positions relative to the transcriptional start site of the mouse Nos2 gene. NF- κ B, nuclear factor kappa B; GAS, gamma-IFN activation sequence; ISRE, interferon-stimulated responsive element; NF-IL-6, nuclear factor IL-6; OCT, octamer transcription factor; TATA, TATA-box. RAW264.7 cells were transiently transfected with wild-type or mutant Nos2 luciferase reporter constructs. Twenty-four hours after transfection, the cells were either left untreated (UT) or treated with IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 8 hours before measurement of the luciferase activity. The relative luciferase activities are shown as percentages of the activity in cells transfected with the wild-type construct (pNOS-996) and stimulated with IFN γ and LPS. Each column and bar represents the mean ± SEM of five independent experiments. The asterisks denote a statistically significant difference compared to the cultures with IL-4 ( P

    Techniques Used: Luciferase, Construct, Activation Assay, Sequencing, Transfection, Mutagenesis, Activity Assay

    IL-4 inhibits IFN γ - and/or LPS-induced NO production and Nos2 mRNA expression in RAW264.7 cells. (a) RAW264.7 cells were treated with medium alone (untreated, UT) or IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL). The culture supernatants were harvested and assessed for NO production by the Griess assay. The protein concentrations of the residual cells in the cultures were also determined. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures with IL-4, (* P
    Figure Legend Snippet: IL-4 inhibits IFN γ - and/or LPS-induced NO production and Nos2 mRNA expression in RAW264.7 cells. (a) RAW264.7 cells were treated with medium alone (untreated, UT) or IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL). The culture supernatants were harvested and assessed for NO production by the Griess assay. The protein concentrations of the residual cells in the cultures were also determined. Each column and bar represents the mean ± SEM of three independent experiments. The asterisks denote a statistically significant difference compared to the cultures with IL-4, (* P

    Techniques Used: Expressing, Griess Assay

    Analysis of OCT DNA-binding activity in nuclear extracts from RAW264.7 cells. (a) Effect of IL-4 treatment on OCT DNA-binding activity. RAW264.7 cells were treated with medium alone or IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 4 hours before the preparation of nuclear extracts. The OCT binding activity was assessed by EMSA. (b) OCT DNA-binding activity in nuclear extracts from RAW264.7 cells treated with IFN γ and LPS. RAW264.7 cells were cultured in the presence of IFN γ (10 ng/mL) and LPS (100 ng/mL) for the indicated time prior to the preparation of nuclear extracts. In total, 10 μ g of each nuclear extract was analyzed for OCT binding activity by EMSA. (c) Analysis of OCT DNA-binding affinity to Nos2 OCT by an oligonucleotide competition assay. Nuclear extracts were prepared from RAW264.7 cells stimulated with IFN γ (10 ng/mL) and LPS (100 ng/mL) for 30 min. The OCT DNA-binding activity was determined by EMSA using radio-labeled OCT oligonucleotides corresponding to the Nos2 OCT (NOS OCT) site or the immunoglobulin κ chain OCT site (Igk OCT) in the presence or absence of a 25-fold excess of unlabeled wild-type (wt) or mutant (mut) oligonucleotide, as indicated. (d) Antibody super-shift assay for Nos2 OCT. Nuclear extracts (NE) from RAW264.7 cells stimulated with IFN γ (10 ng/mL) and LPS (100 ng/mL) for 30 min were incubated with the indicated antibodies (1 μ g each) before analysis of the binding activity, as described above.
    Figure Legend Snippet: Analysis of OCT DNA-binding activity in nuclear extracts from RAW264.7 cells. (a) Effect of IL-4 treatment on OCT DNA-binding activity. RAW264.7 cells were treated with medium alone or IL-4 (10 ng/mL) for 30 min prior to stimulation with IFN γ (10 ng/mL) and/or LPS (100 ng/mL) for 4 hours before the preparation of nuclear extracts. The OCT binding activity was assessed by EMSA. (b) OCT DNA-binding activity in nuclear extracts from RAW264.7 cells treated with IFN γ and LPS. RAW264.7 cells were cultured in the presence of IFN γ (10 ng/mL) and LPS (100 ng/mL) for the indicated time prior to the preparation of nuclear extracts. In total, 10 μ g of each nuclear extract was analyzed for OCT binding activity by EMSA. (c) Analysis of OCT DNA-binding affinity to Nos2 OCT by an oligonucleotide competition assay. Nuclear extracts were prepared from RAW264.7 cells stimulated with IFN γ (10 ng/mL) and LPS (100 ng/mL) for 30 min. The OCT DNA-binding activity was determined by EMSA using radio-labeled OCT oligonucleotides corresponding to the Nos2 OCT (NOS OCT) site or the immunoglobulin κ chain OCT site (Igk OCT) in the presence or absence of a 25-fold excess of unlabeled wild-type (wt) or mutant (mut) oligonucleotide, as indicated. (d) Antibody super-shift assay for Nos2 OCT. Nuclear extracts (NE) from RAW264.7 cells stimulated with IFN γ (10 ng/mL) and LPS (100 ng/mL) for 30 min were incubated with the indicated antibodies (1 μ g each) before analysis of the binding activity, as described above.

    Techniques Used: Binding Assay, Activity Assay, Cell Culture, Competitive Binding Assay, Labeling, Mutagenesis, Super-Shift Assay, Incubation

    21) Product Images from "NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2"

    Article Title: NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2

    Journal: Oncotarget

    doi:

    NBAT1 inhibits invasion of breast cancer cells by activating DKK1 expression a. , b . qRT-PCR and western blot analysis for DKK1 in NBAT1-expression MDA-MB-231 cells transfected with siRNA targeting DKK1 (NC, siDKK1-1 and siDKK1-2). c . Representative images of Boyden chamber assay for invaded cells (over-expression NBAT1 while inhibit DKK1). d . Histogram showing that the number of invaded cells with knockdown DKK1 was significantly higher than for NC, and similar to control groups (untreated, mock and vector, mean±SD, n=3, * P
    Figure Legend Snippet: NBAT1 inhibits invasion of breast cancer cells by activating DKK1 expression a. , b . qRT-PCR and western blot analysis for DKK1 in NBAT1-expression MDA-MB-231 cells transfected with siRNA targeting DKK1 (NC, siDKK1-1 and siDKK1-2). c . Representative images of Boyden chamber assay for invaded cells (over-expression NBAT1 while inhibit DKK1). d . Histogram showing that the number of invaded cells with knockdown DKK1 was significantly higher than for NC, and similar to control groups (untreated, mock and vector, mean±SD, n=3, * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Multiple Displacement Amplification, Transfection, Boyden Chamber Assay, Over Expression, Plasmid Preparation

    Over-expression of NBAT1 in MDA-MB-231 cells results in global gene expression profile change a . Heatmap representing hierarchical clustering of all dysregulated genes whose relative fold changes are more than 2 times compared MDA-MB-231/NBAT1 with MDA-MB-231/vector cells. b . Pathway-network analysis of the significant pathways of the differential expression genes. (Lines represent the relationship between the pathways, red to white represents the P value; the smaller the P value is, the deeper the red is.) c . The expression levels of DKK1, PRLR, NUPR1, PTGS2, WNT11 were determined in MDA-MB-231 with over-expression NBAT1 by qRT-PCR (mean±SD, n=3, *** p
    Figure Legend Snippet: Over-expression of NBAT1 in MDA-MB-231 cells results in global gene expression profile change a . Heatmap representing hierarchical clustering of all dysregulated genes whose relative fold changes are more than 2 times compared MDA-MB-231/NBAT1 with MDA-MB-231/vector cells. b . Pathway-network analysis of the significant pathways of the differential expression genes. (Lines represent the relationship between the pathways, red to white represents the P value; the smaller the P value is, the deeper the red is.) c . The expression levels of DKK1, PRLR, NUPR1, PTGS2, WNT11 were determined in MDA-MB-231 with over-expression NBAT1 by qRT-PCR (mean±SD, n=3, *** p

    Techniques Used: Over Expression, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Quantitative RT-PCR

    NBAT1 inhibits invasion of breast cancer cells via EZH2 a . Binding of NBAT1 to EZH2 complex in MDA-MB-231 cells, shown by RNA immunoprecipitation followed qRT-PCR (mean±SD, n=3, *** p
    Figure Legend Snippet: NBAT1 inhibits invasion of breast cancer cells via EZH2 a . Binding of NBAT1 to EZH2 complex in MDA-MB-231 cells, shown by RNA immunoprecipitation followed qRT-PCR (mean±SD, n=3, *** p

    Techniques Used: Binding Assay, Multiple Displacement Amplification, Immunoprecipitation, Quantitative RT-PCR

    22) 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

    23) Product Images from "Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line"

    Article Title: Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line

    Journal: Iranian Journal of Cancer Prevention

    doi: 10.17795/ijcp-3446

    Stemness Genes Expression of Spheroid and Parental Cells Have Assessed by Real-Time PCR Spheroid cells have shown higher expression of stemness genes, in comparison with parental cells (P
    Figure Legend Snippet: Stemness Genes Expression of Spheroid and Parental Cells Have Assessed by Real-Time PCR Spheroid cells have shown higher expression of stemness genes, in comparison with parental cells (P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    24) Product Images from "Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems"

    Article Title: Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

    Journal: Scientific Reports

    doi: 10.1038/srep30110

    Nanostring Microarray Analysis of Changes in microRNA Content in the Panc-1 Exosomes after Transfection with miR-155 and miR-125b Expressing Plasmid DNA. ( A ) Relative fold change as shown in the water-fall plot in microRNAs composition from Panc-1 exosomes after miR-155 transfection as compared to non-transfected exosomes. ( B ) Relative fold change in microRNAs composition from Panc-1 exosomes after miR-125b-2 transfection as compared to non-transfected exosomes. ( C ) The expression level of miR-155 transcript in miR-155 plasmid DNA transfected Panc-1 cells and exosomes by specific miR-155 Taqman primer ( D ) The expression level of miR-125b-2 transcript in miR-125b-2 plasmid DNA transfected Panc-1 cells and exosomes by specific miR-125b-2 Taqman primer. n = 3, *p
    Figure Legend Snippet: Nanostring Microarray Analysis of Changes in microRNA Content in the Panc-1 Exosomes after Transfection with miR-155 and miR-125b Expressing Plasmid DNA. ( A ) Relative fold change as shown in the water-fall plot in microRNAs composition from Panc-1 exosomes after miR-155 transfection as compared to non-transfected exosomes. ( B ) Relative fold change in microRNAs composition from Panc-1 exosomes after miR-125b-2 transfection as compared to non-transfected exosomes. ( C ) The expression level of miR-155 transcript in miR-155 plasmid DNA transfected Panc-1 cells and exosomes by specific miR-155 Taqman primer ( D ) The expression level of miR-125b-2 transcript in miR-125b-2 plasmid DNA transfected Panc-1 cells and exosomes by specific miR-125b-2 Taqman primer. n = 3, *p

    Techniques Used: Microarray, Transfection, Expressing, Plasmid Preparation

    Characterization of HA-PEI/HA-PEG Nanoparticles and Transfection of miR-155 and miR-125b in Panc-1 Cells using miR-Expressing Plasmid DNA. ( A ) HA-PEI/HA-PEG nanoparticle size distribution and zeta potential (surface charge) measurements of blank HA-PEI/HA-PEG in PBS by dynamic light scattering. ( B ) Transmission electron microscopy (TEM) image of blank HA-PEI/HA-PEG in PBS. ( C ) Size distribution and zeta potential measurements of miR125b-2 encapsulated HA-PEI/HA-PEG in PBS (9:1) by dynamic light scattering. D. TEM image of miR-125b-2 encapsulated HA-PEI/HA-PEG in PBS (9:1). ( E ) Fluorescent confocal microscopy images of miR-155/GFP plasmid DNA expression in Panc-1 cells at 12 h, 24 h, and 48 h post-transfection. ( F ) Quantitative analysis of miR125b-2 plasmid DNA transfection studies in Panc-1 cells and exosomes upon transfection with Lipofectamine ® and HA-PEI/HA-PEG nanoparticles. pPCR analysis was performed by miR125b-2 Taqman gene expression assay with specific miR-125b-2 Taqman primer. n = 3, *p
    Figure Legend Snippet: Characterization of HA-PEI/HA-PEG Nanoparticles and Transfection of miR-155 and miR-125b in Panc-1 Cells using miR-Expressing Plasmid DNA. ( A ) HA-PEI/HA-PEG nanoparticle size distribution and zeta potential (surface charge) measurements of blank HA-PEI/HA-PEG in PBS by dynamic light scattering. ( B ) Transmission electron microscopy (TEM) image of blank HA-PEI/HA-PEG in PBS. ( C ) Size distribution and zeta potential measurements of miR125b-2 encapsulated HA-PEI/HA-PEG in PBS (9:1) by dynamic light scattering. D. TEM image of miR-125b-2 encapsulated HA-PEI/HA-PEG in PBS (9:1). ( E ) Fluorescent confocal microscopy images of miR-155/GFP plasmid DNA expression in Panc-1 cells at 12 h, 24 h, and 48 h post-transfection. ( F ) Quantitative analysis of miR125b-2 plasmid DNA transfection studies in Panc-1 cells and exosomes upon transfection with Lipofectamine ® and HA-PEI/HA-PEG nanoparticles. pPCR analysis was performed by miR125b-2 Taqman gene expression assay with specific miR-125b-2 Taqman primer. n = 3, *p

