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

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

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

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01259

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

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

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

    Techniques Used: Expressing, Quantitative RT-PCR

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

    Techniques Used: Expressing, Quantitative RT-PCR

    2) Product Images from "Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice"

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061786

    Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p
    Figure Legend Snippet: Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p

    Techniques Used: Expressing, Quantitative RT-PCR, Staining

    Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p
    Figure Legend Snippet: Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p

    Techniques Used: Expressing, Derivative Assay, Concentration Assay, Quantitative RT-PCR, Mouse Assay, Injection, Labeling, Activity Assay

    3) Product Images from "Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer"

    Article Title: Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-4-14

    Expression of Hsulf-1 in pancreatic cancer cell lines (A) Quantification of Hsulf-1 mRNA levels in pancreatic cancer cell lines by real time QRT-PCR as described in the Materials and Methods section. Values are normalized to housekeeping genes (cyclopilin B and HRPT), and presented as mean ± SD. (B/C) Panc-1 cells were stable transfected with a Hsulf-1 sense expression plasmid as described in the Material and Methods section. (B) Hsulf-1 sense RNA expression in Panc-1 cells was verified by Northern blot analysis using a radiolabeled Hsulf-1 antisense riboprobe. A sample Northern blot of 2 controls and 2 transfected clones is shown. (C) Expression of c-myc tagged Hsulf-1 (arrow) by immunoblot analysis as described in the Materials and Methods section. Equal loading of the protein samples was confirmed using anti-γ-tubulin antibodies. (D) Sulfatase activity was measured as described in Material and Methods section in control and positive transfected clones. Data are expressed as relative fluorescence and presented as mean ± SD.
    Figure Legend Snippet: Expression of Hsulf-1 in pancreatic cancer cell lines (A) Quantification of Hsulf-1 mRNA levels in pancreatic cancer cell lines by real time QRT-PCR as described in the Materials and Methods section. Values are normalized to housekeeping genes (cyclopilin B and HRPT), and presented as mean ± SD. (B/C) Panc-1 cells were stable transfected with a Hsulf-1 sense expression plasmid as described in the Material and Methods section. (B) Hsulf-1 sense RNA expression in Panc-1 cells was verified by Northern blot analysis using a radiolabeled Hsulf-1 antisense riboprobe. A sample Northern blot of 2 controls and 2 transfected clones is shown. (C) Expression of c-myc tagged Hsulf-1 (arrow) by immunoblot analysis as described in the Materials and Methods section. Equal loading of the protein samples was confirmed using anti-γ-tubulin antibodies. (D) Sulfatase activity was measured as described in Material and Methods section in control and positive transfected clones. Data are expressed as relative fluorescence and presented as mean ± SD.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, RNA Expression, Northern Blot, Clone Assay, Activity Assay, Fluorescence

    Expression of Hsulf-1 mRNA in pancreatic tissues and cell lines Quantification of Hsuf-1 mRNA levels in the normal pancreas, chronic pancreatitis (CP), pancreatic cancer tissues (PC), by real time QRT-PCR as described in the Material and Methods section. Values are normalized to housekeeping genes (cyclophilin B and HRPT), and presented as single values and mean (horizontal line).
    Figure Legend Snippet: Expression of Hsulf-1 mRNA in pancreatic tissues and cell lines Quantification of Hsuf-1 mRNA levels in the normal pancreas, chronic pancreatitis (CP), pancreatic cancer tissues (PC), by real time QRT-PCR as described in the Material and Methods section. Values are normalized to housekeeping genes (cyclophilin B and HRPT), and presented as single values and mean (horizontal line).

    Techniques Used: Expressing, Quantitative RT-PCR

    4) Product Images from "CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells"

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052682

    CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time qRT-PCR analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p
    Figure Legend Snippet: CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time qRT-PCR analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p

    Techniques Used: Expressing, Quantitative RT-PCR

    5) Product Images from "Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2"

    Article Title: Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2

    Journal: Molecular Cytogenetics

    doi: 10.1186/1755-8166-7-42

    Expression analysis. a) MSI2 and b) HOXA9 are over-expressed in the present patient with NUP98/HOXA13 . BC1 and BC2: two other cases of Ph + blast crisis CML with additional karyotypic aberrations over-expressing MSI2 but not HOXA9 . Expression values were referred to the average of two references. Fluorescence data were analyzed with the Second Derivative Maximum method; gene expression was expressed as Cp (Crossing point) values. c) Significance for MSI2 expression was tested by Mann–Whitney test (*p
    Figure Legend Snippet: Expression analysis. a) MSI2 and b) HOXA9 are over-expressed in the present patient with NUP98/HOXA13 . BC1 and BC2: two other cases of Ph + blast crisis CML with additional karyotypic aberrations over-expressing MSI2 but not HOXA9 . Expression values were referred to the average of two references. Fluorescence data were analyzed with the Second Derivative Maximum method; gene expression was expressed as Cp (Crossing point) values. c) Significance for MSI2 expression was tested by Mann–Whitney test (*p

    Techniques Used: Expressing, Fluorescence, MANN-WHITNEY

    Chromatin Immunoprecipitation. NUP98/HOXA13 binds both MSI2 and HOXA9 promoters. ChIP was performed on both NUP98/HOXA13 sample and a non-malignant disease sample (wt). 1,5 μg of rat IgG (Millipore Normal Rat IgG Polyclonal Antibody) and No Antibody (not shown) were used as negative controls. a) Semi-quantitative PCR showed an enrichment in NUP98/HOXA13 sample compared to controls. b) qPCR confirmed this result; data are presented as fold increase relative to the control sample (wt) based on the formula 2 −ΔΔ C p [ 23 ]. One out of three (for MSI2 ) or two (for HOXA9 ) ChIP experiments is shown. The results shown are the mean ± S.E.M. (error bars) of two independent qPCR experiments. c) NUP98/HOXA13 binds both MSI2 and HOXA9 promoters. HOXA9 binds MSI2 promoter. Protein structure: homeodomain (HD). Gene structure: exons (numbered boxes), transcription start site (TSS; +1), direction of transcription (flag), putative HOX binding element 1 kb upstream of TSS (oval).
    Figure Legend Snippet: Chromatin Immunoprecipitation. NUP98/HOXA13 binds both MSI2 and HOXA9 promoters. ChIP was performed on both NUP98/HOXA13 sample and a non-malignant disease sample (wt). 1,5 μg of rat IgG (Millipore Normal Rat IgG Polyclonal Antibody) and No Antibody (not shown) were used as negative controls. a) Semi-quantitative PCR showed an enrichment in NUP98/HOXA13 sample compared to controls. b) qPCR confirmed this result; data are presented as fold increase relative to the control sample (wt) based on the formula 2 −ΔΔ C p [ 23 ]. One out of three (for MSI2 ) or two (for HOXA9 ) ChIP experiments is shown. The results shown are the mean ± S.E.M. (error bars) of two independent qPCR experiments. c) NUP98/HOXA13 binds both MSI2 and HOXA9 promoters. HOXA9 binds MSI2 promoter. Protein structure: homeodomain (HD). Gene structure: exons (numbered boxes), transcription start site (TSS; +1), direction of transcription (flag), putative HOX binding element 1 kb upstream of TSS (oval).

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    6) 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

    7) 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

    8) Product Images from "Stanniocalcin-1 Controls Ion Regulation Functions of Ion-transporting Epithelium Other than Calcium Balance"

    Article Title: Stanniocalcin-1 Controls Ion Regulation Functions of Ion-transporting Epithelium Other than Calcium Balance

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.10773

    STC-1 negatively regulates foxi3a expression at the tail-bud stage. One-cell stage embryos were injected with an stc-1 morpholino (MO) (1.3 ng/embryo) or stc-1 cRNA (40 pg/embryo), and foxi3a mRNA was subsequently detected using in situ hybridization and qRT-PCR at the tail-bud stage. Mismatched- MO (MIS) and 1x Danieau solution (Control) were used as controls. The number of foxi3a -expressing cells in the surface of yolk and foxi3a mRNA expression were significantly increased by stc-1 MO (A-D), and significantly decreased by stc-1 cRNA (E-H). qRT-PCR values were normalized to that of beta-actin. Mean ± SEM ( n = 10 or 6). * Indicates a significant difference from the control (Student's t -test, p
    Figure Legend Snippet: STC-1 negatively regulates foxi3a expression at the tail-bud stage. One-cell stage embryos were injected with an stc-1 morpholino (MO) (1.3 ng/embryo) or stc-1 cRNA (40 pg/embryo), and foxi3a mRNA was subsequently detected using in situ hybridization and qRT-PCR at the tail-bud stage. Mismatched- MO (MIS) and 1x Danieau solution (Control) were used as controls. The number of foxi3a -expressing cells in the surface of yolk and foxi3a mRNA expression were significantly increased by stc-1 MO (A-D), and significantly decreased by stc-1 cRNA (E-H). qRT-PCR values were normalized to that of beta-actin. Mean ± SEM ( n = 10 or 6). * Indicates a significant difference from the control (Student's t -test, p

    Techniques Used: Expressing, Injection, In Situ Hybridization, Quantitative RT-PCR

    STC-1 regulates the functions of ionocytes. The embryos were injected with stc-1 cRNA (40 pg/embryo) and the whole-body contents of Ca 2+ , Na + , and Cl - ions and the H + secretion of zebrafish embryos that were incubated in fresh water for 72 h. Overexpression of stc-1 caused a significant decrease of whole-body Ca 2+ , Na + , and Cl - ion contents in stc-1 cRNA injected embryos compared to those in control embryos injected with 1x Danieau solution (A). The stc-1 cRNA injected embryos also showed a lower H + secretion ability than control embryos (B). Mean ± SEM ( n = 13-16). * Indicates a significant difference from the control (Student's t -test, p
    Figure Legend Snippet: STC-1 regulates the functions of ionocytes. The embryos were injected with stc-1 cRNA (40 pg/embryo) and the whole-body contents of Ca 2+ , Na + , and Cl - ions and the H + secretion of zebrafish embryos that were incubated in fresh water for 72 h. Overexpression of stc-1 caused a significant decrease of whole-body Ca 2+ , Na + , and Cl - ion contents in stc-1 cRNA injected embryos compared to those in control embryos injected with 1x Danieau solution (A). The stc-1 cRNA injected embryos also showed a lower H + secretion ability than control embryos (B). Mean ± SEM ( n = 13-16). * Indicates a significant difference from the control (Student's t -test, p

    Techniques Used: Injection, Incubation, Over Expression

    STC-1 suppresses the mRNA expressions of ion transporter genes. One-cell stage embryos were injected with stc-1 cRNA (40 pg/embryo) and the mRNA expressions of atp6v1a (H + -ATPase), trpv6 (ECaC), and slc12a10.2 (NC) were analyzed by qRT-PCR. The gene expressions of three ion transporters in stc-1 cRNA injected embryos showed similar levels compared with control embryos injected with 1x Danieau solution at 1 dpf, but were significantly down-regulated at 2 and 3 dpf. qRT-PCR values were normalized to that of beta-actin. Mean ± SEM ( n = 4-5). * ( p
    Figure Legend Snippet: STC-1 suppresses the mRNA expressions of ion transporter genes. One-cell stage embryos were injected with stc-1 cRNA (40 pg/embryo) and the mRNA expressions of atp6v1a (H + -ATPase), trpv6 (ECaC), and slc12a10.2 (NC) were analyzed by qRT-PCR. The gene expressions of three ion transporters in stc-1 cRNA injected embryos showed similar levels compared with control embryos injected with 1x Danieau solution at 1 dpf, but were significantly down-regulated at 2 and 3 dpf. qRT-PCR values were normalized to that of beta-actin. Mean ± SEM ( n = 4-5). * ( p

    Techniques Used: Injection, Quantitative RT-PCR

    STC-1 negatively modulates the number of ionocytes in zebrafish skin. One-cell stage embryos were injected with either an stc-1 morpholino (MO) (1.3 ng/embryo) or stc-1 cRNA (40 pg/embryo). NaR cells (NaRC), HR cells (HRC), and NC cells (NCC) were subsequently detected at 72 hpf, by antibody labeling of Na + -K + -ATPase, H + -ATPase, and Na + -Cl - cotransporter, respectively. Mismatched-MO (MIS) and 1x Danieau solution (control) were used as controls. NaRC, HRC, and NC cell density in the yolk sac skin of embryos were significantly higher in stc-1 morphants (the embryos injected with MO) than in mismatched-MO-injected embryos (A-G). stc-1 cRNA injection significantly decreased ionocyte cell densities (H-N). Mean ± SEM ( n = 12). * Indicates a significant difference from the control (Student's t -test, p
    Figure Legend Snippet: STC-1 negatively modulates the number of ionocytes in zebrafish skin. One-cell stage embryos were injected with either an stc-1 morpholino (MO) (1.3 ng/embryo) or stc-1 cRNA (40 pg/embryo). NaR cells (NaRC), HR cells (HRC), and NC cells (NCC) were subsequently detected at 72 hpf, by antibody labeling of Na + -K + -ATPase, H + -ATPase, and Na + -Cl - cotransporter, respectively. Mismatched-MO (MIS) and 1x Danieau solution (control) were used as controls. NaRC, HRC, and NC cell density in the yolk sac skin of embryos were significantly higher in stc-1 morphants (the embryos injected with MO) than in mismatched-MO-injected embryos (A-G). stc-1 cRNA injection significantly decreased ionocyte cell densities (H-N). Mean ± SEM ( n = 12). * Indicates a significant difference from the control (Student's t -test, p

    Techniques Used: Injection, Antibody Labeling

    STC-1 does not affect epidermal stem cells. One-cell stage embryos were injected with an STC-1 morpholino (MO) (1.3 ng/embryo) and epidermis stem cells were subsequently detected by antibody labeling. The densities of epidermis stem cells (A-C) in the central part of yolk sac skin were unaffected by STC-1 MO (Student's t -test, p
    Figure Legend Snippet: STC-1 does not affect epidermal stem cells. One-cell stage embryos were injected with an STC-1 morpholino (MO) (1.3 ng/embryo) and epidermis stem cells were subsequently detected by antibody labeling. The densities of epidermis stem cells (A-C) in the central part of yolk sac skin were unaffected by STC-1 MO (Student's t -test, p

    Techniques Used: Injection, Antibody Labeling

    Different environments affect expression of STC genes in zebrafish embryos. Zebrafish embryos were incubated with high-Ca 2+ or acidic (pH=4) media for 4 d or double-deionized water for 1 d, and the mRNA levels of four STC genes were then measured by qRT-PCR. (A) stc-1 (STC1) and stc-2 (STC2) mRNAs were significantly decreased by low-Ca 2+ (A), acidic (B) and double-deionized water (DDW)(C) treatments, while that of stc-1 like (STC1L) and stc-2 like (STC2L) were unaffected. Low-Ca 2+ (A), acidic (B) and double-deionized water (C) treatments caused greater reductions of stc-1 mRNA (87%, 87% and 60%, respectively) than stc-2 mRNA (17%, 32% and 17%, respectively). qRT-PCR values were normalized to that of rpl13a . Mean ± SEM ( n = 4-5). * ( p
    Figure Legend Snippet: Different environments affect expression of STC genes in zebrafish embryos. Zebrafish embryos were incubated with high-Ca 2+ or acidic (pH=4) media for 4 d or double-deionized water for 1 d, and the mRNA levels of four STC genes were then measured by qRT-PCR. (A) stc-1 (STC1) and stc-2 (STC2) mRNAs were significantly decreased by low-Ca 2+ (A), acidic (B) and double-deionized water (DDW)(C) treatments, while that of stc-1 like (STC1L) and stc-2 like (STC2L) were unaffected. Low-Ca 2+ (A), acidic (B) and double-deionized water (C) treatments caused greater reductions of stc-1 mRNA (87%, 87% and 60%, respectively) than stc-2 mRNA (17%, 32% and 17%, respectively). qRT-PCR values were normalized to that of rpl13a . Mean ± SEM ( n = 4-5). * ( p

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR

    9) 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

    10) Product Images from "Vitamin D3-dependent VDR signaling delays ron-mediated breast tumorigenesis through suppression of β-catenin activity"

    Article Title: Vitamin D3-dependent VDR signaling delays ron-mediated breast tumorigenesis through suppression of β-catenin activity

    Journal: Oncotarget

    doi:

    Vitamin D 3 -dependent VDR signaling induces DKK-1 expression and binds to β-catenin to disrupt interaction at consensus sequences within promoters of TCF/LEF target genes A. qRT-PCR mRNA expression of DKK-1 in R7 cells treated with the designated concentrations of 1,25D 3 for 72 hours. Data represent mean values from three independent experiments ± SE. B. qRT-PCR mRNA expression of DKK-1 in tumors and mammary glands (MG) from 8 month-old MMTV-Ron VDR+/+ and VDR−/− mice demonstrating a reduction in DKK-1 levels in tumors with loss of VDR. Data represent mean values from three independent experiments ± SE. C. Western blot demonstrating loss of DKK-1 protein expression with siRNA-mediated silencing in R7 cells. D. R7 cells transfected with siRNA against DKK-1 were treated with the designated concentrations of 1,25D 3 for 72 hours and cell viability/number was determined by crystal violet assays. Data are normalized to the respective vehicle treated cells set at 1 and represent mean values from two independent experiments performed in quadruplicate ± SE. E. Chromatin immunoprecipitation (ChIP) assays with a mouse IgG isotype control and an anti-active β-catenin (ABC) antibody. ChIP-ABC quantitative real time PCR (qRT-PCR) analysis of R7 cells treated with 100 nM 1,25D 3 for 72 hours. The graph shows qRT-PCR on DNA purified from ChIP-ABC, using primers designed to the LEF-1 binding sequence within the mouse cyclin D1 promoter and relative to the respective input controls. Vitamin D 3 treatment significantly reduces enrichment compared to the vehicle control (EtOH). Data represent mean values from three independent experiments ± SE. F. Representative agarose gel from ChIP-ABC PCR showing reduced cyclin D1 promoter enrichment with vitamin D 3 treatment in R7 cells. Negative control primers designed to a site 4000 bp upstream of the LEF-1 binding sequence within the cyclin D1 promoter (Off-Target) verify specificity of cyclin D1 primers to the sheared DNA product. G. Densitometry analysis of PCR products from three separate ChIP-ABC experiments supporting loss of ABC interaction with the cyclin D1 promoter. Error bars represent SE. H. Re-ChIP qRT-PCR analysis of R7 cells treated with 100 nM 1,25D 3 for 72 hours and sequentially immunoprecipitated with anti-ABC then anti-VDR antibodies. The graph shows qRT-PCR of DNA purified from ChIP-ABC-VDR, using primers designed to the LEF-1 binding sequence within the mouse cyclin D1 promoter, and showing less interaction of the ABC-VDR complex at the cyclin D1 promoter. Data represent the relative mean CT values from two experiments ± standard deviation. * P
    Figure Legend Snippet: Vitamin D 3 -dependent VDR signaling induces DKK-1 expression and binds to β-catenin to disrupt interaction at consensus sequences within promoters of TCF/LEF target genes A. qRT-PCR mRNA expression of DKK-1 in R7 cells treated with the designated concentrations of 1,25D 3 for 72 hours. Data represent mean values from three independent experiments ± SE. B. qRT-PCR mRNA expression of DKK-1 in tumors and mammary glands (MG) from 8 month-old MMTV-Ron VDR+/+ and VDR−/− mice demonstrating a reduction in DKK-1 levels in tumors with loss of VDR. Data represent mean values from three independent experiments ± SE. C. Western blot demonstrating loss of DKK-1 protein expression with siRNA-mediated silencing in R7 cells. D. R7 cells transfected with siRNA against DKK-1 were treated with the designated concentrations of 1,25D 3 for 72 hours and cell viability/number was determined by crystal violet assays. Data are normalized to the respective vehicle treated cells set at 1 and represent mean values from two independent experiments performed in quadruplicate ± SE. E. Chromatin immunoprecipitation (ChIP) assays with a mouse IgG isotype control and an anti-active β-catenin (ABC) antibody. ChIP-ABC quantitative real time PCR (qRT-PCR) analysis of R7 cells treated with 100 nM 1,25D 3 for 72 hours. The graph shows qRT-PCR on DNA purified from ChIP-ABC, using primers designed to the LEF-1 binding sequence within the mouse cyclin D1 promoter and relative to the respective input controls. Vitamin D 3 treatment significantly reduces enrichment compared to the vehicle control (EtOH). Data represent mean values from three independent experiments ± SE. F. Representative agarose gel from ChIP-ABC PCR showing reduced cyclin D1 promoter enrichment with vitamin D 3 treatment in R7 cells. Negative control primers designed to a site 4000 bp upstream of the LEF-1 binding sequence within the cyclin D1 promoter (Off-Target) verify specificity of cyclin D1 primers to the sheared DNA product. G. Densitometry analysis of PCR products from three separate ChIP-ABC experiments supporting loss of ABC interaction with the cyclin D1 promoter. Error bars represent SE. H. Re-ChIP qRT-PCR analysis of R7 cells treated with 100 nM 1,25D 3 for 72 hours and sequentially immunoprecipitated with anti-ABC then anti-VDR antibodies. The graph shows qRT-PCR of DNA purified from ChIP-ABC-VDR, using primers designed to the LEF-1 binding sequence within the mouse cyclin D1 promoter, and showing less interaction of the ABC-VDR complex at the cyclin D1 promoter. Data represent the relative mean CT values from two experiments ± standard deviation. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Western Blot, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Purification, Binding Assay, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Immunoprecipitation, Standard Deviation