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Confocal Microscopy

    Macrophage Re-programming with Exosomes Harvested from miR-155 and miR-125b-2 Transfected in Panc-1 Cells. Quantitative determination of ( A ) IL-1β and ( B ) Arg1 expression in M2 J774A.1 macrophages after treating 160 μg of miR-155/miR-125b-2-modified exosomes for 48 h. ( C ) IL-1β/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages treated with miR-155/miR-125b-2-modified exosomes for 48 h. Quantitative determination of ( D ) iNOS and ( E ) Arg1 expression in M2 J774A.1 macrophages after treating 160 μg of miR-155/miR-125b-2-modified exosomes for 48 h. ( F ) iNOS/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages treated with miR-155/miR-125b-2-modified exosomes for 48 h. n = 3, *p
    Figure Legend Snippet: Macrophage Re-programming with Exosomes Harvested from miR-155 and miR-125b-2 Transfected in Panc-1 Cells. Quantitative determination of ( A ) IL-1β and ( B ) Arg1 expression in M2 J774A.1 macrophages after treating 160 μg of miR-155/miR-125b-2-modified exosomes for 48 h. ( C ) IL-1β/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages treated with miR-155/miR-125b-2-modified exosomes for 48 h. Quantitative determination of ( D ) iNOS and ( E ) Arg1 expression in M2 J774A.1 macrophages after treating 160 μg of miR-155/miR-125b-2-modified exosomes for 48 h. ( F ) iNOS/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages treated with miR-155/miR-125b-2-modified exosomes for 48 h. n = 3, *p

    Techniques Used: Transfection, Expressing, Modification

    Macrophage Reprogramming with Exosomes Transferred in Transwell Co-Culture System from miR-155 and miR-125b-2 Transfected Panc-1 Cells. Quantitative determination of ( A ) IL-1β and ( B ) Arg1 expression in M2 J774A.1 macrophages after co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. ( C ) IL-1β/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. Quantitative Determination of ( D ) iNOS and ( E ) Arg1 expression in M2 J774A.1 macrophages co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. ( F ) iNOS/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. n = 3, *p
    Figure Legend Snippet: Macrophage Reprogramming with Exosomes Transferred in Transwell Co-Culture System from miR-155 and miR-125b-2 Transfected Panc-1 Cells. Quantitative determination of ( A ) IL-1β and ( B ) Arg1 expression in M2 J774A.1 macrophages after co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. ( C ) IL-1β/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. Quantitative Determination of ( D ) iNOS and ( E ) Arg1 expression in M2 J774A.1 macrophages co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. ( F ) iNOS/Arg1 (M1/M2) ratio of M2 J774A.1 macrophages co-culturing with miR-155/miR-125b-2-modified transfected panc-1 cells for 48 h. n = 3, *p

    Techniques Used: Co-Culture Assay, Transfection, Expressing, Modification

    25) Product Images from "ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production"

    Article Title: ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005568

    Identification of ABHD5 as a new host factor for HCV production. ( a, b ) A rational siRNA screen was designed to identify host factors involved in the lipid metabolism and participating in the HCV replication cycle (see S2 Fig ) with readouts for HCV entry and replication ( a ) or assembly and release ( b ). For each graph, the p-value is plotted against the median score. A maximal p-value of 0.05 together with a mean score superior to 2 (blue dots, antiviral factors) or inferior to -2 (green dots, proviral factors) was considered highly significant. CD81, PI4KA and APOE controls are shown in red in the relevant graph and the non-targeting negative control siRNAs in grey. ABHD5 is depicted with a diamond. Yellow dotted lines indicate our statistical thresholds. ( c, d ) ABHD5-specific siRNAs used in the initial screen as a pool (panels a and b) were transfected individually into HCV RNA-transfected cells. Their specific effect on HCV RNA replication (panel c , corrected for cell viability effects) and progeny virion production (panel d, corrected for HCV RNA replication effects) is depicted after normalisation to the average value of two non-targeting siRNAs. Note that statistics were performed at the gene level. ( e- g ) Effect of ABHD5-specific shRNAs on ABHD5 gene expression ( e ), HCV entry and replication ( f ) and HCV assembly and release ( g ). ( e ) ABHD5 mRNA levels were quantified by qRT-PCR at the time of virus harvest. ( f ) HCV entry and replication were determined by the RLuc activity in the producer cell lysates at the same time point and corrected for the effects on cell viability. ( g ) The efficiency of HCV production was evaluated by the RLuc activity in target cells infected with the supernatant of shRNA-transduced and JcR-2a-infected producer cells, and corrected for the shRNA effects on HCV entry and replication.
    Figure Legend Snippet: Identification of ABHD5 as a new host factor for HCV production. ( a, b ) A rational siRNA screen was designed to identify host factors involved in the lipid metabolism and participating in the HCV replication cycle (see S2 Fig ) with readouts for HCV entry and replication ( a ) or assembly and release ( b ). For each graph, the p-value is plotted against the median score. A maximal p-value of 0.05 together with a mean score superior to 2 (blue dots, antiviral factors) or inferior to -2 (green dots, proviral factors) was considered highly significant. CD81, PI4KA and APOE controls are shown in red in the relevant graph and the non-targeting negative control siRNAs in grey. ABHD5 is depicted with a diamond. Yellow dotted lines indicate our statistical thresholds. ( c, d ) ABHD5-specific siRNAs used in the initial screen as a pool (panels a and b) were transfected individually into HCV RNA-transfected cells. Their specific effect on HCV RNA replication (panel c , corrected for cell viability effects) and progeny virion production (panel d, corrected for HCV RNA replication effects) is depicted after normalisation to the average value of two non-targeting siRNAs. Note that statistics were performed at the gene level. ( e- g ) Effect of ABHD5-specific shRNAs on ABHD5 gene expression ( e ), HCV entry and replication ( f ) and HCV assembly and release ( g ). ( e ) ABHD5 mRNA levels were quantified by qRT-PCR at the time of virus harvest. ( f ) HCV entry and replication were determined by the RLuc activity in the producer cell lysates at the same time point and corrected for the effects on cell viability. ( g ) The efficiency of HCV production was evaluated by the RLuc activity in target cells infected with the supernatant of shRNA-transduced and JcR-2a-infected producer cells, and corrected for the shRNA effects on HCV entry and replication.

    Techniques Used: Negative Control, Transfection, Expressing, Quantitative RT-PCR, Activity Assay, Infection, shRNA

    26) Product Images from "Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination"

    Article Title: Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination

    Journal: Retrovirology

    doi: 10.1186/s12977-016-0257-x

    Impact of UNG2 and RPA32 on HIV-1 replication in PBMCs. a – c Viral replication. Replication-competent viruses were produced in shLuc- ( black curves and bars ), shUNG2- ( red curves and bars ) or shRPA32- ( green curves and bars ) transduced 293T cells, normalized for p24, and then used for infection in duplicate of PBMCs from five different healthy blood donors. Aliquots of PBMC culture supernatant were collected 2, 4 and 8 days after infection for p24 quantification. In a , the individual kinetics of replication in PBMCs from the five healthy donors are shown. In b , results are expressed as the percentage of p24 production at each time point relative to that of PBMCs infected with viruses produced in shLuc-transduced ( black bars ) 293T cells. Values are the means of two independent experiments performed on PBMCs from the five healthy donors. In c , PBMCs were collected 7 h after infection, subjected to DNA purification, and total viral DNA was quantified via qPCR using specific primers for U5 - gag . Results are expressed as the percentage of total viral DNA relative to that of PBMCs infected with viruses produced in shLuc-transduced ( black bar ) cells. d Virus infectivity. GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells as indicated, normalized for p24, and then used to infect PBMCs from three different donors. The percentage of GFP-positive infected cells was then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced 293T cells. Error bars represent the SEM. Statistical significance was determined by using the Students t test (ns, p > 0.05; *p
    Figure Legend Snippet: Impact of UNG2 and RPA32 on HIV-1 replication in PBMCs. a – c Viral replication. Replication-competent viruses were produced in shLuc- ( black curves and bars ), shUNG2- ( red curves and bars ) or shRPA32- ( green curves and bars ) transduced 293T cells, normalized for p24, and then used for infection in duplicate of PBMCs from five different healthy blood donors. Aliquots of PBMC culture supernatant were collected 2, 4 and 8 days after infection for p24 quantification. In a , the individual kinetics of replication in PBMCs from the five healthy donors are shown. In b , results are expressed as the percentage of p24 production at each time point relative to that of PBMCs infected with viruses produced in shLuc-transduced ( black bars ) 293T cells. Values are the means of two independent experiments performed on PBMCs from the five healthy donors. In c , PBMCs were collected 7 h after infection, subjected to DNA purification, and total viral DNA was quantified via qPCR using specific primers for U5 - gag . Results are expressed as the percentage of total viral DNA relative to that of PBMCs infected with viruses produced in shLuc-transduced ( black bar ) cells. d Virus infectivity. GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells as indicated, normalized for p24, and then used to infect PBMCs from three different donors. The percentage of GFP-positive infected cells was then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced 293T cells. Error bars represent the SEM. Statistical significance was determined by using the Students t test (ns, p > 0.05; *p

    Techniques Used: Produced, Infection, DNA Purification, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Impact of UNG2 and RPA32 for dissemination of cell-free virus particles between MDMs and Jurkat T cells. a Schematic representation of the experimental system. MDMs were infected with replication-competent HIV-1 (NL4.3 strain) co-expressing the VSV-G envelope, and the cell culture supernatant was then collected 8 days later. After p24 normalization, cell-free viruses produced by MDMs were used for infection of shLuc-, shUNG2- or shRPA32-transduced Jurkat cells, and virus production was monitored after infection. b and c Replication in Jurkat cells of MDMs-produced viruses. Replication-competent viruses were produced in MDMs and then used for infection of shLuc- ( black lines and bars ), shUNG2- ( red lines and bars ) or shRPA32- ( green lines and bars ) transduced Jurkat cells. Aliquots of cell culture supernatants were collected 2, 4 and 8 days after infection for p24 quantification. In b , the individual kinetics of replication in shRNA-tranduced Jurkat cells of viruses produced in MDMs from five different donors are shown. In c , results are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced Jurkat cells. Values are the means of two independent experiments performed with virus produced in MDMs from five different donors. Error bars represent the SEM. Statistical significance was determined using Students t test (ns, p > 0.05; *p
    Figure Legend Snippet: Impact of UNG2 and RPA32 for dissemination of cell-free virus particles between MDMs and Jurkat T cells. a Schematic representation of the experimental system. MDMs were infected with replication-competent HIV-1 (NL4.3 strain) co-expressing the VSV-G envelope, and the cell culture supernatant was then collected 8 days later. After p24 normalization, cell-free viruses produced by MDMs were used for infection of shLuc-, shUNG2- or shRPA32-transduced Jurkat cells, and virus production was monitored after infection. b and c Replication in Jurkat cells of MDMs-produced viruses. Replication-competent viruses were produced in MDMs and then used for infection of shLuc- ( black lines and bars ), shUNG2- ( red lines and bars ) or shRPA32- ( green lines and bars ) transduced Jurkat cells. Aliquots of cell culture supernatants were collected 2, 4 and 8 days after infection for p24 quantification. In b , the individual kinetics of replication in shRNA-tranduced Jurkat cells of viruses produced in MDMs from five different donors are shown. In c , results are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced Jurkat cells. Values are the means of two independent experiments performed with virus produced in MDMs from five different donors. Error bars represent the SEM. Statistical significance was determined using Students t test (ns, p > 0.05; *p

    Techniques Used: Infection, Expressing, Cell Culture, Produced, shRNA

    Impact of UNG2 and RPA32 on HIV-1 replication in macrophages. a – c Wild-type ( a ) or Δ vpr ( b ) replication-competent viruses were produced in shLuc- ( black curves and bars ), shUNG2- ( red curves and bars ) or shRPA32- ( green curves and bars ) transduced 293T cells, normalized for p24, and then used for infection in duplicate of MDMs from 3 different healthy donors. In a and b , aliquots of MDM cell culture supernatants were collected 4 and 8 days after infection for p24 quantification. The individual kinetics of replication in PBMCs from the three healthy donors are shown. In c , results are expressed as the percentage of p24 production at each time point relative to that of MDMs infected with wt or Δ vpr viruses produced in shLuc-transduced ( black bars ) cells. Values are the means of two independent experiments performed on MDMs from the two donors. d Virus infectivity in MDMs. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect MDMs from three different donors. The percentages of GFP-positive infected cells were then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced ( black bars ) 293T cells. e Replication-competent viruses were produced in shLuc-, shUNG2- or shRRA32-transduced 293T cells, normalized for p24, and then used for infection of MDMs from three different donors. MDM samples were collected 72 h after infection, subjected to DNA purification, and total viral DNA was quantified via qPCR using specific primers for U5 - gag . Results are expressed as the percentage of total viral DNA relative to that of MDMs infected with viruses produced in shLuc-transduced ( black bar ) cells. f Double-depletion of UNG2 and RPA32 expression in virus-producing 293T cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2 or Luciferase and containing the gene for puromycin resistance, and with lentiviral vectors expressing shRNA against RPA32 or Luciferase and the GFP reporter gene. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32 and anti-β-actin antibodies. g Replication-competent viruses were produced in shLuc/shLuc-GFP ( black bar ), in shUNG2/shLuc-GFP ( red bar ) or in shUNG2/shRPA32-GFP ( red and green hatched bar ) 293T cells, normalized for p24, and then used for infection of MDMs from three different healthy donors. The concentration of p24 after 8 days of infection was expressed as the percentage of p24 production relative to that of MDMs infected with viruses produced in shLuc-transduced ( black bar ) cells. Error bars represent the SEM. Statistical significance was determined using Students t test (ns, p > 0.05; *p
    Figure Legend Snippet: Impact of UNG2 and RPA32 on HIV-1 replication in macrophages. a – c Wild-type ( a ) or Δ vpr ( b ) replication-competent viruses were produced in shLuc- ( black curves and bars ), shUNG2- ( red curves and bars ) or shRPA32- ( green curves and bars ) transduced 293T cells, normalized for p24, and then used for infection in duplicate of MDMs from 3 different healthy donors. In a and b , aliquots of MDM cell culture supernatants were collected 4 and 8 days after infection for p24 quantification. The individual kinetics of replication in PBMCs from the three healthy donors are shown. In c , results are expressed as the percentage of p24 production at each time point relative to that of MDMs infected with wt or Δ vpr viruses produced in shLuc-transduced ( black bars ) cells. Values are the means of two independent experiments performed on MDMs from the two donors. d Virus infectivity in MDMs. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect MDMs from three different donors. The percentages of GFP-positive infected cells were then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced ( black bars ) 293T cells. e Replication-competent viruses were produced in shLuc-, shUNG2- or shRRA32-transduced 293T cells, normalized for p24, and then used for infection of MDMs from three different donors. MDM samples were collected 72 h after infection, subjected to DNA purification, and total viral DNA was quantified via qPCR using specific primers for U5 - gag . Results are expressed as the percentage of total viral DNA relative to that of MDMs infected with viruses produced in shLuc-transduced ( black bar ) cells. f Double-depletion of UNG2 and RPA32 expression in virus-producing 293T cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2 or Luciferase and containing the gene for puromycin resistance, and with lentiviral vectors expressing shRNA against RPA32 or Luciferase and the GFP reporter gene. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32 and anti-β-actin antibodies. g Replication-competent viruses were produced in shLuc/shLuc-GFP ( black bar ), in shUNG2/shLuc-GFP ( red bar ) or in shUNG2/shRPA32-GFP ( red and green hatched bar ) 293T cells, normalized for p24, and then used for infection of MDMs from three different healthy donors. The concentration of p24 after 8 days of infection was expressed as the percentage of p24 production relative to that of MDMs infected with viruses produced in shLuc-transduced ( black bar ) cells. Error bars represent the SEM. Statistical significance was determined using Students t test (ns, p > 0.05; *p

    Techniques Used: Produced, Infection, Cell Culture, Flow Cytometry, Cytometry, DNA Purification, Real-time Polymerase Chain Reaction, Expressing, Transduction, shRNA, Luciferase, Western Blot, Concentration Assay

    Impact of UNG2 and RPA32 for dissemination of cell-free virus particles between T cells and MDMs. a Schematic representation of the experimental system. shLuc, shUNG2- or shRPA32-transduced Jurkat cells were infected with HIV-1 (YU2 strain) expressing the VSV-G envelope, and the cell culture supernatant was then collected 3 days later. After p24 normalization, cell-free viruses produced by shRNA-transduced Jurkat cells were used for infection of MDMs, and virus production was monitored after infection. b and c Replication in MDMs of Jurkat cells-produced viruses. Replication-competent viruses were produced in shLuc-, shUNG2- or shRPA32-transduced Jurkat cells and then used to infect MDMs. Aliquots of cell culture supernatants were collected 4 and 8 days after infection for p24 quantification. In b , the individual kinetics of replication in MDMs from three different donors of viruses produced in shUNG2- or shRPA32-tranduced Jurkat cells are shown. In c , results are expressed as the percentage of p24 production at each time point relative to that of MDM cells infected with control viruses. Values are the means of two independent experiments performed with MDMs from three different donors. Error bars represent the SEM. Statistical significance was determined using Students t test (ns, p > 0.05; *p
    Figure Legend Snippet: Impact of UNG2 and RPA32 for dissemination of cell-free virus particles between T cells and MDMs. a Schematic representation of the experimental system. shLuc, shUNG2- or shRPA32-transduced Jurkat cells were infected with HIV-1 (YU2 strain) expressing the VSV-G envelope, and the cell culture supernatant was then collected 3 days later. After p24 normalization, cell-free viruses produced by shRNA-transduced Jurkat cells were used for infection of MDMs, and virus production was monitored after infection. b and c Replication in MDMs of Jurkat cells-produced viruses. Replication-competent viruses were produced in shLuc-, shUNG2- or shRPA32-transduced Jurkat cells and then used to infect MDMs. Aliquots of cell culture supernatants were collected 4 and 8 days after infection for p24 quantification. In b , the individual kinetics of replication in MDMs from three different donors of viruses produced in shUNG2- or shRPA32-tranduced Jurkat cells are shown. In c , results are expressed as the percentage of p24 production at each time point relative to that of MDM cells infected with control viruses. Values are the means of two independent experiments performed with MDMs from three different donors. Error bars represent the SEM. Statistical significance was determined using Students t test (ns, p > 0.05; *p

    Techniques Used: Infection, Expressing, Cell Culture, Produced, shRNA

    Impact of UNG2 and RPA32 depletion on HIV-1 replication in HeLa-CD4 cells. a Depletion of UNG2 and RPA32 in 293T ( left panels ) and HeLa-CD4 ( right panels ) cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2, RPA32 or Luciferase (Luc) used as a control. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32, anti-RPA70 and anti-β-actin antibodies. b , c Virus replication in UNG2- or RPA32-depleted cells. Replication-competent viruses were produced in UNG2-, RPA32-depleted or in control shLuc 293T cells, normalized for viral p24, and then used for infection of UNG2-depleted ( red line and bars ), RPA32-depleted ( green line and bars ) or control shLuc ( black line and bars ) HeLa-CD4 cells. Aliquots of cell culture supernatant were collected 2, 4, and 8 days after infection for p24 quantification. In b , the kinetic of replication shown is representative of four independent experiments. In c , results are the means of the four independent experiments and are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced HeLa-CD4 cells infected with control viruses. d Virus infectivity. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect shUNG2-, shRPA32- or shLuc-transduced HeLa-CD4 cells as indicated. The percentage of GFP-positive infected cells was then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in control 293T cells and measured on control HeLa-CD4 as target cells. e Quantification of total viral DNA. Infected HeLa-CD4 cells were collected 7 h after infection, subjected to DNA purification, and the total viral DNA was quantified by qPCR using specific primers for U5 - gag . Results are expressed as the percentage of total viral DNA relative to that of shLuc-transduced HeLa-CD4 cells infected with control viruses produced in shLuc-transduced 293T cells. Values are the means of at least three independent experiments. Error bars represent 1 SEM (standard error of the mean). Statistical significance was determined using Students t test (ns, p > 0.05; *p
    Figure Legend Snippet: Impact of UNG2 and RPA32 depletion on HIV-1 replication in HeLa-CD4 cells. a Depletion of UNG2 and RPA32 in 293T ( left panels ) and HeLa-CD4 ( right panels ) cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2, RPA32 or Luciferase (Luc) used as a control. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32, anti-RPA70 and anti-β-actin antibodies. b , c Virus replication in UNG2- or RPA32-depleted cells. Replication-competent viruses were produced in UNG2-, RPA32-depleted or in control shLuc 293T cells, normalized for viral p24, and then used for infection of UNG2-depleted ( red line and bars ), RPA32-depleted ( green line and bars ) or control shLuc ( black line and bars ) HeLa-CD4 cells. Aliquots of cell culture supernatant were collected 2, 4, and 8 days after infection for p24 quantification. In b , the kinetic of replication shown is representative of four independent experiments. In c , results are the means of the four independent experiments and are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced HeLa-CD4 cells infected with control viruses. d Virus infectivity. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect shUNG2-, shRPA32- or shLuc-transduced HeLa-CD4 cells as indicated. The percentage of GFP-positive infected cells was then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in control 293T cells and measured on control HeLa-CD4 as target cells. e Quantification of total viral DNA. Infected HeLa-CD4 cells were collected 7 h after infection, subjected to DNA purification, and the total viral DNA was quantified by qPCR using specific primers for U5 - gag . Results are expressed as the percentage of total viral DNA relative to that of shLuc-transduced HeLa-CD4 cells infected with control viruses produced in shLuc-transduced 293T cells. Values are the means of at least three independent experiments. Error bars represent 1 SEM (standard error of the mean). Statistical significance was determined using Students t test (ns, p > 0.05; *p

    Techniques Used: Transduction, Expressing, shRNA, Luciferase, Western Blot, Produced, Infection, Cell Culture, Flow Cytometry, Cytometry, DNA Purification, Real-time Polymerase Chain Reaction

    Impact of UNG2 and RPA32 on HIV-1 replication in Jurkat T cells. a Depletion of UNG2 and RPA32 in Jurkat cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2, RPA32 or Luciferase. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32, anti-RPA70 and anti-β-actin antibodies. b – e Virus replication in UNG2- or RPA32-depleted Jurkat cells. Replication-competent viruses were produced in UNG2- (B and D) or RPA32- ( c , e ) depleted cells or in control shLuc-transduced 293T cells, normalized for p24, and then used for infection of shLuc-, shUNG2- or shRPA32-transduced Jurkat cells. Aliquots of cell culture supernatant were collected 2, 4, and 8 days after infection for p24 quantification. In b , c , the kinetic of replication shown is representative of four independent experiments. In d , e , values are the means of the four independent experiments. Results are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced Jurkat cells infected with viruses produced in shLuc-transduced 293T cells. f Virus infectivity in UNG2- and RPA32-depleted cells. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect shUNG2-, shRPA32- or shLuc-transduced Jurkat cells as indicated. The percentage of GFP-positive infected cells was then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced 293T cells and measured on shLuc-transduced Jurkat as target cells. Error bars represent the SEM. Statistical significance was determined by using the Students t test (ns, p > 0.05; *p
    Figure Legend Snippet: Impact of UNG2 and RPA32 on HIV-1 replication in Jurkat T cells. a Depletion of UNG2 and RPA32 in Jurkat cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2, RPA32 or Luciferase. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32, anti-RPA70 and anti-β-actin antibodies. b – e Virus replication in UNG2- or RPA32-depleted Jurkat cells. Replication-competent viruses were produced in UNG2- (B and D) or RPA32- ( c , e ) depleted cells or in control shLuc-transduced 293T cells, normalized for p24, and then used for infection of shLuc-, shUNG2- or shRPA32-transduced Jurkat cells. Aliquots of cell culture supernatant were collected 2, 4, and 8 days after infection for p24 quantification. In b , c , the kinetic of replication shown is representative of four independent experiments. In d , e , values are the means of the four independent experiments. Results are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced Jurkat cells infected with viruses produced in shLuc-transduced 293T cells. f Virus infectivity in UNG2- and RPA32-depleted cells. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect shUNG2-, shRPA32- or shLuc-transduced Jurkat cells as indicated. The percentage of GFP-positive infected cells was then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced 293T cells and measured on shLuc-transduced Jurkat as target cells. Error bars represent the SEM. Statistical significance was determined by using the Students t test (ns, p > 0.05; *p