    11) Product Images from "Pleiotropic Anti-Angiogenic and Anti-Oncogenic Activities of the Novel Mithralog Demycarosyl-3D-ß-D-Digitoxosyl-Mithramycin SK (EC-8042)"

    Article Title: Pleiotropic Anti-Angiogenic and Anti-Oncogenic Activities of the Novel Mithralog Demycarosyl-3D-ß-D-Digitoxosyl-Mithramycin SK (EC-8042)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0140786

    Analysis of the expression of key anti-angiogenic genes in ECs treated with MTA or DIG-MSK. qRT-PCR analysis of the expression of several key anti-angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared to untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate in HUVEC (a) or HMEC-1 (b) endothelial cells. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p
    Figure Legend Snippet: Analysis of the expression of key anti-angiogenic genes in ECs treated with MTA or DIG-MSK. qRT-PCR analysis of the expression of several key anti-angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared to untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate in HUVEC (a) or HMEC-1 (b) endothelial cells. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p

    Techniques Used: Expressing, Quantitative RT-PCR

    MSK and DIG-MSK modulate the expression of key angiogenic genes in ovarian carcinoma cells. a) qRT-PCR analysis of the expression of several key angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared with untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p
    Figure Legend Snippet: MSK and DIG-MSK modulate the expression of key angiogenic genes in ovarian carcinoma cells. a) qRT-PCR analysis of the expression of several key angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared with untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of MTA and DIG-MSK on tube formation by human microvascular endothelial cells. a) HMEC-1 cells were seeded onto Matrigel-coated wells and incubated with DMSO or various concentrations of MTA and DIG-MSK for 48 hours. Capillary-like structures formation was captured and processed with Angiodraw Software. Angiogenic index was calculated as the number of branch points in a field. The bars represent the mean ± SEM of the angiogenic index of four independent experiments (*p
    Figure Legend Snippet: Effect of MTA and DIG-MSK on tube formation by human microvascular endothelial cells. a) HMEC-1 cells were seeded onto Matrigel-coated wells and incubated with DMSO or various concentrations of MTA and DIG-MSK for 48 hours. Capillary-like structures formation was captured and processed with Angiodraw Software. Angiogenic index was calculated as the number of branch points in a field. The bars represent the mean ± SEM of the angiogenic index of four independent experiments (*p

    Techniques Used: Incubation, Software

    DIG-MSK regulates the expression of SP1 and key oncogenic genes in ovarian tumor cells. (a) Relative changes in luciferase activity of a transfected Sp1-reported vector in the presence of 200 nM MTA or DIG-MSK compared to untreated ovarian cancer cells. qRT-PCR analysis of the expression of SP1 gene and several key Sp1-regulated oncogenic genes in ovarian cancer cells treated or untreated with 200 nM MTA or DIG-MSK. Data represent the mean ± SEM of Ct values obtained from at least three independent experiments made by duplicate. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p
    Figure Legend Snippet: DIG-MSK regulates the expression of SP1 and key oncogenic genes in ovarian tumor cells. (a) Relative changes in luciferase activity of a transfected Sp1-reported vector in the presence of 200 nM MTA or DIG-MSK compared to untreated ovarian cancer cells. qRT-PCR analysis of the expression of SP1 gene and several key Sp1-regulated oncogenic genes in ovarian cancer cells treated or untreated with 200 nM MTA or DIG-MSK. Data represent the mean ± SEM of Ct values obtained from at least three independent experiments made by duplicate. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p

    Techniques Used: Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR

    Effect of MTA and DIG-MSK on the secretion of VEGF and the surface expression of VEGFR1 and VEGFR2. a) Soluble VEGF levels were measured by ELISA in supernatants of ovarian carcinoma cells (A2780, OVCAR-3 and IGROV-1) treated with 200 nM MTA or DIG-MSK compared with untreated cells. b) The surface expression of VEGFR1 and VEGFR2 was analyzed by flow cytometry in ECs treated with 200 nM MTA or DIG-MSK relative to DMSO treated cells. Data represent the mean ± SEM of levels obtained from at least three independent experiments. (*p
    Figure Legend Snippet: Effect of MTA and DIG-MSK on the secretion of VEGF and the surface expression of VEGFR1 and VEGFR2. a) Soluble VEGF levels were measured by ELISA in supernatants of ovarian carcinoma cells (A2780, OVCAR-3 and IGROV-1) treated with 200 nM MTA or DIG-MSK compared with untreated cells. b) The surface expression of VEGFR1 and VEGFR2 was analyzed by flow cytometry in ECs treated with 200 nM MTA or DIG-MSK relative to DMSO treated cells. Data represent the mean ± SEM of levels obtained from at least three independent experiments. (*p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    Cell cycle distribution and pro-apoptotic effect on microvascular endothelial cells upon exposure to MTA and DIG-MSK. a) HUVEC and HMEC-1 cells treated with MTA (200 nM) or DIG-MSK (200 nM) or DMSO were stained with propidium iodide (PI) and the cell cycle distribution was analyzed by flow cytometry. A representative cytometric profile of HUVEC cells is shown. b) Cell death was analyzed by flow cytometry in ECs (HUVEC and HMEC-1 cells) treated with 200 nM MTA, DIG-MSK or in untreated cells after staining them with Annexin-V and 7-AAD. The bars represent the mean ± SEM of the specific cell death. At least three independent experiments were analyzed (*p
    Figure Legend Snippet: Cell cycle distribution and pro-apoptotic effect on microvascular endothelial cells upon exposure to MTA and DIG-MSK. a) HUVEC and HMEC-1 cells treated with MTA (200 nM) or DIG-MSK (200 nM) or DMSO were stained with propidium iodide (PI) and the cell cycle distribution was analyzed by flow cytometry. A representative cytometric profile of HUVEC cells is shown. b) Cell death was analyzed by flow cytometry in ECs (HUVEC and HMEC-1 cells) treated with 200 nM MTA, DIG-MSK or in untreated cells after staining them with Annexin-V and 7-AAD. The bars represent the mean ± SEM of the specific cell death. At least three independent experiments were analyzed (*p

    Techniques Used: Staining, Flow Cytometry, Cytometry

    Pro-apoptotic activity of MTA and DIG-MSK in ovarian and mononuclear blood cells. a) Ovarian cells and PBMCs were treated with MTA (200 nM), DIG-MSK (200 nM) or DMSO and cell death was analyzed by flow cytometry by staining the treated cells with Annexin-V-FITC and 7-AAD. b) The bars represent the mean ± SEM of the specific cell death of at least three independent experiments. PBMCs were obtained from six unrelated donors. c and d) Analysis of caspase-3 and -9 activities in ovarian cells and PBMCs treated with 200 nM MTA or DIG-MSK compared to vehicle (DMSO) treated cells. The bars represent the mean ± SEM of the units (U) of caspase activity obtained from at least three independent experiments (*p
    Figure Legend Snippet: Pro-apoptotic activity of MTA and DIG-MSK in ovarian and mononuclear blood cells. a) Ovarian cells and PBMCs were treated with MTA (200 nM), DIG-MSK (200 nM) or DMSO and cell death was analyzed by flow cytometry by staining the treated cells with Annexin-V-FITC and 7-AAD. b) The bars represent the mean ± SEM of the specific cell death of at least three independent experiments. PBMCs were obtained from six unrelated donors. c and d) Analysis of caspase-3 and -9 activities in ovarian cells and PBMCs treated with 200 nM MTA or DIG-MSK compared to vehicle (DMSO) treated cells. The bars represent the mean ± SEM of the units (U) of caspase activity obtained from at least three independent experiments (*p

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Staining

    Cell cycle distribution in ovarian cancer cells treated with MTA and DIG-MSK. a) Ovarian tumor cells were treated with MTA (200 nM), DIG-MSK (200 nM) or DMSO, and the cell cycle distribution was analyzed by propidium iodide (PI) staining and flow cytometry analysis. Representative histograms of OVCAR-3 cells are shown. b) The bars represent the mean ± SEM of the percentage of cells in the different phases of the cell cycle in IGROV-1, A2780 and OVCAR-3 ovarian carcinoma cells. The results are obtained from at least three independent experiments (drug vs. DMSO; *p
    Figure Legend Snippet: Cell cycle distribution in ovarian cancer cells treated with MTA and DIG-MSK. a) Ovarian tumor cells were treated with MTA (200 nM), DIG-MSK (200 nM) or DMSO, and the cell cycle distribution was analyzed by propidium iodide (PI) staining and flow cytometry analysis. Representative histograms of OVCAR-3 cells are shown. b) The bars represent the mean ± SEM of the percentage of cells in the different phases of the cell cycle in IGROV-1, A2780 and OVCAR-3 ovarian carcinoma cells. The results are obtained from at least three independent experiments (drug vs. DMSO; *p

    Techniques Used: Staining, Flow Cytometry, Cytometry

    12) Product Images from "Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice"

    Article Title: Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17040499

    Classification of miRNAs during pollen development: ( A , B ) the total number of miRNAs detected during different pollen development stages of Taichung65-2x ( A ) and Taichung65-4x ( B ); ( C , D ) Venn analysis of miRNAs expressed in Taichung65-2x ( C ) and Taichung65-4x during pollen development ( D ); and ( E , F ) specifically up- and down-regulated miRNAs during each adjacent stage of pollen development in Taichung65-2x and Taichung65-4x. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.
    Figure Legend Snippet: Classification of miRNAs during pollen development: ( A , B ) the total number of miRNAs detected during different pollen development stages of Taichung65-2x ( A ) and Taichung65-4x ( B ); ( C , D ) Venn analysis of miRNAs expressed in Taichung65-2x ( C ) and Taichung65-4x during pollen development ( D ); and ( E , F ) specifically up- and down-regulated miRNAs during each adjacent stage of pollen development in Taichung65-2x and Taichung65-4x. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.

    Techniques Used:

    Abundance of siRNAs transposable elements associated with Taichung65-4x and Taichung65-2x during pollen development stages: ( A ) Ty3-gypsy type of class I; ( B ) unclassified type of class I; ( C ) En/Spm type of class II; and ( D ) unclassified type of class II. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.
    Figure Legend Snippet: Abundance of siRNAs transposable elements associated with Taichung65-4x and Taichung65-2x during pollen development stages: ( A ) Ty3-gypsy type of class I; ( B ) unclassified type of class I; ( C ) En/Spm type of class II; and ( D ) unclassified type of class II. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.

    Techniques Used:

    Analysis of DEM (differentially expressed miRNAs) in Taichung65-2x and Taichung65-4x during pollen development. ( A ) Hierarchical cluster analysis of DEM. The hierarchical clustering tree of 172 DEM in different libraries of pollen development was constructed by MultiExperiment View (version 4.9). Each column represents the difference between Taichung65-2x and Taichung65-4x in each stage. Red and green represent the up- and down-regulated miRNAs, respectively. The scale bar indicates the relative expression levels of miRNAs (log 2 ); ( B ) The number of DEM at different pollen development stages; ( C , D ) Venn analysis of DEM during pollen development. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.
    Figure Legend Snippet: Analysis of DEM (differentially expressed miRNAs) in Taichung65-2x and Taichung65-4x during pollen development. ( A ) Hierarchical cluster analysis of DEM. The hierarchical clustering tree of 172 DEM in different libraries of pollen development was constructed by MultiExperiment View (version 4.9). Each column represents the difference between Taichung65-2x and Taichung65-4x in each stage. Red and green represent the up- and down-regulated miRNAs, respectively. The scale bar indicates the relative expression levels of miRNAs (log 2 ); ( B ) The number of DEM at different pollen development stages; ( C , D ) Venn analysis of DEM during pollen development. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.

    Techniques Used: Construct, Expressing

    The relative expression levels of miR2118 ( A ) and miR2275 ( B ) families between Taichung65-4x and Taichung65-2x. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.
    Figure Legend Snippet: The relative expression levels of miR2118 ( A ) and miR2275 ( B ) families between Taichung65-4x and Taichung65-2x. PMA, MA and SCP represent pre-meiotic interphase, meiosis and single microspore stage, respectively. “4x” and “2x” represent the autotetraploid and diploid rice, respectively.

    Techniques Used: Expressing

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

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

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-016-0340-1

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

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

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

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

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

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

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

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

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

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

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

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

    14) Product Images from "Prognostic Significance of Erythropoietin in Pancreatic Adenocarcinoma"

    Article Title: Prognostic Significance of Erythropoietin in Pancreatic Adenocarcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023151

    Expression of EpoR in PDAC cells. (A) qRT-PCR analysis revealed constitutive EpoR mRNA expression in all studied cancer cell lines and its strong up-regulation under hypoxic conditions. Nx = normoxia, Hx = hypoxia. (B) EpoR was detectable on the surface of hEpoR-transduced Ba/F3 cells and PANC-1 cells by flow cytometry analysis using an FITC-labeled antibody. Mouse IgG2b was used as a negative control. (C) Left panel: detection of the full-length EpoR protein in cell lysates of hEpoR-transduced NIH/3T3 cells and the recombinant sol-EpoR protein using the 3D10-antibody by Western blot analysis (with GAPDH as loading control). Right panel: detection of EpoR in pancreatic tumor cells and hEpoR-NIH/3T3 by immunoprecipitation with the 3D10-antibody followed by immunoblotting with the C-20 antibody.
    Figure Legend Snippet: Expression of EpoR in PDAC cells. (A) qRT-PCR analysis revealed constitutive EpoR mRNA expression in all studied cancer cell lines and its strong up-regulation under hypoxic conditions. Nx = normoxia, Hx = hypoxia. (B) EpoR was detectable on the surface of hEpoR-transduced Ba/F3 cells and PANC-1 cells by flow cytometry analysis using an FITC-labeled antibody. Mouse IgG2b was used as a negative control. (C) Left panel: detection of the full-length EpoR protein in cell lysates of hEpoR-transduced NIH/3T3 cells and the recombinant sol-EpoR protein using the 3D10-antibody by Western blot analysis (with GAPDH as loading control). Right panel: detection of EpoR in pancreatic tumor cells and hEpoR-NIH/3T3 by immunoprecipitation with the 3D10-antibody followed by immunoblotting with the C-20 antibody.

    Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Labeling, Negative Control, Recombinant, Western Blot, Immunoprecipitation

    Activation of PI3K/Akt but not Jak2/STAT5 signaling in PDAC cells exposed to Epo. (A) Phosphorylation status of Stat5 was assessed in serum-starved pancreatic tumor cells and hEpoR-transduced NIH/3T3 cells stimulated with 50 U/ml erythropoietin (Epo) for 10 min. Clear accumulation of pSTAT5 was observed in hEpoR-NIH/3T3 but not in mock-transduced NIH/3T3 cells or in PDAC cells, independent of the level of constitutive pSTAT5 activation. (B) Phosphorylation status of Akt was assessed in serum-starved (left panel) or non-starved (right panel) PANC-1 cells consequently stimulated with Epo at 0–50 U/ml for 15 min. Epo-enhanced pAkt phosporylation was detected only under conditions of serum starvation and could be specifically inhibited by anti-EpoR antibody (+Ab) or phosphatidylinositol-3-OH kinase (PI3K) inhibitor LY2940020 (+LY). (C) Accumulation of mRNA coding for soluble EpoR isoform (upper panel; primers: EpoR_S5/6, table 2 ) as compared to mRNAs coding for full-length isoforms (two middle panels) and further related to expression of ß-actin (lower panel). 3′-end-based detection of EpoR mRNA was performed with antisense primers binding after (EpoR_FL1/2) or prior to a stop codon (EpoR_FL3/4) as visualized by a hEpoR plasmid carrying only the coding sequence for a full-length isoform.
    Figure Legend Snippet: Activation of PI3K/Akt but not Jak2/STAT5 signaling in PDAC cells exposed to Epo. (A) Phosphorylation status of Stat5 was assessed in serum-starved pancreatic tumor cells and hEpoR-transduced NIH/3T3 cells stimulated with 50 U/ml erythropoietin (Epo) for 10 min. Clear accumulation of pSTAT5 was observed in hEpoR-NIH/3T3 but not in mock-transduced NIH/3T3 cells or in PDAC cells, independent of the level of constitutive pSTAT5 activation. (B) Phosphorylation status of Akt was assessed in serum-starved (left panel) or non-starved (right panel) PANC-1 cells consequently stimulated with Epo at 0–50 U/ml for 15 min. Epo-enhanced pAkt phosporylation was detected only under conditions of serum starvation and could be specifically inhibited by anti-EpoR antibody (+Ab) or phosphatidylinositol-3-OH kinase (PI3K) inhibitor LY2940020 (+LY). (C) Accumulation of mRNA coding for soluble EpoR isoform (upper panel; primers: EpoR_S5/6, table 2 ) as compared to mRNAs coding for full-length isoforms (two middle panels) and further related to expression of ß-actin (lower panel). 3′-end-based detection of EpoR mRNA was performed with antisense primers binding after (EpoR_FL1/2) or prior to a stop codon (EpoR_FL3/4) as visualized by a hEpoR plasmid carrying only the coding sequence for a full-length isoform.