    Techniques Used: Transduction, Expressing, shRNA, Luciferase, Western Blot, Produced, Infection, Cell Culture, Flow Cytometry, Cytometry

    27) Product Images from "Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans"

    Article Title: Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005590

    The mRNA deadenylase Ccr4 regulates extracellular matrix production in biofilms. (A) qPCR analysis of the expression of mitochondrial biogenesis genes in biofilms grown for 48 h in Spider medium. SCR1 RNA was used for normalization. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: ***
    Figure Legend Snippet: The mRNA deadenylase Ccr4 regulates extracellular matrix production in biofilms. (A) qPCR analysis of the expression of mitochondrial biogenesis genes in biofilms grown for 48 h in Spider medium. SCR1 RNA was used for normalization. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: ***

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    28) Product Images from "Expression and clinical significance of the long non-coding RNA PVT1 in human gastric cancer"

    Article Title: Expression and clinical significance of the long non-coding RNA PVT1 in human gastric cancer

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S68854

    Ratio of relative PVT1 expression in cancerous tissues versus adjacent noncancerous tissues from 31 patients with gastric cancer, examined by quantitative reverse transcriptase polymerase chain reaction. N indicates the number of patients with lymph node metastasis.
    Figure Legend Snippet: Ratio of relative PVT1 expression in cancerous tissues versus adjacent noncancerous tissues from 31 patients with gastric cancer, examined by quantitative reverse transcriptase polymerase chain reaction. N indicates the number of patients with lymph node metastasis.

    Techniques Used: Expressing, Polymerase Chain Reaction

    29) Product Images from "Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, survivin, and Bcl-2"

    Article Title: Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, survivin, and Bcl-2

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    doi:

    Bcl-2 mRNA expression of ONHA and TMC after treatment with minocycline 20 μM and 40 μM only or after additional treatment with 600 mM H 2 O 2 or TGFβ-2, as investigated by quantitative RT-PCR. All tests were performed in triplicate and repeated three times. The mRNA levels were normalized to those of 18S rRNA and expressed as the ratio of Bcl-2 mRNA/18S rRNA. ONHA treated with minocycline concentrations showed a significant increase in Bcl-2 mRNA expression compared with the control in both cell lines. TGFβ-2 and H 2 O 2 treatment decreased Bcl-2 expression. When ONHA cells were treated with minocycline 20 μM and 40 μM and TGFβ-2 or H 2 O 2 , Bcl-2 expression was increased compared with ONHA treated only with TGFβ-2 or H 2 O 2 . Similarly, TMC treated with minocycline 20 μM and TGFβ-2 or H 2 O 2 showed an increased expression of Bcl-2 mRNA compared with TMC treated only with TGFβ-2 or H 2 O 2 . All differences between the two minocycline concentrations and the corresponding controls are statistically significant. Key: x-axis, RR of Bcl-2 mRNA normalized to 18s rRNA expressed in decimal format; y-axis, tested concentrations of minocycline. Abbreviations: TMC, trabecular meshwork cells; ONHA; optic nerve head astrocytes; RT-PCR, real time polymerase chain reaction; RR, relative ratio; TGFβ-2, transforming growth factor-beta-2.
    Figure Legend Snippet: Bcl-2 mRNA expression of ONHA and TMC after treatment with minocycline 20 μM and 40 μM only or after additional treatment with 600 mM H 2 O 2 or TGFβ-2, as investigated by quantitative RT-PCR. All tests were performed in triplicate and repeated three times. The mRNA levels were normalized to those of 18S rRNA and expressed as the ratio of Bcl-2 mRNA/18S rRNA. ONHA treated with minocycline concentrations showed a significant increase in Bcl-2 mRNA expression compared with the control in both cell lines. TGFβ-2 and H 2 O 2 treatment decreased Bcl-2 expression. When ONHA cells were treated with minocycline 20 μM and 40 μM and TGFβ-2 or H 2 O 2 , Bcl-2 expression was increased compared with ONHA treated only with TGFβ-2 or H 2 O 2 . Similarly, TMC treated with minocycline 20 μM and TGFβ-2 or H 2 O 2 showed an increased expression of Bcl-2 mRNA compared with TMC treated only with TGFβ-2 or H 2 O 2 . All differences between the two minocycline concentrations and the corresponding controls are statistically significant. Key: x-axis, RR of Bcl-2 mRNA normalized to 18s rRNA expressed in decimal format; y-axis, tested concentrations of minocycline. Abbreviations: TMC, trabecular meshwork cells; ONHA; optic nerve head astrocytes; RT-PCR, real time polymerase chain reaction; RR, relative ratio; TGFβ-2, transforming growth factor-beta-2.

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

    Effect of minocycline treatment on Bcl-2 protein expression. ONHA and TMC were treated with minocycline 20 μM only or additionally with TGFβ-2 (1 ng/mL) or 600 μM H 2 O 2 . Western blotting was used to analyze protein expression in control (Co) and treated cell extracts: TGFβ-2 (Tgf), 20 μM minocycline (M20), minocycline 20 μM and TGFβ-2 (M20+Tgf), 600 μM H 2 O 2 (H 2 O 2 ), and minocycline 20μM and 600 μM H 2 O 2 (M20 + H 2 O 2 ). Ten micrograms of protein was loaded per lane. Abbreviations: TMC, trabecular meshwork cells; ONHA; optic nerve head astrocytes; TGFβ-2, transforming growth factor-beta-2.
    Figure Legend Snippet: Effect of minocycline treatment on Bcl-2 protein expression. ONHA and TMC were treated with minocycline 20 μM only or additionally with TGFβ-2 (1 ng/mL) or 600 μM H 2 O 2 . Western blotting was used to analyze protein expression in control (Co) and treated cell extracts: TGFβ-2 (Tgf), 20 μM minocycline (M20), minocycline 20 μM and TGFβ-2 (M20+Tgf), 600 μM H 2 O 2 (H 2 O 2 ), and minocycline 20μM and 600 μM H 2 O 2 (M20 + H 2 O 2 ). Ten micrograms of protein was loaded per lane. Abbreviations: TMC, trabecular meshwork cells; ONHA; optic nerve head astrocytes; TGFβ-2, transforming growth factor-beta-2.

    Techniques Used: Expressing, Western Blot

    30) 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

    31) Product Images from "Galectin-1 Is Implicated in the Protein Kinase C ?/Vimentin-Controlled Trafficking of Integrin-?1 in Glioblastoma Cells"

    Article Title: Galectin-1 Is Implicated in the Protein Kinase C ?/Vimentin-Controlled Trafficking of Integrin-?1 in Glioblastoma Cells

    Journal: Brain Pathology (Zurich, Switzerland)

    doi: 10.1111/j.1750-3639.2008.00227.x

    Integrin-α9 versus integrin-β1 expression . A. Integrin-α9, integrin-β1 and actin expression across eight glioma cell lines by reverse transcription-polymerase chain reaction (RT-PCR) analysis. B. Quantitative RT-PCR analysis of integrin-α9 versus integrin-β1 expression in glioma cell lines Hs683, U87 and U373. C. Hs683 expression levels of integrin-α9 and integrin-β1 under control (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) conditions obtained through microarray analysis using the Affymetrix Human Genome U133 set Plus 2.0.
    Figure Legend Snippet: Integrin-α9 versus integrin-β1 expression . A. Integrin-α9, integrin-β1 and actin expression across eight glioma cell lines by reverse transcription-polymerase chain reaction (RT-PCR) analysis. B. Quantitative RT-PCR analysis of integrin-α9 versus integrin-β1 expression in glioma cell lines Hs683, U87 and U373. C. Hs683 expression levels of integrin-α9 and integrin-β1 under control (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) conditions obtained through microarray analysis using the Affymetrix Human Genome U133 set Plus 2.0.

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

    32) Product Images from "Adaptor Protein 1A Facilitates Dengue Virus Replication"

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0130065

    Silencing of AP-1A reduced virus production. Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. (A) Virus titer in culture supernatants was measured by FFU assay. (B) Cell viability was measured by trypan blue exclusion. Statistical significance was analyzed using unpaired t test. *P
    Figure Legend Snippet: Silencing of AP-1A reduced virus production. Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. (A) Virus titer in culture supernatants was measured by FFU assay. (B) Cell viability was measured by trypan blue exclusion. Statistical significance was analyzed using unpaired t test. *P

    Techniques Used: Transfection, Infection

    AP-1A knockdown affected the DENV replication site. (A) Ultrastructural analysis of Huh7 cells transfected with control siRNA was observed by TEM at 48 h after second transfection. (B) Cells transfected with control siRNA. (C) Cells transfected with AP-1A siRNA. (D) Cells transfected with AP-2 siRNA were infected with DENV-2 at a MOI of 10 for 24 h. Cells were fixed, processed and analyzed by TEM. Ve, virus-induced vesicles (arrow); Vi, virus particles (arrowhead).
    Figure Legend Snippet: AP-1A knockdown affected the DENV replication site. (A) Ultrastructural analysis of Huh7 cells transfected with control siRNA was observed by TEM at 48 h after second transfection. (B) Cells transfected with control siRNA. (C) Cells transfected with AP-1A siRNA. (D) Cells transfected with AP-2 siRNA were infected with DENV-2 at a MOI of 10 for 24 h. Cells were fixed, processed and analyzed by TEM. Ve, virus-induced vesicles (arrow); Vi, virus particles (arrowhead).

    Techniques Used: Transfection, Transmission Electron Microscopy, Infection

    Silencing of AP-1A reduced DENV RNA level. (A) DENV RNA level was measured by real-time RT-PCR at 24 h post-infection. (B) Kinetics of DENV RNA expression were determined by real-time RT-PCR. Relative expression of DENV RNA in AP-1A knockdown cells was compared with control cells. (C) Knockdown efficiency of AP-1A siRNA was examined by real-time RT-PCR. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using the unpaired t test (*P
    Figure Legend Snippet: Silencing of AP-1A reduced DENV RNA level. (A) DENV RNA level was measured by real-time RT-PCR at 24 h post-infection. (B) Kinetics of DENV RNA expression were determined by real-time RT-PCR. Relative expression of DENV RNA in AP-1A knockdown cells was compared with control cells. (C) Knockdown efficiency of AP-1A siRNA was examined by real-time RT-PCR. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using the unpaired t test (*P

    Techniques Used: Quantitative RT-PCR, Infection, RNA Expression, Expressing, Transfection

    Expression of DENV protein was decreased in Huh7 cells transfected with AP-1A siRNA. (A) Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. DENV proteins were examined at 24 h post-infection by western blotting. Band intensity of DENV proteins was quantified using Image J software. (B) Expression of AP-1A, AP-2 or AP-3A in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (C) pRL-SV40 vector, which contains Renilla luciferase gene, was subjected to in vitro transcription. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA. After the second round of siRNA transfection, cells were transfected with 2.5 nM reporter RNA followed by replacement with fresh culture medium at 4 h later. Following 8 h after transfection with reporter RNA, cells were harvested and determined for Renilla luciferase expression using Luciferase Reporter Assay System (Promega).
    Figure Legend Snippet: Expression of DENV protein was decreased in Huh7 cells transfected with AP-1A siRNA. (A) Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. DENV proteins were examined at 24 h post-infection by western blotting. Band intensity of DENV proteins was quantified using Image J software. (B) Expression of AP-1A, AP-2 or AP-3A in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (C) pRL-SV40 vector, which contains Renilla luciferase gene, was subjected to in vitro transcription. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA. After the second round of siRNA transfection, cells were transfected with 2.5 nM reporter RNA followed by replacement with fresh culture medium at 4 h later. Following 8 h after transfection with reporter RNA, cells were harvested and determined for Renilla luciferase expression using Luciferase Reporter Assay System (Promega).