    Techniques Used: Activation Assay, Expressing, Binding Assay, Plasmid Preparation, Sequencing

    15) 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

    16) Product Images from "MEG3, HCN3 and linc01105 influence the proliferation and apoptosis of neuroblastoma cells via the HIF-1α and p53 pathways"

    Article Title: MEG3, HCN3 and linc01105 influence the proliferation and apoptosis of neuroblastoma cells via the HIF-1α and p53 pathways

    Journal: Scientific Reports

    doi: 10.1038/srep36268

    The effect of lncRNAs on HIF-1α, Bid and Noxa protein expression. ( A ) Expression of HIF-1α, Noxa and Bid in the blank, negative control and MEG3 OE groups. ( B ) Expression of HIF-1α, Noxa and Bid in blank, negative control and HCN3 KD groups. ( C ) Expression of HIF-1α, Noxa and Bid in blank, negative control and linc01105 KD groups. B, Blank group; N, Negative control group; H, HCN3 KD group; M, MEG3 OE group; KD, linc01105 KD group. Full-length blots were shown as supplementary figure 6 in supplementary information .
    Figure Legend Snippet: The effect of lncRNAs on HIF-1α, Bid and Noxa protein expression. ( A ) Expression of HIF-1α, Noxa and Bid in the blank, negative control and MEG3 OE groups. ( B ) Expression of HIF-1α, Noxa and Bid in blank, negative control and HCN3 KD groups. ( C ) Expression of HIF-1α, Noxa and Bid in blank, negative control and linc01105 KD groups. B, Blank group; N, Negative control group; H, HCN3 KD group; M, MEG3 OE group; KD, linc01105 KD group. Full-length blots were shown as supplementary figure 6 in supplementary information .

    Techniques Used: Expressing, Negative Control

    17) Product Images from "Effect of Cinnamomum osmophloeum Kanehira Leaf Aqueous Extract on Dermal Papilla Cell Proliferation and Hair Growth"

    Article Title: Effect of Cinnamomum osmophloeum Kanehira Leaf Aqueous Extract on Dermal Papilla Cell Proliferation and Hair Growth

    Journal: Cell Transplantation

    doi: 10.1177/0963689717741139

    Quantitative polymerase chain reaction of growth factor genes in human hair dermal papilla cells (hDPCs). Changes in growth factor messenger RNA (mRNA) expression levels induced after different treatments for 48 h. The growth factor mRNA expression levels were normalized to that of β-actin mRNA expression with the results expressed as fold changes.
    Figure Legend Snippet: Quantitative polymerase chain reaction of growth factor genes in human hair dermal papilla cells (hDPCs). Changes in growth factor messenger RNA (mRNA) expression levels induced after different treatments for 48 h. The growth factor mRNA expression levels were normalized to that of β-actin mRNA expression with the results expressed as fold changes.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    18) 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

    19) Product Images from "EsGLUT4 and CHHBP are involved in the regulation of glucose homeostasis in the crustacean Eriocheir sinensis"

    Article Title: EsGLUT4 and CHHBP are involved in the regulation of glucose homeostasis in the crustacean Eriocheir sinensis

    Journal: Biology Open

    doi: 10.1242/bio.027532

    Expression and purification of the extracellular and intracellular regions of recombinant CHH, EsGLUT4 and the CHHBP protein. The purified recombinant proteins were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Lane M: protein molecular weight standard. Lane 1: CHH protein. Lane 2: extracellular region of EsGLUT4. Lane 3: intracellular region of EsGLUT4. Lane 4: CHHBP protein.
    Figure Legend Snippet: Expression and purification of the extracellular and intracellular regions of recombinant CHH, EsGLUT4 and the CHHBP protein. The purified recombinant proteins were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Lane M: protein molecular weight standard. Lane 1: CHH protein. Lane 2: extracellular region of EsGLUT4. Lane 3: intracellular region of EsGLUT4. Lane 4: CHHBP protein.

    Techniques Used: Expressing, Purification, Recombinant, SDS Page, Staining, Molecular Weight

    Co-expression of CHHBP and EsGLUT4 in Hi5 insect cells. pIZV5-CHHBP-Cherry and pIZV5-EsGLUT4-GFP were cotransfected into Hi5 insect cells using X-tremeGENE HP DNA Transfection reagent. The expression of the reporter genes Cherry and Enhanced Green Fluorescent Protein (EGFP), which represented CHHBP (red) and EsGLUT4 (green), respectively, was observed under a fluorescent confocal microscope at 24 h post-transfection (A,B). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) (C). Merged images showing overlap of CHHBP, EsGLUT4, and DAPI staining (D). Scale bar: 9.9 µm.
    Figure Legend Snippet: Co-expression of CHHBP and EsGLUT4 in Hi5 insect cells. pIZV5-CHHBP-Cherry and pIZV5-EsGLUT4-GFP were cotransfected into Hi5 insect cells using X-tremeGENE HP DNA Transfection reagent. The expression of the reporter genes Cherry and Enhanced Green Fluorescent Protein (EGFP), which represented CHHBP (red) and EsGLUT4 (green), respectively, was observed under a fluorescent confocal microscope at 24 h post-transfection (A,B). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) (C). Merged images showing overlap of CHHBP, EsGLUT4, and DAPI staining (D). Scale bar: 9.9 µm.

    Techniques Used: Expressing, Transfection, Microscopy, Staining

    The effects of EsCHH on hepatopancreatic cells. (A) Hepatopancreatic cells from E. sinensis were cultured in medium for 2 days and examined under an inverted microscope. (B) The glucose concentration in the medium of cultured hepatopancreatic cells from E. sinensis after the addition of EsCHH at different concentrations. (C) EsCHH-induced EsGLUT4 and CHHBP showed different translocation patterns in Hi5 insect cells. CHHBP and EsGLUT4 were co-expressed and their translocation in Hi5 insect cells was monitored by live-cell imaging at different time points after stimulation with CHH. Detection of pIZV5-CHHBP-Cherry at 0, 60, 132, and 216 min (Ca-Cd). Detection of pIZV5-EsGLUT4-GFP at 0, 60, 132, and 216 min (Ce-Ch). Merged images show overlapping fluorescent signals for CHHBP, EsGLUT4, and DAPI (Ci-Cl). (D) The glucose concentration in the medium of cultured Hi5 insect cells at 0, 0.5, 1, and 2 h after stimulation with CHH. Cells were divided into four groups as follows: cells transfected with pIZV5-CHHBP-Cherry, cells transfected with pIZV5-EsGLUT4-GFP, cells cotransfected with pIZV5-CHHBP-Cherry and pIZV5-EsGLUT4-GFP, and cells without any treatment (control). In B and D, data denoted with different lowercase letters indicate significant differences within groups; the capital letters indicate significant differences between groups ( P
    Figure Legend Snippet: The effects of EsCHH on hepatopancreatic cells. (A) Hepatopancreatic cells from E. sinensis were cultured in medium for 2 days and examined under an inverted microscope. (B) The glucose concentration in the medium of cultured hepatopancreatic cells from E. sinensis after the addition of EsCHH at different concentrations. (C) EsCHH-induced EsGLUT4 and CHHBP showed different translocation patterns in Hi5 insect cells. CHHBP and EsGLUT4 were co-expressed and their translocation in Hi5 insect cells was monitored by live-cell imaging at different time points after stimulation with CHH. Detection of pIZV5-CHHBP-Cherry at 0, 60, 132, and 216 min (Ca-Cd). Detection of pIZV5-EsGLUT4-GFP at 0, 60, 132, and 216 min (Ce-Ch). Merged images show overlapping fluorescent signals for CHHBP, EsGLUT4, and DAPI (Ci-Cl). (D) The glucose concentration in the medium of cultured Hi5 insect cells at 0, 0.5, 1, and 2 h after stimulation with CHH. Cells were divided into four groups as follows: cells transfected with pIZV5-CHHBP-Cherry, cells transfected with pIZV5-EsGLUT4-GFP, cells cotransfected with pIZV5-CHHBP-Cherry and pIZV5-EsGLUT4-GFP, and cells without any treatment (control). In B and D, data denoted with different lowercase letters indicate significant differences within groups; the capital letters indicate significant differences between groups ( P

    Techniques Used: Cell Culture, Inverted Microscopy, Concentration Assay, Translocation Assay, Live Cell Imaging, Transfection

    Functional analysis of EsGLUT4 in vivo . Crabs were injected with GFP dsRNA or EsGLUT4 dsRNA. The hepatopancreas tissues were collected to test gene knock-down efficiency. Transcript expression of EsGLUT4, CHHBP, and GS in hepatopancreas at 2 days and 4 days after dsRNA injection was assessed by quantitative real-time PCR (A,D,E). Transcript expression of EsGLUT4 in muscle was also assessed (B). β-actin was employed as an internal reference gene. Hemolymph glucose concentration of crabs after GFP RNAi or EsGLUT4 RNAi injection were measured (C). (F) Changes in glucose level in control (black bars) and EsGLUT4 RNAi group (gray bars) after injection of rCHH. Values are presented as means±s.d. ( n =6). Data denoted with different lowercase letters indicate significant differences ( P
    Figure Legend Snippet: Functional analysis of EsGLUT4 in vivo . Crabs were injected with GFP dsRNA or EsGLUT4 dsRNA. The hepatopancreas tissues were collected to test gene knock-down efficiency. Transcript expression of EsGLUT4, CHHBP, and GS in hepatopancreas at 2 days and 4 days after dsRNA injection was assessed by quantitative real-time PCR (A,D,E). Transcript expression of EsGLUT4 in muscle was also assessed (B). β-actin was employed as an internal reference gene. Hemolymph glucose concentration of crabs after GFP RNAi or EsGLUT4 RNAi injection were measured (C). (F) Changes in glucose level in control (black bars) and EsGLUT4 RNAi group (gray bars) after injection of rCHH. Values are presented as means±s.d. ( n =6). Data denoted with different lowercase letters indicate significant differences ( P

    Techniques Used: Functional Assay, In Vivo, Injection, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay

    20) Product Images from "Adipose-Derived Mesenchymal Stem Cells from the Elderly Exhibit Decreased Migration and Differentiation Abilities with Senescent Properties"

    Article Title: Adipose-Derived Mesenchymal Stem Cells from the Elderly Exhibit Decreased Migration and Differentiation Abilities with Senescent Properties

    Journal: Cell Transplantation

    doi: 10.1177/0963689717721221

    Effect of donor age on human adipose-derived mesenchymal stem cells (hASCs) colony-forming unit fibroblasts (CFU-Fs), proliferation, apoptosis, and cell cycle distribution. (A) CFU-Fs assay showed the number of clone forming cells decreased significantly in elderly group compared with the child group. (B) Donor age had no effect on hASCs proliferation, as evidenced by the MTS assay. (C) qRT-PCR analysis of genes associated with proliferation ( c-Jun and c-Fos ) showing a decreasing trend with age. (D) Apoptosis was analyzed using a Muse Cell Analyzer, showing no significant difference between the different age-groups. (E) Expression of apoptosis-related genes Bcl-2 and caspase-8 were determined by qRT-PCR and did not appear to be significantly affected by donor age. (F) Cell cycle distribution of hASCs in different age-groups analyzed with a Muse Cell Analyzer. The number of cells in S phase in the elderly group was significantly decreased compared with the other 2 groups. (G) qRT-PCR analysis showed that the expression of CHEK1 , a cell cycle-associated gene, was upregulated with increasing age, although the difference was not significant. Experiments were carried out in triplicate. Data are expressed as the mean ± standard error of mean [SEM]. *P
    Figure Legend Snippet: Effect of donor age on human adipose-derived mesenchymal stem cells (hASCs) colony-forming unit fibroblasts (CFU-Fs), proliferation, apoptosis, and cell cycle distribution. (A) CFU-Fs assay showed the number of clone forming cells decreased significantly in elderly group compared with the child group. (B) Donor age had no effect on hASCs proliferation, as evidenced by the MTS assay. (C) qRT-PCR analysis of genes associated with proliferation ( c-Jun and c-Fos ) showing a decreasing trend with age. (D) Apoptosis was analyzed using a Muse Cell Analyzer, showing no significant difference between the different age-groups. (E) Expression of apoptosis-related genes Bcl-2 and caspase-8 were determined by qRT-PCR and did not appear to be significantly affected by donor age. (F) Cell cycle distribution of hASCs in different age-groups analyzed with a Muse Cell Analyzer. The number of cells in S phase in the elderly group was significantly decreased compared with the other 2 groups. (G) qRT-PCR analysis showed that the expression of CHEK1 , a cell cycle-associated gene, was upregulated with increasing age, although the difference was not significant. Experiments were carried out in triplicate. Data are expressed as the mean ± standard error of mean [SEM]. *P

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

    21) Product Images from "A Five-microRNA Signature for Survival Prognosis in Pancreatic Adenocarcinoma based on TCGA Data"

    Article Title: A Five-microRNA Signature for Survival Prognosis in Pancreatic Adenocarcinoma based on TCGA Data

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22493-5

    Aberrant expression of miRNAs in pancreatic adenocarcinoma. Expression of the five-miRNA signature was analyzed by qRT-PCR in pancreatic adenocarcinoma tissues. ( A ) miRNA-203. ( B ) miRNA-1266. ( C ) miR-1293. ( D ) miRNA-424. ( D ) miRNA-4772.
    Figure Legend Snippet: Aberrant expression of miRNAs in pancreatic adenocarcinoma. Expression of the five-miRNA signature was analyzed by qRT-PCR in pancreatic adenocarcinoma tissues. ( A ) miRNA-203. ( B ) miRNA-1266. ( C ) miR-1293. ( D ) miRNA-424. ( D ) miRNA-4772.

    Techniques Used: Expressing, Quantitative RT-PCR

    22) Product Images from "miR-17-5p suppresses cell proliferation and invasion by targeting ETV1 in triple-negative breast cancer"

    Article Title: miR-17-5p suppresses cell proliferation and invasion by targeting ETV1 in triple-negative breast cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-017-3674-x

    Features of miR-17-5p and ETV1 expression in TNBC cells and clinical samples. a , b , QRT-PCR analyses of miR-17-5p and ETV1 expression levels in TNBC cells. c , d , The expression levels of miR-17-5p and ETV1 in clinical TNBC samples evaluated by qRT-PCR. Data are expressed as the mean ± SEM of three independent experiments. ** P
    Figure Legend Snippet: Features of miR-17-5p and ETV1 expression in TNBC cells and clinical samples. a , b , QRT-PCR analyses of miR-17-5p and ETV1 expression levels in TNBC cells. c , d , The expression levels of miR-17-5p and ETV1 in clinical TNBC samples evaluated by qRT-PCR. Data are expressed as the mean ± SEM of three independent experiments. ** P

    Techniques Used: Expressing, Quantitative RT-PCR

    ETV1 is a direct target of miR-17-5p in TNBC cells. a , Target sequences of miR-17-5p in ETV1 3′-UTR and mutant sites in 3′-UTR. b , Relative luciferase activity of ETV1 3′-UTR and mutant in the miR-17-5p mimic-transfected 293 T cells. c , d , The effect of miR-17-5p on ETV1 expression in MDA-MB-231 and BT549 cells was detected by qRT-PCR and western blotting after the cells were transfected with miR-17-5p mimic or inhibitor, respectively. e , The effect of miR-17-5p on ETV1 expression was also observed in MCF10A cells by co-transfecting with GV141-ETV1 and miR-17-5p inhibitor. Data are expressed as the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: ETV1 is a direct target of miR-17-5p in TNBC cells. a , Target sequences of miR-17-5p in ETV1 3′-UTR and mutant sites in 3′-UTR. b , Relative luciferase activity of ETV1 3′-UTR and mutant in the miR-17-5p mimic-transfected 293 T cells. c , d , The effect of miR-17-5p on ETV1 expression in MDA-MB-231 and BT549 cells was detected by qRT-PCR and western blotting after the cells were transfected with miR-17-5p mimic or inhibitor, respectively. e , The effect of miR-17-5p on ETV1 expression was also observed in MCF10A cells by co-transfecting with GV141-ETV1 and miR-17-5p inhibitor. Data are expressed as the mean ± SEM of three independent experiments. * P

    Techniques Used: Mutagenesis, Luciferase, Activity Assay, Transfection, Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot

    23) Product Images from "Design of live attenuated bacterial vaccines based on D-glutamate auxotrophy"

    Article Title: Design of live attenuated bacterial vaccines based on D-glutamate auxotrophy

    Journal: Nature Communications

    doi: 10.1038/ncomms15480

    D-Glu auxotrophy produces cell wall degeneration and bacterial lysis. ( a – c ) Different atypical morphologies, progressive degeneration of the cell wall, membrane-bursting events and lysis of D-Glu auxotrophic strains after being kept in the absence of D-Glu (red dotted arrows). Micrographs were taken with a TEM at different scales. ( a ) A. baumannii ATCC 17978 (Ab) and Ab Δ murI1 Δ murI2 . ( b ) P. aeruginosa PAO1 (Pa) and Pa Δ murI . ( c ) S. aureus 132 (Sa) and Sa Δ murI Δ dat . ( d ) Proposed schematic mechanism of cell wall degeneration and bacterial lysis of (1) gram-negative and (2) gram-positive bacteria auxotrophic for D-Glu.
    Figure Legend Snippet: D-Glu auxotrophy produces cell wall degeneration and bacterial lysis. ( a – c ) Different atypical morphologies, progressive degeneration of the cell wall, membrane-bursting events and lysis of D-Glu auxotrophic strains after being kept in the absence of D-Glu (red dotted arrows). Micrographs were taken with a TEM at different scales. ( a ) A. baumannii ATCC 17978 (Ab) and Ab Δ murI1 Δ murI2 . ( b ) P. aeruginosa PAO1 (Pa) and Pa Δ murI . ( c ) S. aureus 132 (Sa) and Sa Δ murI Δ dat . ( d ) Proposed schematic mechanism of cell wall degeneration and bacterial lysis of (1) gram-negative and (2) gram-positive bacteria auxotrophic for D-Glu.