    Techniques Used: Expressing, Transfection, Infection, Western Blot, Software, Quantitative RT-PCR, Plasmid Preparation, Luciferase, In Vitro, Reporter Assay

    AP-1A was not involved in DENV binding and internalization. (A) Knockdown efficiency of AP-1A siRNA in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (B) AP-1A protein was measured by western blotting. (C) Quantification of DENV binding on Huh7 cells transfected with AP-1A siRNA. Cells transfected with control siRNA and AP-1A siRNA were incubated with DENV-2 at a MOI of 1 for 30 min on ice. Cells were surface stained with antibody to DENV E, followed by staining with the rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate. The surface E-positive cells were analyzed by flow cytometry. (D) Viral internalization was determined by detecting DENV RNA at 2 h post-infection using real-time RT-PCR. Statistical significance was analyzed using unpaired t test (*P
    Figure Legend Snippet: AP-1A was not involved in DENV binding and internalization. (A) Knockdown efficiency of AP-1A siRNA in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (B) AP-1A protein was measured by western blotting. (C) Quantification of DENV binding on Huh7 cells transfected with AP-1A siRNA. Cells transfected with control siRNA and AP-1A siRNA were incubated with DENV-2 at a MOI of 1 for 30 min on ice. Cells were surface stained with antibody to DENV E, followed by staining with the rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate. The surface E-positive cells were analyzed by flow cytometry. (D) Viral internalization was determined by detecting DENV RNA at 2 h post-infection using real-time RT-PCR. Statistical significance was analyzed using unpaired t test (*P

    Techniques Used: Binding Assay, Quantitative RT-PCR, Transfection, Western Blot, Incubation, Staining, Flow Cytometry, Cytometry, Infection

    Depletion of AP-1A impaired DENV genome replication. (A) Effect of AP-1A siRNA on DENV RNA synthesis. Cells transfected with control and AP-1A siRNA were transfected with 0.5 μg DENV RNA using Lipofectamine 2000. Viral RNA level was determined by real-time RT-PCR at 6, 12 and 24 h post-transfection. (B) Quantification of virions released from cells transfected with DENV RNA. At 24 h and 48 h post-transfection, culture supernatants were collected for titration by FFU assay. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using unpaired t test (*P
    Figure Legend Snippet: Depletion of AP-1A impaired DENV genome replication. (A) Effect of AP-1A siRNA on DENV RNA synthesis. Cells transfected with control and AP-1A siRNA were transfected with 0.5 μg DENV RNA using Lipofectamine 2000. Viral RNA level was determined by real-time RT-PCR at 6, 12 and 24 h post-transfection. (B) Quantification of virions released from cells transfected with DENV RNA. At 24 h and 48 h post-transfection, culture supernatants were collected for titration by FFU assay. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using unpaired t test (*P

    Techniques Used: Transfection, Quantitative RT-PCR, Titration

    AP-1A was partially co-localized with dsRNA in DENV-infected cells. Huh7 cells were plated on coverslips, transfected with a plasmid containing AP-1A [ 47 ] and infected with DENV for 24 h. The cells were fixed and incubated with anti-dsRNA antibody and anti-GFP antibody, Upon removal of primary antibodies, cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-mouse IgG Hoechst 33342 was used to stain nuclei of the cells. The cells were visualized by a confocal laser-scanning microscope (LSM 510 Meta).
    Figure Legend Snippet: AP-1A was partially co-localized with dsRNA in DENV-infected cells. Huh7 cells were plated on coverslips, transfected with a plasmid containing AP-1A [ 47 ] and infected with DENV for 24 h. The cells were fixed and incubated with anti-dsRNA antibody and anti-GFP antibody, Upon removal of primary antibodies, cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-mouse IgG Hoechst 33342 was used to stain nuclei of the cells. The cells were visualized by a confocal laser-scanning microscope (LSM 510 Meta).

    Techniques Used: Infection, Transfection, Plasmid Preparation, Incubation, Staining, Laser-Scanning Microscopy

    33) Product Images from "Adaptor Protein 1A Facilitates Dengue Virus Replication"

    Article Title: Adaptor Protein 1A Facilitates Dengue Virus Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0130065

    Silencing of AP-1A reduced virus production. Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. (A) Virus titer in culture supernatants was measured by FFU assay. (B) Cell viability was measured by trypan blue exclusion. Statistical significance was analyzed using unpaired t test. *P
    Figure Legend Snippet: Silencing of AP-1A reduced virus production. Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. (A) Virus titer in culture supernatants was measured by FFU assay. (B) Cell viability was measured by trypan blue exclusion. Statistical significance was analyzed using unpaired t test. *P

    Techniques Used: Transfection, Infection

    AP-1A knockdown affected the DENV replication site. (A) Ultrastructural analysis of Huh7 cells transfected with control siRNA was observed by TEM at 48 h after second transfection. (B) Cells transfected with control siRNA. (C) Cells transfected with AP-1A siRNA. (D) Cells transfected with AP-2 siRNA were infected with DENV-2 at a MOI of 10 for 24 h. Cells were fixed, processed and analyzed by TEM. Ve, virus-induced vesicles (arrow); Vi, virus particles (arrowhead).
    Figure Legend Snippet: AP-1A knockdown affected the DENV replication site. (A) Ultrastructural analysis of Huh7 cells transfected with control siRNA was observed by TEM at 48 h after second transfection. (B) Cells transfected with control siRNA. (C) Cells transfected with AP-1A siRNA. (D) Cells transfected with AP-2 siRNA were infected with DENV-2 at a MOI of 10 for 24 h. Cells were fixed, processed and analyzed by TEM. Ve, virus-induced vesicles (arrow); Vi, virus particles (arrowhead).

    Techniques Used: Transfection, Transmission Electron Microscopy, Infection

    Silencing of AP-1A reduced DENV RNA level. (A) DENV RNA level was measured by real-time RT-PCR at 24 h post-infection. (B) Kinetics of DENV RNA expression were determined by real-time RT-PCR. Relative expression of DENV RNA in AP-1A knockdown cells was compared with control cells. (C) Knockdown efficiency of AP-1A siRNA was examined by real-time RT-PCR. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using the unpaired t test (*P
    Figure Legend Snippet: Silencing of AP-1A reduced DENV RNA level. (A) DENV RNA level was measured by real-time RT-PCR at 24 h post-infection. (B) Kinetics of DENV RNA expression were determined by real-time RT-PCR. Relative expression of DENV RNA in AP-1A knockdown cells was compared with control cells. (C) Knockdown efficiency of AP-1A siRNA was examined by real-time RT-PCR. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using the unpaired t test (*P

    Techniques Used: Quantitative RT-PCR, Infection, RNA Expression, Expressing, Transfection

    Expression of DENV protein was decreased in Huh7 cells transfected with AP-1A siRNA. (A) Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. DENV proteins were examined at 24 h post-infection by western blotting. Band intensity of DENV proteins was quantified using Image J software. (B) Expression of AP-1A, AP-2 or AP-3A in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (C) pRL-SV40 vector, which contains Renilla luciferase gene, was subjected to in vitro transcription. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA. After the second round of siRNA transfection, cells were transfected with 2.5 nM reporter RNA followed by replacement with fresh culture medium at 4 h later. Following 8 h after transfection with reporter RNA, cells were harvested and determined for Renilla luciferase expression using Luciferase Reporter Assay System (Promega).
    Figure Legend Snippet: Expression of DENV protein was decreased in Huh7 cells transfected with AP-1A siRNA. (A) Huh7 cells were transfected with control siRNA and AP-1A siRNA and infected with DENV-2 for 24 h. DENV proteins were examined at 24 h post-infection by western blotting. Band intensity of DENV proteins was quantified using Image J software. (B) Expression of AP-1A, AP-2 or AP-3A in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (C) pRL-SV40 vector, which contains Renilla luciferase gene, was subjected to in vitro transcription. To determine the effect of AP-1A knockdown on translation, Huh7 cells were transfected twice with AP-1A-specific siRNA or control siRNA. After the second round of siRNA transfection, cells were transfected with 2.5 nM reporter RNA followed by replacement with fresh culture medium at 4 h later. Following 8 h after transfection with reporter RNA, cells were harvested and determined for Renilla luciferase expression using Luciferase Reporter Assay System (Promega).

    Techniques Used: Expressing, Transfection, Infection, Western Blot, Software, Quantitative RT-PCR, Plasmid Preparation, Luciferase, In Vitro, Reporter Assay

    AP-1A was not involved in DENV binding and internalization. (A) Knockdown efficiency of AP-1A siRNA in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (B) AP-1A protein was measured by western blotting. (C) Quantification of DENV binding on Huh7 cells transfected with AP-1A siRNA. Cells transfected with control siRNA and AP-1A siRNA were incubated with DENV-2 at a MOI of 1 for 30 min on ice. Cells were surface stained with antibody to DENV E, followed by staining with the rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate. The surface E-positive cells were analyzed by flow cytometry. (D) Viral internalization was determined by detecting DENV RNA at 2 h post-infection using real-time RT-PCR. Statistical significance was analyzed using unpaired t test (*P
    Figure Legend Snippet: AP-1A was not involved in DENV binding and internalization. (A) Knockdown efficiency of AP-1A siRNA in Huh7 cells was examined by real-time RT-PCR at 48 h after second transfection. (B) AP-1A protein was measured by western blotting. (C) Quantification of DENV binding on Huh7 cells transfected with AP-1A siRNA. Cells transfected with control siRNA and AP-1A siRNA were incubated with DENV-2 at a MOI of 1 for 30 min on ice. Cells were surface stained with antibody to DENV E, followed by staining with the rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate. The surface E-positive cells were analyzed by flow cytometry. (D) Viral internalization was determined by detecting DENV RNA at 2 h post-infection using real-time RT-PCR. Statistical significance was analyzed using unpaired t test (*P

    Techniques Used: Binding Assay, Quantitative RT-PCR, Transfection, Western Blot, Incubation, Staining, Flow Cytometry, Cytometry, Infection

    Depletion of AP-1A impaired DENV genome replication. (A) Effect of AP-1A siRNA on DENV RNA synthesis. Cells transfected with control and AP-1A siRNA were transfected with 0.5 μg DENV RNA using Lipofectamine 2000. Viral RNA level was determined by real-time RT-PCR at 6, 12 and 24 h post-transfection. (B) Quantification of virions released from cells transfected with DENV RNA. At 24 h and 48 h post-transfection, culture supernatants were collected for titration by FFU assay. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using unpaired t test (*P
    Figure Legend Snippet: Depletion of AP-1A impaired DENV genome replication. (A) Effect of AP-1A siRNA on DENV RNA synthesis. Cells transfected with control and AP-1A siRNA were transfected with 0.5 μg DENV RNA using Lipofectamine 2000. Viral RNA level was determined by real-time RT-PCR at 6, 12 and 24 h post-transfection. (B) Quantification of virions released from cells transfected with DENV RNA. At 24 h and 48 h post-transfection, culture supernatants were collected for titration by FFU assay. The results were plotted relative to cells transfected with control siRNA. Statistical significance was analyzed using unpaired t test (*P

    Techniques Used: Transfection, Quantitative RT-PCR, Titration

    AP-1A was partially co-localized with dsRNA in DENV-infected cells. Huh7 cells were plated on coverslips, transfected with a plasmid containing AP-1A [ 47 ] and infected with DENV for 24 h. The cells were fixed and incubated with anti-dsRNA antibody and anti-GFP antibody, Upon removal of primary antibodies, cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-mouse IgG Hoechst 33342 was used to stain nuclei of the cells. The cells were visualized by a confocal laser-scanning microscope (LSM 510 Meta).
    Figure Legend Snippet: AP-1A was partially co-localized with dsRNA in DENV-infected cells. Huh7 cells were plated on coverslips, transfected with a plasmid containing AP-1A [ 47 ] and infected with DENV for 24 h. The cells were fixed and incubated with anti-dsRNA antibody and anti-GFP antibody, Upon removal of primary antibodies, cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-mouse IgG Hoechst 33342 was used to stain nuclei of the cells. The cells were visualized by a confocal laser-scanning microscope (LSM 510 Meta).

    Techniques Used: Infection, Transfection, Plasmid Preparation, Incubation, Staining, Laser-Scanning Microscopy

    34) Product Images from "Interleukin-33 (IL-33) Increases Hyperoxia-Induced Bronchopulmonary Dysplasia in Newborn Mice by Regulation of Inflammatory Mediators"

    Article Title: Interleukin-33 (IL-33) Increases Hyperoxia-Induced Bronchopulmonary Dysplasia in Newborn Mice by Regulation of Inflammatory Mediators

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.910851

    Effect of interleukin-33 (IL-33) inhibition of apoptosis and pulmonary inflammation. ( A ) Western blot analysis shows protein expression patterns of Bcl-2, Bax, and cleaved caspase-3 in the lungs of 12-day-old mouse pups in each group. ( B, C ) Quantitative real-time polymerase chain reaction (qRT-PCR) data shows that Bcl-2 and Bax mRNA expression was altered after treatment with mechanical ventilation with oxygen-rich air MV-O 2 and IL-33 inhibition. ( D ) Quantitative analysis of cleaved caspase-3 band intensity in each group. ( E ) Western blot analysis comparing the concentrations of three cytokines (IL-1β, CXCL-1, and MCP-1) in the lungs of pups treated as indicated. ( F–H ) Comparison of interleukin (IL)-1β, chemokine (CC motif) ligand 1 (CXCL-1), and monocyte chemoattractant protein-1 (MCP-1) mRNA expression, expressed relative to β-actin mRNA, in 14-day-old pups treated as indicated. * Represents p
    Figure Legend Snippet: Effect of interleukin-33 (IL-33) inhibition of apoptosis and pulmonary inflammation. ( A ) Western blot analysis shows protein expression patterns of Bcl-2, Bax, and cleaved caspase-3 in the lungs of 12-day-old mouse pups in each group. ( B, C ) Quantitative real-time polymerase chain reaction (qRT-PCR) data shows that Bcl-2 and Bax mRNA expression was altered after treatment with mechanical ventilation with oxygen-rich air MV-O 2 and IL-33 inhibition. ( D ) Quantitative analysis of cleaved caspase-3 band intensity in each group. ( E ) Western blot analysis comparing the concentrations of three cytokines (IL-1β, CXCL-1, and MCP-1) in the lungs of pups treated as indicated. ( F–H ) Comparison of interleukin (IL)-1β, chemokine (CC motif) ligand 1 (CXCL-1), and monocyte chemoattractant protein-1 (MCP-1) mRNA expression, expressed relative to β-actin mRNA, in 14-day-old pups treated as indicated. * Represents p

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

    Mechanical ventilation with oxygen (MV-O 2 ) increased apoptosis in mouse lungs. ( A–C ) Western blot was performed to assess Bcl-2 and Bax expression and cleaved caspase-3 protein expression in newborn mice compared with the on-air group after 8 h of mechanical ventilation with oxygen-rich air (MV-O 2 ). ( D, E ) Quantitative real-time polymerase chain reaction (qRT-PCR) data shows that Bcl-2 and Bax mRNA expression was altered by MV-O 2 . ( F ) Quantitative analysis of the cleaved caspase-3 band intensity in each group. ( G ) Immunofluorescence (IF) analysis of lung tissue shows increased dual staining for cleaved caspase-3 (red) and Bcl-2 (green) in the lungs of 6-day-old to 7-day-old mice after 8 h MV-O 2 compared with the on-air group. Magnification ×400. ** Represents p
    Figure Legend Snippet: Mechanical ventilation with oxygen (MV-O 2 ) increased apoptosis in mouse lungs. ( A–C ) Western blot was performed to assess Bcl-2 and Bax expression and cleaved caspase-3 protein expression in newborn mice compared with the on-air group after 8 h of mechanical ventilation with oxygen-rich air (MV-O 2 ). ( D, E ) Quantitative real-time polymerase chain reaction (qRT-PCR) data shows that Bcl-2 and Bax mRNA expression was altered by MV-O 2 . ( F ) Quantitative analysis of the cleaved caspase-3 band intensity in each group. ( G ) Immunofluorescence (IF) analysis of lung tissue shows increased dual staining for cleaved caspase-3 (red) and Bcl-2 (green) in the lungs of 6-day-old to 7-day-old mice after 8 h MV-O 2 compared with the on-air group. Magnification ×400. ** Represents p

    Techniques Used: Western Blot, Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining

    35) Product Images from "A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein"

    Article Title: A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01253-16

    Antiviral activity of BDAA enantiomers. (A) Structures of BDAA and its enantiomers. (B) Determination of the antiviral activities of BDAA and its enantiomers from a qRT-PCR assay. Huh7.5 cells were cultured and treated as described for . YFV RNAs
    Figure Legend Snippet: Antiviral activity of BDAA enantiomers. (A) Structures of BDAA and its enantiomers. (B) Determination of the antiviral activities of BDAA and its enantiomers from a qRT-PCR assay. Huh7.5 cells were cultured and treated as described for . YFV RNAs

    Techniques Used: Activity Assay, Quantitative RT-PCR, Cell Culture

    36) Product Images from "Differentially expressed mRNAs, lncRNAs, and miRNAs with associated co-expression and ceRNA networks in ankylosing spondylitis"

    Article Title: Differentially expressed mRNAs, lncRNAs, and miRNAs with associated co-expression and ceRNA networks in ankylosing spondylitis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22708

    Validation of microarray data by qRT-PCR ( A ) Five lncRNAs, ( B ) four miRNAs, and ( C ) five mRNAs were validated by qRT-PCR between AS and control groups. The relative expression level of each RNA was normalized. The data displayed in histograms are expressed as means ± standard deviation. * P
    Figure Legend Snippet: Validation of microarray data by qRT-PCR ( A ) Five lncRNAs, ( B ) four miRNAs, and ( C ) five mRNAs were validated by qRT-PCR between AS and control groups. The relative expression level of each RNA was normalized. The data displayed in histograms are expressed as means ± standard deviation. * P

    Techniques Used: Microarray, Quantitative RT-PCR, Expressing, Standard Deviation

    MiR-17-5p and miR-27b-3p were differentially expressed during osteogenic differentiation of human ligament fibroblasts ( A ) ALP in fibroblasts was stained using the BCIP/NBT kit after the cells were incubated in OM for 7 and 14 days. Scale bar = 200 μm. ( B ) The ALP activity of fibroblasts was measured after they were incubated in OM for 7 and 14 days. ( C ) Fibroblasts were incubated in OM for 21 days, and then the mineralized nodules were stained using alizarin red S (ARS). Scale bar = 200 μm. ( D ) Mineralization was quantified by extraction of ARS dye with 10% cetylpyridinium chloride. ( E ) The total RNA was isolated on days 7 and 14. Runx2, ALP, and COL1A1 mRNA levels were determined using qRT-PCR and normalized to GAPDH. ( F ) The total RNA was isolated on days 7 and 14. Levels of miR-17-5p and miR-27b-3p were determined using qRT-PCR and normalized to U6. * P
    Figure Legend Snippet: MiR-17-5p and miR-27b-3p were differentially expressed during osteogenic differentiation of human ligament fibroblasts ( A ) ALP in fibroblasts was stained using the BCIP/NBT kit after the cells were incubated in OM for 7 and 14 days. Scale bar = 200 μm. ( B ) The ALP activity of fibroblasts was measured after they were incubated in OM for 7 and 14 days. ( C ) Fibroblasts were incubated in OM for 21 days, and then the mineralized nodules were stained using alizarin red S (ARS). Scale bar = 200 μm. ( D ) Mineralization was quantified by extraction of ARS dye with 10% cetylpyridinium chloride. ( E ) The total RNA was isolated on days 7 and 14. Runx2, ALP, and COL1A1 mRNA levels were determined using qRT-PCR and normalized to GAPDH. ( F ) The total RNA was isolated on days 7 and 14. Levels of miR-17-5p and miR-27b-3p were determined using qRT-PCR and normalized to U6. * P

    Techniques Used: ALP Assay, Staining, Incubation, Activity Assay, Isolation, Quantitative RT-PCR

    37) Product Images from "NF-κB-direct activation of microRNAs with repressive effects on monocyte-specific genes is critical for osteoclast differentiation"

    Article Title: NF-κB-direct activation of microRNAs with repressive effects on monocyte-specific genes is critical for osteoclast differentiation

    Journal: Genome Biology

    doi: 10.1186/s13059-014-0561-5

    Comparison of changes in the expression levels of the miRNAs within the miR-99b/125a/let7e and miR-132/212 clusters during osteoclast differentiation, and changes during monocyte-to-macrophage and monocyte-to-dendritic cell differentiation. (A) Diagram depicting the three differentiation models used in this experiment. GM-CSF, granulocyte-macrophage colony-stimulating factor. (B) Relative expression levels of the miRNAs in matching samples of MOs (grey), immature dendritic cells (iDC, red), macrophages (green,) and monocytes stimulated with RANKL/M-CSF after 1, 2 and 4 days (immature OCs (iOC) (blue)). qRT-PCR data were normalized with respect to miR-103. (C) Relative expression levels of miRNA targets in the same set of samples. qRT-PCR data were normalized with respect to the RPL38 gene. Error bars correspond to standard deviation of three independent measurements.
    Figure Legend Snippet: Comparison of changes in the expression levels of the miRNAs within the miR-99b/125a/let7e and miR-132/212 clusters during osteoclast differentiation, and changes during monocyte-to-macrophage and monocyte-to-dendritic cell differentiation. (A) Diagram depicting the three differentiation models used in this experiment. GM-CSF, granulocyte-macrophage colony-stimulating factor. (B) Relative expression levels of the miRNAs in matching samples of MOs (grey), immature dendritic cells (iDC, red), macrophages (green,) and monocytes stimulated with RANKL/M-CSF after 1, 2 and 4 days (immature OCs (iOC) (blue)). qRT-PCR data were normalized with respect to miR-103. (C) Relative expression levels of miRNA targets in the same set of samples. qRT-PCR data were normalized with respect to the RPL38 gene. Error bars correspond to standard deviation of three independent measurements.

    Techniques Used: Expressing, Cell Differentiation, Quantitative RT-PCR, Standard Deviation

    NF-κB dependence of miRNA expression changes. (A) Analysis of the presence of NF-κB subunit binding motifs (from TRANSFAC database) in a 1,000-bp window centered around the estimated TSS of the miRNAs. (B) ChIP assays for selected miRNAs showing the binding of NF-κB p65 near the TSS 2 and 4 days after RANKL/M-CSF stimulation of MOs. Each graph contains the relative enrichment of samples immunoprecipitated with the anti-p65 antibody and an IgG as a control. MO samples were tested at 0, 2 and 4 days after M-CSF/RANKL stimulation. On top of each graph the sequence analyzed is indicated. p65 putative binding sites are indicated with a blue dot. Primers used for amplification are indicated with arrows around p65 binding sites. (C) Effects of the two NF-κB inhibitors (10 μM BAY 11-7082 (BAY11) and 100 μM sodium aurothiomalate (SATM) on the phosphorylation levels of p65 as determined by western blotting. p65 and H3 total levels are used as controls. (D) Effects of two NF-κB inhibitors (BAY11 and SATM) on the levels of miRNAs within the miR-99b/125a/let7e and miR-132/212 clusters measured by a time-course analysis of MOs stimulated with RANKL/M-CSF. (E) Effects of treatment with BAY11 and SATM on markers of OC differentiation ( ACP5 , CTSK , TM7SF4 , MMP9 ) as estimated by qRT-PCR. Data relative to DMSO-treated samples and normalized with RPL38 expression levels. (F) Effects of treatment with BAY 11 and SATM on miRNA targets ( IGF1R , TNFAIP3 , ITGA4 , THBS , IL15 and PTGS2 ) as estimated by qRT-PCR. Data are relative to DMSO-treated samples and are normalized with respect to RPL38 expression levels. Error bars correspond to the standard deviation of three independent measurements; *corresponds to P -value
    Figure Legend Snippet: NF-κB dependence of miRNA expression changes. (A) Analysis of the presence of NF-κB subunit binding motifs (from TRANSFAC database) in a 1,000-bp window centered around the estimated TSS of the miRNAs. (B) ChIP assays for selected miRNAs showing the binding of NF-κB p65 near the TSS 2 and 4 days after RANKL/M-CSF stimulation of MOs. Each graph contains the relative enrichment of samples immunoprecipitated with the anti-p65 antibody and an IgG as a control. MO samples were tested at 0, 2 and 4 days after M-CSF/RANKL stimulation. On top of each graph the sequence analyzed is indicated. p65 putative binding sites are indicated with a blue dot. Primers used for amplification are indicated with arrows around p65 binding sites. (C) Effects of the two NF-κB inhibitors (10 μM BAY 11-7082 (BAY11) and 100 μM sodium aurothiomalate (SATM) on the phosphorylation levels of p65 as determined by western blotting. p65 and H3 total levels are used as controls. (D) Effects of two NF-κB inhibitors (BAY11 and SATM) on the levels of miRNAs within the miR-99b/125a/let7e and miR-132/212 clusters measured by a time-course analysis of MOs stimulated with RANKL/M-CSF. (E) Effects of treatment with BAY11 and SATM on markers of OC differentiation ( ACP5 , CTSK , TM7SF4 , MMP9 ) as estimated by qRT-PCR. Data relative to DMSO-treated samples and normalized with RPL38 expression levels. (F) Effects of treatment with BAY 11 and SATM on miRNA targets ( IGF1R , TNFAIP3 , ITGA4 , THBS , IL15 and PTGS2 ) as estimated by qRT-PCR. Data are relative to DMSO-treated samples and are normalized with respect to RPL38 expression levels. Error bars correspond to the standard deviation of three independent measurements; *corresponds to P -value

    Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Sequencing, Amplification, Western Blot, Quantitative RT-PCR, Standard Deviation

    MicroRNA expression profiling during monocyte-to-osteoclast differentiation. (A) Validation of the presence of OCs by TRAP and phalloidin staining, showing the presence of TRAP activity/multiple nuclei and the actin ring, respectively. (B) Molecular characterization of OC differentiation. Several OC markers are upregulated ( CA2 , CTSK , MMP9 , ACP5 / TRAP , and TM7SF4 / DCSTAMP ), and the MO marker CX3CR1 is silenced. Data for MOs, MOs 48 h after M-CSF and RANKL treatment and OCs at 21 days are presented. RPL38 gene expression levels were used for normalization. Error bars correspond to the standard deviation of three individual measurements. (C) Heatmap showing expression array data from the miRNA expression screening. miRNAs were subdivided into eight groups (I to VIII) according to their expression profile (diagram); the number of miRNAs in each group is indicated inside the expression dynamics diagram. Scale shown at the bottom, whereby normalized expression units ranges from -1 (blue) to +1 (red). (D) Representation of the genomic distribution of miR-99b/125a/let7e and miR-132/212 clusters, including the TSS (indicated with an arrow). (E) Validation of array data by quantitative PCR in independent biological replicates. Analysis in MOs, MOs incubated 48 h with RANKL/M-CSF and fully differentiated OCs. Data normalized with respect to miR-103. (F) Expression dynamics of the indicated miRNAs during OC differentiation, also normalized with respect to miR-103.
    Figure Legend Snippet: MicroRNA expression profiling during monocyte-to-osteoclast differentiation. (A) Validation of the presence of OCs by TRAP and phalloidin staining, showing the presence of TRAP activity/multiple nuclei and the actin ring, respectively. (B) Molecular characterization of OC differentiation. Several OC markers are upregulated ( CA2 , CTSK , MMP9 , ACP5 / TRAP , and TM7SF4 / DCSTAMP ), and the MO marker CX3CR1 is silenced. Data for MOs, MOs 48 h after M-CSF and RANKL treatment and OCs at 21 days are presented. RPL38 gene expression levels were used for normalization. Error bars correspond to the standard deviation of three individual measurements. (C) Heatmap showing expression array data from the miRNA expression screening. miRNAs were subdivided into eight groups (I to VIII) according to their expression profile (diagram); the number of miRNAs in each group is indicated inside the expression dynamics diagram. Scale shown at the bottom, whereby normalized expression units ranges from -1 (blue) to +1 (red). (D) Representation of the genomic distribution of miR-99b/125a/let7e and miR-132/212 clusters, including the TSS (indicated with an arrow). (E) Validation of array data by quantitative PCR in independent biological replicates. Analysis in MOs, MOs incubated 48 h with RANKL/M-CSF and fully differentiated OCs. Data normalized with respect to miR-103. (F) Expression dynamics of the indicated miRNAs during OC differentiation, also normalized with respect to miR-103.

    Techniques Used: Expressing, Staining, Activity Assay, Marker, Standard Deviation, Real-time Polymerase Chain Reaction, Incubation

    NF-κB p65 has a direct role in changes in miRNA expression levels. (A) Diagram depicting the region of the p65 gene in exon 11 targeted by the siRNA used in this study. Effects of siRNA experiments on p65 levels in MOs stimulated with RANKL/M-CSF after 1, 2, and 4 days, as analyzed by western blotting (bottom and central panels, normalized with respect to histone H3 levels) and qRT-PCR (left panel, relative to RPL38 expression levels). (B) Effect of p65 depletion on its recruitment near the TSS of the coding sequence of the miRNAs, as demonstrated by ChIP assays. The scheme on top of each graph depicts the region analyzed, indicating the p65 binding site (dot) and the primers used (arrows around the p65 binding site). (C) Effects of p65 siRNA experiments on miR-99b, miR-125a and miR-let7e after 1, 2, and 4 days. Data relative to miR-103 levels. (D) Effects of p65 downregulation on expression of genes upregulated during osteoclastogenesis ( CTSK , MMP9 , ACP5 , TM7SF4 ). (E) Effects of p65 downregulation on the levels of the miRNA targets TNFAIP3 and IGF1R . Expression data compared with MO samples treated with control siRNA and values relative to RPL38 expression levels. Error bars correspond to the standard deviation of three independent measurements; *corresponds to P -value
    Figure Legend Snippet: NF-κB p65 has a direct role in changes in miRNA expression levels. (A) Diagram depicting the region of the p65 gene in exon 11 targeted by the siRNA used in this study. Effects of siRNA experiments on p65 levels in MOs stimulated with RANKL/M-CSF after 1, 2, and 4 days, as analyzed by western blotting (bottom and central panels, normalized with respect to histone H3 levels) and qRT-PCR (left panel, relative to RPL38 expression levels). (B) Effect of p65 depletion on its recruitment near the TSS of the coding sequence of the miRNAs, as demonstrated by ChIP assays. The scheme on top of each graph depicts the region analyzed, indicating the p65 binding site (dot) and the primers used (arrows around the p65 binding site). (C) Effects of p65 siRNA experiments on miR-99b, miR-125a and miR-let7e after 1, 2, and 4 days. Data relative to miR-103 levels. (D) Effects of p65 downregulation on expression of genes upregulated during osteoclastogenesis ( CTSK , MMP9 , ACP5 , TM7SF4 ). (E) Effects of p65 downregulation on the levels of the miRNA targets TNFAIP3 and IGF1R . Expression data compared with MO samples treated with control siRNA and values relative to RPL38 expression levels. Error bars correspond to the standard deviation of three independent measurements; *corresponds to P -value

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Sequencing, Chromatin Immunoprecipitation, Binding Assay, Standard Deviation

    38) Product Images from "Antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro"

    Article Title: Antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro

    Journal: Research in Veterinary Science

    doi: 10.1016/j.rvsc.2018.03.002

    Inhibitory Effects of LiCl on different stages of PEDV infection. A. Relative viral RNA levels of cells treated with LiCl at the viral attachment, entry and replication stages as determined by real-time PCR. B. Viral titers (log10TCID50/ml) of cells treated with LiCl at the viral attachment, entry and replication stages as calculated by the method of Reed and Muench. C. Inhibitory effects of LiCl on PEDV observed at the entry stage using immunofluorescence assays (IFA). D. Inhibitory effects of LiCl on PEDV observed at the replication stage using immunofluorescence assay (IFA). Values represent the mean ± SD for three independent experiments. Green fluorescence represents the PEDV distribution, and the blue fluorescence represents the nuclear distribution. ns, no significant difference; * P
    Figure Legend Snippet: Inhibitory Effects of LiCl on different stages of PEDV infection. A. Relative viral RNA levels of cells treated with LiCl at the viral attachment, entry and replication stages as determined by real-time PCR. B. Viral titers (log10TCID50/ml) of cells treated with LiCl at the viral attachment, entry and replication stages as calculated by the method of Reed and Muench. C. Inhibitory effects of LiCl on PEDV observed at the entry stage using immunofluorescence assays (IFA). D. Inhibitory effects of LiCl on PEDV observed at the replication stage using immunofluorescence assay (IFA). Values represent the mean ± SD for three independent experiments. Green fluorescence represents the PEDV distribution, and the blue fluorescence represents the nuclear distribution. ns, no significant difference; * P

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence

    39) Product Images from "High-Frequency Near-Infrared Diode Laser Irradiation Attenuates IL-1β-Induced Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Human Primary Chondrocytes"

    Article Title: High-Frequency Near-Infrared Diode Laser Irradiation Attenuates IL-1β-Induced Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Human Primary Chondrocytes

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm9030881

    Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of inflammatory cytokines in Normal Human Articular Chondrocyte-Knee (NHAC-Kn) cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time Polymerase Chain Reaction (PCR) analysis. mRNA expression of ( A ) IL-1β, ( B ) IL-6, and ( C ) tumor necrosis factor (TNF) -α are represented as mean ± SEM of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of inflammatory cytokines in Normal Human Articular Chondrocyte-Knee (NHAC-Kn) cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time Polymerase Chain Reaction (PCR) analysis. mRNA expression of ( A ) IL-1β, ( B ) IL-6, and ( C ) tumor necrosis factor (TNF) -α are represented as mean ± SEM of three independent experiments ( n = 3). * p

    Techniques Used: Irradiation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of matrix metalloproteinases (MMPs) in NHAC-Kn cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time polymerase chain reaction (PCR) analysis. mRNA expression of ( A ) MMP-1, ( B ) MMP-3, ( C ) MMP-9, and ( D ) MMP-13 are represented as mean ± SEM of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of matrix metalloproteinases (MMPs) in NHAC-Kn cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time polymerase chain reaction (PCR) analysis. mRNA expression of ( A ) MMP-1, ( B ) MMP-3, ( C ) MMP-9, and ( D ) MMP-13 are represented as mean ± SEM of three independent experiments ( n = 3). * p

    Techniques Used: Irradiation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    40) Product Images from "High-Frequency Near-Infrared Diode Laser Irradiation Attenuates IL-1β-Induced Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Human Primary Chondrocytes"

    Article Title: High-Frequency Near-Infrared Diode Laser Irradiation Attenuates IL-1β-Induced Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Human Primary Chondrocytes

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm9030881

    Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of inflammatory cytokines in Normal Human Articular Chondrocyte-Knee (NHAC-Kn) cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time Polymerase Chain Reaction (PCR) analysis. mRNA expression of ( A ) IL-1β, ( B ) IL-6, and ( C ) tumor necrosis factor (TNF) -α are represented as mean ± SEM of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of inflammatory cytokines in Normal Human Articular Chondrocyte-Knee (NHAC-Kn) cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time Polymerase Chain Reaction (PCR) analysis. mRNA expression of ( A ) IL-1β, ( B ) IL-6, and ( C ) tumor necrosis factor (TNF) -α are represented as mean ± SEM of three independent experiments ( n = 3). * p

    Techniques Used: Irradiation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of matrix metalloproteinases (MMPs) in NHAC-Kn cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time polymerase chain reaction (PCR) analysis. mRNA expression of ( A ) MMP-1, ( B ) MMP-3, ( C ) MMP-9, and ( D ) MMP-13 are represented as mean ± SEM of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Effect of high-frequency near-infrared diode laser irradiation on interleukin (IL) -1β-induced gene expression of matrix metalloproteinases (MMPs) in NHAC-Kn cells. The chondrocytes were treated with IL-1β (10 pg/mL) and laser irradiated (0, 4, 8 J/cm 2 ) for 4 h, followed by real-time polymerase chain reaction (PCR) analysis. mRNA expression of ( A ) MMP-1, ( B ) MMP-3, ( C ) MMP-9, and ( D ) MMP-13 are represented as mean ± SEM of three independent experiments ( n = 3). * p

    Techniques Used: Irradiation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