    Techniques Used: Lysis, Transmission Electron Microscopy

    Vaccination with D-Glu auxotrophic strains triggers cytokine-secreting T-cells. ( a ) Number of spot-forming cells per 4 × 10 5 splenocytes collected at day 82 from mice vaccinated twice with A. baumannii ATCC 17978 Δ murI1 Δ murI2 (α-Ab vaccine) (6 × 10 7 CFU) ( n =6) and control mice ( n =7) after being restimulated ex vivo with α-Ab vaccine (4 × 10 5 CFU). ( b ) Number of spot-forming cells per 8 × 10 5 splenocytes collected at day 68 from mice vaccinated twice with P. aeruginosa PAO1 Δ murI (α-Pa strain) (2 × 10 7 CFU) ( n =6) and control mice ( n =6) after being restimulated ex vivo with α-Pa vaccine (8 × 10 4 CFU). ( c ) Number of spot-forming cells per 8 × 10 5 splenocytes collected at day 50 from mice vaccinated twice with S. aureus 132 Δ murI Δ dat (α-Sa vaccine) (3 × 10 7 CFU) ( n =6) and control mice ( n =6) after being restimulated ex vivo with α-Sa vaccine (3 × 10 6 CFU). ( a – c ) S, saline; V, vaccinated. * P
    Figure Legend Snippet: Vaccination with D-Glu auxotrophic strains triggers cytokine-secreting T-cells. ( a ) Number of spot-forming cells per 4 × 10 5 splenocytes collected at day 82 from mice vaccinated twice with A. baumannii ATCC 17978 Δ murI1 Δ murI2 (α-Ab vaccine) (6 × 10 7 CFU) ( n =6) and control mice ( n =7) after being restimulated ex vivo with α-Ab vaccine (4 × 10 5 CFU). ( b ) Number of spot-forming cells per 8 × 10 5 splenocytes collected at day 68 from mice vaccinated twice with P. aeruginosa PAO1 Δ murI (α-Pa strain) (2 × 10 7 CFU) ( n =6) and control mice ( n =6) after being restimulated ex vivo with α-Pa vaccine (8 × 10 4 CFU). ( c ) Number of spot-forming cells per 8 × 10 5 splenocytes collected at day 50 from mice vaccinated twice with S. aureus 132 Δ murI Δ dat (α-Sa vaccine) (3 × 10 7 CFU) ( n =6) and control mice ( n =6) after being restimulated ex vivo with α-Sa vaccine (3 × 10 6 CFU). ( a – c ) S, saline; V, vaccinated. * P

    Techniques Used: Mouse Assay, Ex Vivo

    Vaccination with D-Glu auxotrophic strains elicits early and long-term antibody memory against parental and heterologous unrelated strains. ( a ) Antibody titres against A. baumannii ATCC 17978 ( n =5–13), P. aeruginosa PAO1 ( n =4–10) and S. aureus 132 Δ spa ( n =5–10) in vaccinated and control mice after one or two injections with ATCC 17978 Δ murI1 Δ murI2 (α-Ab vaccine) (6 × 10 7 CFU), PAO1 Δ murI (α-Pa vaccine) (2 × 10 7 CFU) and 132 Δ murI Δ dat (α-Sa vaccine) (3 × 10 7 CFU), respectively, or saline. ( b ) Antibody titres against PAO1 in vaccinated and control mice ( n =8) after three intramuscular (IM) injections with α-Pa vaccine (2 × 10 7 CFU), or saline, respectively. ( c ) Antibody titres against ATCC 17978 in vaccinated and control mice ( n =5–8) after α-Ab vaccine (4 × 10 3 CFU), or saline administration, respectively. ( d ) IgG titres against different A. baumannii ( n =6–10), P. aeruginosa ( n =5–10) and S. aureus ( n =7–10) strains in vaccinated and control mice after two injections with α-Ab (6 × 10 7 CFU), α-Pa (2 × 10 7 CFU) and α-Sa (3 × 10 7 CFU) vaccines, respectively, or saline. ( a – d ) S, saline; D, day. * P
    Figure Legend Snippet: Vaccination with D-Glu auxotrophic strains elicits early and long-term antibody memory against parental and heterologous unrelated strains. ( a ) Antibody titres against A. baumannii ATCC 17978 ( n =5–13), P. aeruginosa PAO1 ( n =4–10) and S. aureus 132 Δ spa ( n =5–10) in vaccinated and control mice after one or two injections with ATCC 17978 Δ murI1 Δ murI2 (α-Ab vaccine) (6 × 10 7 CFU), PAO1 Δ murI (α-Pa vaccine) (2 × 10 7 CFU) and 132 Δ murI Δ dat (α-Sa vaccine) (3 × 10 7 CFU), respectively, or saline. ( b ) Antibody titres against PAO1 in vaccinated and control mice ( n =8) after three intramuscular (IM) injections with α-Pa vaccine (2 × 10 7 CFU), or saline, respectively. ( c ) Antibody titres against ATCC 17978 in vaccinated and control mice ( n =5–8) after α-Ab vaccine (4 × 10 3 CFU), or saline administration, respectively. ( d ) IgG titres against different A. baumannii ( n =6–10), P. aeruginosa ( n =5–10) and S. aureus ( n =7–10) strains in vaccinated and control mice after two injections with α-Ab (6 × 10 7 CFU), α-Pa (2 × 10 7 CFU) and α-Sa (3 × 10 7 CFU) vaccines, respectively, or saline. ( a – d ) S, saline; D, day. * P

    Techniques Used: Mouse Assay

    24) Product Images from "β-Sitosterol Reduces the Expression of Chemotactic Cytokine Genes in Cystic Fibrosis Bronchial Epithelial Cells"

    Article Title: β-Sitosterol Reduces the Expression of Chemotactic Cytokine Genes in Cystic Fibrosis Bronchial Epithelial Cells

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00236

    Effect of BSS on IL-8 mRNA, bacterial growth and cell viability in CuFi-1 cells. Cells were treated for 16 h with BSS (100 nM) and infected with PAO1 for further 4 h. IL-8 mRNA expression was measured as indicated in the legend of Figure 2 . (A) Basal IL-8 mRNA expression. Data are expressed as relative to the expression of GAPDH housekeeping gene. (B) PAO1-stimulated mRNA expression. Data are expressed as relative to not infected cells. (C) PAO1 growth. Bacteria were cultured overnight at 37°C in the presence of solvent or ranging doses (0.1– 200 μM) of BSS. Bacterial growth was monitored by absorbance measures at 660 nm. A representative experiment performed in duplicate is shown. (D) Cell viability. CuFi-1 cells were treated with solvent alone or BSS (0.01–200 μM) for 24 and 48 h. Cell viability was recorded by cytometer analysis. Data are expressed as % control (solvent) and are relative to a representative experiment performed in duplicate. Dashed line corresponds to cells treated with solvent alone.
    Figure Legend Snippet: Effect of BSS on IL-8 mRNA, bacterial growth and cell viability in CuFi-1 cells. Cells were treated for 16 h with BSS (100 nM) and infected with PAO1 for further 4 h. IL-8 mRNA expression was measured as indicated in the legend of Figure 2 . (A) Basal IL-8 mRNA expression. Data are expressed as relative to the expression of GAPDH housekeeping gene. (B) PAO1-stimulated mRNA expression. Data are expressed as relative to not infected cells. (C) PAO1 growth. Bacteria were cultured overnight at 37°C in the presence of solvent or ranging doses (0.1– 200 μM) of BSS. Bacterial growth was monitored by absorbance measures at 660 nm. A representative experiment performed in duplicate is shown. (D) Cell viability. CuFi-1 cells were treated with solvent alone or BSS (0.01–200 μM) for 24 and 48 h. Cell viability was recorded by cytometer analysis. Data are expressed as % control (solvent) and are relative to a representative experiment performed in duplicate. Dashed line corresponds to cells treated with solvent alone.

    Techniques Used: Infection, Expressing, Cell Culture, Cytometry

    Effect of BSS on expression of neutrophil chemokines in CuFi-1 cells. (A) CuFi-1 cells were treated for 16 h with solvent alone or 100 nM BSS and then infected by PAO1 (10–50 CFU/cell) for 4 h. mRNA expression was measured as indicated in the legend of Figure 2 . (B–D) Release of the neutrophil chemokines IL-8, Groα, and GROβ in the supernatants of CuFi-1 cells were measured by Bio-plex assay. CuFi-1 cells were treated as described for (A) . Representative of at least three experiments performed in duplicate.
    Figure Legend Snippet: Effect of BSS on expression of neutrophil chemokines in CuFi-1 cells. (A) CuFi-1 cells were treated for 16 h with solvent alone or 100 nM BSS and then infected by PAO1 (10–50 CFU/cell) for 4 h. mRNA expression was measured as indicated in the legend of Figure 2 . (B–D) Release of the neutrophil chemokines IL-8, Groα, and GROβ in the supernatants of CuFi-1 cells were measured by Bio-plex assay. CuFi-1 cells were treated as described for (A) . Representative of at least three experiments performed in duplicate.

    Techniques Used: Expressing, Infection, Plex Assay

    25) Product Images from "Specific expression of heme oxygenase-1 by myeloid cells modulates renal ischemia-reperfusion injury"

    Article Title: Specific expression of heme oxygenase-1 by myeloid cells modulates renal ischemia-reperfusion injury

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00220-w

    Myeloid-restricted deletion of HO-1 in HO-1 M-KO mice. ( A ) qRT-PCR analysis of HO-1 mRNA levels and ( B ) representative images of western blot analysis for HO-1 in BMDMs generated from LT (white bars) and HO-1 M-KO (grey bars) mice in resting and after LPS stimulation (100 ng/ml) for 24 hours. Results are expressed as the mean ± SEM, ★★ p
    Figure Legend Snippet: Myeloid-restricted deletion of HO-1 in HO-1 M-KO mice. ( A ) qRT-PCR analysis of HO-1 mRNA levels and ( B ) representative images of western blot analysis for HO-1 in BMDMs generated from LT (white bars) and HO-1 M-KO (grey bars) mice in resting and after LPS stimulation (100 ng/ml) for 24 hours. Results are expressed as the mean ± SEM, ★★ p

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Western Blot, Generated

    26) Product Images from "Non-viral-mediated suppression of AMIGO3 promotes disinhibited NT3-mediated regeneration of spinal cord dorsal column axons"

    Article Title: Non-viral-mediated suppression of AMIGO3 promotes disinhibited NT3-mediated regeneration of spinal cord dorsal column axons

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29124-z

    NT3 levels were overexpressed in DRGN after injection of in vivo-jet PEI transduced plasmids encoding shAMIGO3. ( A ) Low levels of nt3 mRNA were detected in IC, DC and DC + PEI- gfp groups, whilst significantly higher levels (P
    Figure Legend Snippet: NT3 levels were overexpressed in DRGN after injection of in vivo-jet PEI transduced plasmids encoding shAMIGO3. ( A ) Low levels of nt3 mRNA were detected in IC, DC and DC + PEI- gfp groups, whilst significantly higher levels (P