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  • 94
    Roche pcr qpcr
    TgAP2X-5 depletion prevents TgAP2XI-5 binding to a number of promoters. ( A ) ChIP-on-chip data are represented as the log 2 ratio of the hybridization signal given by DNA immunoprecipitated over the signal given by the non-enriched input DNA. The log 2 ratio of each oligonucleotide present on the tiled microarray has been plotted at the respective genomic position. Genes above the horizontal axis are encoded the forward strand whereas those below are encoded the reverse strand. The scale of the Y -axis was kept identical for all representation to allow the comparison of signals. TgAP2XI-5 ChIP-chip assay is represented for the TgAP2XI-5 protein in the parental strain (RH ΔhxgprtΔku80 , green, negative control) or TgAP2XI-5 tagged strain (red, positive control) or TgAP2XI-5 tagged in the iKD TgAP2X-5 strain in presence of ATc (orange). The associated peaks identified by the mPeak algorithm are also provided. A portion of the Toxoplasma gondii chromosome III is represented. The boxed promoter presents a lower enrichment of TgAP2XI-5 in the iKD TgAP2X-5 strain in presence of ATc. ( B ) Representation of the ChIP-chip experiments for a portion of chromosome IV which encompasses the TgROP15 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( C ) Representation of the ChIP-chip experiments for a portion of chromosome III which encompasses the TgROP24 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( D ) Quantitative <t>PCR</t> was performed on the TgAP2XI-5 ChIP assays performed in the iKD TgAP2X-5/TgAP2XI-5 strain (orange), iKD TgAP2X-5 complemented/TgAP2XI-5 strain (yellow), TgAP2XI-5-tagged strain (red) and parental strain (negative control, green). Amplification was carried out on regions within the promoter of each of the six genes listed. The enrichment for corresponding ChIP DNA samples is presented as a percentage of INPUT for each target.
    Pcr Qpcr, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche pcr qpcr one step reverse transcription
    Characterization of HBV particles released from apoptotic PHH. Supernatants of HBV-infected PHH cultures were collected 24 h after anti-CD95 MAb (130 ng/ml) or mock treatment and subjected to CsCl gradient centrifugation. Twelve fractions (F) with decreasing densities (F1 to F12) were collected; F3 to F10 were used for further characterization. (a) HBsAg ELISA indicating enveloped HBV particles. Average values from three experiments ± standard errors of the means are given. (b) Qualitative and quantitative analysis of encapsidated viral nucleic acids using one-step <t>RT-PCR.</t> To discriminate between DNA and RNA, DNase digestion was performed. (Left) Mock treatment. (Right) Anti-CD95 treatment. The total amount of HBV nucleic acids (DNA plus RNA) (gray bars) detected in F8 of mock-treated cells was set to 100%. HBV RNA levels (continued line) of all samples are reported as parts per thousand. (c) Amplification products of <t>qPCR</t> runs of F4 to F6 with the indicated treatments were separated on a 2% agarose gel and visualized by ethidium bromide staining.
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    Roche real time quantitative pcr
    Expression profile of <t>Catsper1au</t> in male and female mouse organs. A cDNA panel was prepared from different organs and cells to amplify the Catsper1au 5′ region through <t>PCR</t> based on the sequences obtained using 5′-RACE. A product of 496 bp
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    TgAP2X-5 depletion prevents TgAP2XI-5 binding to a number of promoters. ( A ) ChIP-on-chip data are represented as the log 2 ratio of the hybridization signal given by DNA immunoprecipitated over the signal given by the non-enriched input DNA. The log 2 ratio of each oligonucleotide present on the tiled microarray has been plotted at the respective genomic position. Genes above the horizontal axis are encoded the forward strand whereas those below are encoded the reverse strand. The scale of the Y -axis was kept identical for all representation to allow the comparison of signals. TgAP2XI-5 ChIP-chip assay is represented for the TgAP2XI-5 protein in the parental strain (RH ΔhxgprtΔku80 , green, negative control) or TgAP2XI-5 tagged strain (red, positive control) or TgAP2XI-5 tagged in the iKD TgAP2X-5 strain in presence of ATc (orange). The associated peaks identified by the mPeak algorithm are also provided. A portion of the Toxoplasma gondii chromosome III is represented. The boxed promoter presents a lower enrichment of TgAP2XI-5 in the iKD TgAP2X-5 strain in presence of ATc. ( B ) Representation of the ChIP-chip experiments for a portion of chromosome IV which encompasses the TgROP15 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( C ) Representation of the ChIP-chip experiments for a portion of chromosome III which encompasses the TgROP24 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( D ) Quantitative PCR was performed on the TgAP2XI-5 ChIP assays performed in the iKD TgAP2X-5/TgAP2XI-5 strain (orange), iKD TgAP2X-5 complemented/TgAP2XI-5 strain (yellow), TgAP2XI-5-tagged strain (red) and parental strain (negative control, green). Amplification was carried out on regions within the promoter of each of the six genes listed. The enrichment for corresponding ChIP DNA samples is presented as a percentage of INPUT for each target.

    Journal: Nucleic Acids Research

    Article Title: Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii

    doi: 10.1093/nar/gky373

    Figure Lengend Snippet: TgAP2X-5 depletion prevents TgAP2XI-5 binding to a number of promoters. ( A ) ChIP-on-chip data are represented as the log 2 ratio of the hybridization signal given by DNA immunoprecipitated over the signal given by the non-enriched input DNA. The log 2 ratio of each oligonucleotide present on the tiled microarray has been plotted at the respective genomic position. Genes above the horizontal axis are encoded the forward strand whereas those below are encoded the reverse strand. The scale of the Y -axis was kept identical for all representation to allow the comparison of signals. TgAP2XI-5 ChIP-chip assay is represented for the TgAP2XI-5 protein in the parental strain (RH ΔhxgprtΔku80 , green, negative control) or TgAP2XI-5 tagged strain (red, positive control) or TgAP2XI-5 tagged in the iKD TgAP2X-5 strain in presence of ATc (orange). The associated peaks identified by the mPeak algorithm are also provided. A portion of the Toxoplasma gondii chromosome III is represented. The boxed promoter presents a lower enrichment of TgAP2XI-5 in the iKD TgAP2X-5 strain in presence of ATc. ( B ) Representation of the ChIP-chip experiments for a portion of chromosome IV which encompasses the TgROP15 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( C ) Representation of the ChIP-chip experiments for a portion of chromosome III which encompasses the TgROP24 gene (yellow). Two repeats (R1 and R2) are presented for each ChIP experiment along with the mPeak identified peaks. ( D ) Quantitative PCR was performed on the TgAP2XI-5 ChIP assays performed in the iKD TgAP2X-5/TgAP2XI-5 strain (orange), iKD TgAP2X-5 complemented/TgAP2XI-5 strain (yellow), TgAP2XI-5-tagged strain (red) and parental strain (negative control, green). Amplification was carried out on regions within the promoter of each of the six genes listed. The enrichment for corresponding ChIP DNA samples is presented as a percentage of INPUT for each target.

    Article Snippet: Libraries were validated using a Fragment Analyzer and quantified by quantitative PCR (qPCR) (ROCHE LightCycler 480).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Hybridization, Immunoprecipitation, Microarray, Negative Control, Positive Control, Real-time Polymerase Chain Reaction, Amplification

    Characterization of HBV particles released from apoptotic PHH. Supernatants of HBV-infected PHH cultures were collected 24 h after anti-CD95 MAb (130 ng/ml) or mock treatment and subjected to CsCl gradient centrifugation. Twelve fractions (F) with decreasing densities (F1 to F12) were collected; F3 to F10 were used for further characterization. (a) HBsAg ELISA indicating enveloped HBV particles. Average values from three experiments ± standard errors of the means are given. (b) Qualitative and quantitative analysis of encapsidated viral nucleic acids using one-step RT-PCR. To discriminate between DNA and RNA, DNase digestion was performed. (Left) Mock treatment. (Right) Anti-CD95 treatment. The total amount of HBV nucleic acids (DNA plus RNA) (gray bars) detected in F8 of mock-treated cells was set to 100%. HBV RNA levels (continued line) of all samples are reported as parts per thousand. (c) Amplification products of qPCR runs of F4 to F6 with the indicated treatments were separated on a 2% agarose gel and visualized by ethidium bromide staining.

    Journal: Journal of Virology

    Article Title: Apoptosis of Hepatitis B Virus-Infected Hepatocytes Prevents Release of Infectious Virus ▿

    doi: 10.1128/JVI.00653-10

    Figure Lengend Snippet: Characterization of HBV particles released from apoptotic PHH. Supernatants of HBV-infected PHH cultures were collected 24 h after anti-CD95 MAb (130 ng/ml) or mock treatment and subjected to CsCl gradient centrifugation. Twelve fractions (F) with decreasing densities (F1 to F12) were collected; F3 to F10 were used for further characterization. (a) HBsAg ELISA indicating enveloped HBV particles. Average values from three experiments ± standard errors of the means are given. (b) Qualitative and quantitative analysis of encapsidated viral nucleic acids using one-step RT-PCR. To discriminate between DNA and RNA, DNase digestion was performed. (Left) Mock treatment. (Right) Anti-CD95 treatment. The total amount of HBV nucleic acids (DNA plus RNA) (gray bars) detected in F8 of mock-treated cells was set to 100%. HBV RNA levels (continued line) of all samples are reported as parts per thousand. (c) Amplification products of qPCR runs of F4 to F6 with the indicated treatments were separated on a 2% agarose gel and visualized by ethidium bromide staining.

    Article Snippet: Total nucleic acids were extracted from the respective fractions with a QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany) and analyzed by quantitative PCR (qPCR) (one-step reverse transcription [RT]-PCR LightCycler RNA Master SYBR green I kit; Roche Diagnostics, Mannheim, Germany) using HBV-specific primers ( ).

    Techniques: Infection, Gradient Centrifugation, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Expression profile of Catsper1au in male and female mouse organs. A cDNA panel was prepared from different organs and cells to amplify the Catsper1au 5′ region through PCR based on the sequences obtained using 5′-RACE. A product of 496 bp

    Journal: Scientific Reports

    Article Title: Catsper1 promoter is bidirectional and regulates the expression of a novel lncRNA

    doi: 10.1038/s41598-017-13867-2

    Figure Lengend Snippet: Expression profile of Catsper1au in male and female mouse organs. A cDNA panel was prepared from different organs and cells to amplify the Catsper1au 5′ region through PCR based on the sequences obtained using 5′-RACE. A product of 496 bp

    Article Snippet: The relative expression of Catsper1au RNA and Catsper1 was measured by real-time quantitative PCR (LightCycler® 480 System, Roche) using SYBR® Premix assay (Roche) with the primers NGFORW225RT (F)-REVD3 (R) and Cat1(F)-Cat1(R).

    Techniques: Expressing, Polymerase Chain Reaction

    NuRD occupancy at RARβ2 promoter is independent of PRC2 complex. (A) Western blot analysis of mock and Suz12 interference U937-PR9 cells. Equal amounts of cell extract from mock and RNAi-MBD3 cells were blotted with the indicated antibodies (left panel). ChIP analysis was performed using antibodies indicated in the figure. The RARβ2 promoter was amplified by real-time PCR. Errors bars indicate the standard deviation obtained from three independent experiments (right panel). (B) Polycomb knockdown reduces DNA methylation of the RARβ2 gene. DNA extracted from mock (RNAi-Scr) and RNAi-Suz12 U937-PR9 cells untreated or treated with 100 μM Zn for 24 h was used for bisulfite genomic sequencing. The methylation status of each CpG dinucleotide in each sequenced cloned is depicted by a red square if the position was methylated or a white square if it was not.

    Journal: Molecular and Cellular Biology

    Article Title: MBD3, a Component of the NuRD Complex, Facilitates Chromatin Alteration and Deposition of Epigenetic Marks ▿

    doi: 10.1128/MCB.00467-08

    Figure Lengend Snippet: NuRD occupancy at RARβ2 promoter is independent of PRC2 complex. (A) Western blot analysis of mock and Suz12 interference U937-PR9 cells. Equal amounts of cell extract from mock and RNAi-MBD3 cells were blotted with the indicated antibodies (left panel). ChIP analysis was performed using antibodies indicated in the figure. The RARβ2 promoter was amplified by real-time PCR. Errors bars indicate the standard deviation obtained from three independent experiments (right panel). (B) Polycomb knockdown reduces DNA methylation of the RARβ2 gene. DNA extracted from mock (RNAi-Scr) and RNAi-Suz12 U937-PR9 cells untreated or treated with 100 μM Zn for 24 h was used for bisulfite genomic sequencing. The methylation status of each CpG dinucleotide in each sequenced cloned is depicted by a red square if the position was methylated or a white square if it was not.

    Article Snippet: The immunoprecipitated DNA was quantified by real-time quantitative PCR (Roche LightCycler).

    Techniques: Western Blot, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Standard Deviation, DNA Methylation Assay, Genomic Sequencing, Methylation, Clone Assay