    Techniques Used: Injection, In Vivo

    GFP + (green) DRGN in DRG in the DC + PEI- gfp group ( A ( i , ii )); high power to show GFP + DRGN, ( iii ); high power GFP + DRGN with DAPI counterstained (blue) nuclei), ( B ) the proportion of GFP + small (0–29 μm), medium (30–59 μm) and large ( > 60 μm) diameter DRGN (green bar); % total GFP + + GFP − small, medium and large DRGN (black bar). ( C ) GFP + DRGN in the DC + PEI-shAMIGO3 /gfp group ( C ( i ),( ii )), high power to show GFP + DRGN, ( iii ); high power GFP + DRGN with DAPI counterstained (blue) nuclei), ( D ) the proportion of GFP + small (0–29 μm), medium (30–59 μm) and large ( > 60 μm) diameter DRGN (green bar); % total GFP + /GFP − small, medium and large diameter DRGN (black bar). ( E ) GFP + DRGN in the DC + PEI-nt3 /gfp group ( E ( i ),( ii )), high power to show GFP + DRGN, ( iii ); high power GFP + DRGN with DAPI counterstained (blue) nuclei), ( F ) the proportion of GFP + small (0–29 μm), medium (30–59 μm) and large ( > 60 μm) diameter DRGN (green bar); % total GFP + /GFP − small, medium and large diameter DRGN (black bar). Scale bar in ( A ( i )), ( C ( i )) and ( E ( i )) = 400 μm, in ( A ( ii )), (A( iii )), ( C ( ii )) and ( C ( iii )), ( E ( ii )) and E(iii) = 100 μm.
    Figure Legend Snippet: GFP + (green) DRGN in DRG in the DC + PEI- gfp group ( A ( i , ii )); high power to show GFP + DRGN, ( iii ); high power GFP + DRGN with DAPI counterstained (blue) nuclei), ( B ) the proportion of GFP + small (0–29 μm), medium (30–59 μm) and large ( > 60 μm) diameter DRGN (green bar); % total GFP + + GFP − small, medium and large DRGN (black bar). ( C ) GFP + DRGN in the DC + PEI-shAMIGO3 /gfp group ( C ( i ),( ii )), high power to show GFP + DRGN, ( iii ); high power GFP + DRGN with DAPI counterstained (blue) nuclei), ( D ) the proportion of GFP + small (0–29 μm), medium (30–59 μm) and large ( > 60 μm) diameter DRGN (green bar); % total GFP + /GFP − small, medium and large diameter DRGN (black bar). ( E ) GFP + DRGN in the DC + PEI-nt3 /gfp group ( E ( i ),( ii )), high power to show GFP + DRGN, ( iii ); high power GFP + DRGN with DAPI counterstained (blue) nuclei), ( F ) the proportion of GFP + small (0–29 μm), medium (30–59 μm) and large ( > 60 μm) diameter DRGN (green bar); % total GFP + /GFP − small, medium and large diameter DRGN (black bar). Scale bar in ( A ( i )), ( C ( i )) and ( E ( i )) = 400 μm, in ( A ( ii )), (A( iii )), ( C ( ii )) and ( C ( iii )), ( E ( ii )) and E(iii) = 100 μm.

    Techniques Used:

    Suppression of AMIGO3 in DRGN preserved the spinal compound action potentials (CAP) across the lesion site. ( A ) Superimposed CAP traces from representative Sham controls, DC + PEI-shAMIGO3/ gfp DC + PEI- nt3 / gfp and DC + shAMIGO3/ nt3 groups. ( B ) Compared to Sham controls, negative CAP amplitudes (mV) were highly attenuated in DC + PEI-shAMIGO3/ gfp and DC + PEI- nt3 / gfp groups but was significantly improved in DC + PEI-shAMIGO3/ nt3 groups (P
    Figure Legend Snippet: Suppression of AMIGO3 in DRGN preserved the spinal compound action potentials (CAP) across the lesion site. ( A ) Superimposed CAP traces from representative Sham controls, DC + PEI-shAMIGO3/ gfp DC + PEI- nt3 / gfp and DC + shAMIGO3/ nt3 groups. ( B ) Compared to Sham controls, negative CAP amplitudes (mV) were highly attenuated in DC + PEI-shAMIGO3/ gfp and DC + PEI- nt3 / gfp groups but was significantly improved in DC + PEI-shAMIGO3/ nt3 groups (P

    Techniques Used:

    Suppression of AMIGO3 in DRGN promoted axon regeneration in the cord rostral to the lesion. There were no GAP43 + regenerating axons in the DC of the ( A ) PEI + shAMIGO3/ gfp and ( B ) DC + PEI- nt3 / gfp groups with a large cavity (*) present at the lesion site. ( C ) Knockdown of AMIGO3 and co-incident up-regulation of NT3 in the DC + shAMIGO3/ nt3 group promoted GAP43 + DRGN axon (arrowheads) regeneration through the DC lesion site. (( D ), high power view of axons coursing rostrally from the lesion site (arrowheads)), with the absence of a cavity in the lesion site. Scale bar in A–C = 500 μm; in D = 50 μm. ( E ) GAP43 + axon fiber counts at specific distances rostral and caudal to the lesion site showed a significant proportion of axons present at 2, 4 and 6 mm rostral to the lesion site. **P
    Figure Legend Snippet: Suppression of AMIGO3 in DRGN promoted axon regeneration in the cord rostral to the lesion. There were no GAP43 + regenerating axons in the DC of the ( A ) PEI + shAMIGO3/ gfp and ( B ) DC + PEI- nt3 / gfp groups with a large cavity (*) present at the lesion site. ( C ) Knockdown of AMIGO3 and co-incident up-regulation of NT3 in the DC + shAMIGO3/ nt3 group promoted GAP43 + DRGN axon (arrowheads) regeneration through the DC lesion site. (( D ), high power view of axons coursing rostrally from the lesion site (arrowheads)), with the absence of a cavity in the lesion site. Scale bar in A–C = 500 μm; in D = 50 μm. ( E ) GAP43 + axon fiber counts at specific distances rostral and caudal to the lesion site showed a significant proportion of axons present at 2, 4 and 6 mm rostral to the lesion site. **P

    Techniques Used:

    Knockdown of AMIGO3 and NT3 over-expression by PEI-delivered plasmid DNA disinhibited DRGN neurite outgrowth. ( A ) Increasing concentrations of plasmid DNA encoding shAMIGO3/ nt3 efficiently suppressed AMIGO3 mRNA in cultured DRGN. ( B ) Plasmids encoding nt3 significantly increased the titres of NT3 in DRGN culture media. ( C ) Representative images show that in the presence of CME, plasmid DNA encoding gfp , nt3 or shAMIGO3/ gfp did not, but that plasmids encoding shAMIGO3 and nt3 did promote DRGN neurite outgrowth. Note: DRGN do not have neurites due to the presence of inhibitory concentrations of CME, which does not affect their survival. ( D ) Quantification of the mean DRGN neurite length and ( E ) the proportion of DRGN with neurites showed that AMIGO3 suppression combined with nt3 overexpression promoted significant disinhibited DRGN neurite outgrowth. Scale bars in C = 50 μm. ***P
    Figure Legend Snippet: Knockdown of AMIGO3 and NT3 over-expression by PEI-delivered plasmid DNA disinhibited DRGN neurite outgrowth. ( A ) Increasing concentrations of plasmid DNA encoding shAMIGO3/ nt3 efficiently suppressed AMIGO3 mRNA in cultured DRGN. ( B ) Plasmids encoding nt3 significantly increased the titres of NT3 in DRGN culture media. ( C ) Representative images show that in the presence of CME, plasmid DNA encoding gfp , nt3 or shAMIGO3/ gfp did not, but that plasmids encoding shAMIGO3 and nt3 did promote DRGN neurite outgrowth. Note: DRGN do not have neurites due to the presence of inhibitory concentrations of CME, which does not affect their survival. ( D ) Quantification of the mean DRGN neurite length and ( E ) the proportion of DRGN with neurites showed that AMIGO3 suppression combined with nt3 overexpression promoted significant disinhibited DRGN neurite outgrowth. Scale bars in C = 50 μm. ***P

    Techniques Used: Over Expression, Plasmid Preparation, Cell Culture

    Response to PEI-delivered plasmids. ( A ) CD68 and GFAP immunoreactivity in intact and after PEI-delivered plasmids in section of DRG to show a lack of induction of immunological responses to these markers. In contrast, T7-sip75 NTR invoked significant changes in CD68 and GFAP + immunoreactivity in DRG. ( B ) Lack of activation of off-target cytokines and innate immunity related genes in DRG after PEI-delivered plasmid transduction in DRG. Once again, T7-sip75 NTR showed significant upregulation of mRNA for all of these genes. ( C ) ED1 immunoreactivity was low in DC + PEI-nt3/ gfp -treated animals whilst abundant ED1 + cells were present within the lesion site (*) in DC + PEI-shAMIGO3/ nt3 -treated animals. ( D ) GFAP + astrocytes were restricted to the lesion edge in DC + PEI-nt3/ gfp -treated animals whilst GFAP + astrocytes invaded the lesion site in DC + PEI-shAMIGO3/ nt3 -treated animals. Scale bars in A = 100 µm; C and D = 500 µm.
    Figure Legend Snippet: Response to PEI-delivered plasmids. ( A ) CD68 and GFAP immunoreactivity in intact and after PEI-delivered plasmids in section of DRG to show a lack of induction of immunological responses to these markers. In contrast, T7-sip75 NTR invoked significant changes in CD68 and GFAP + immunoreactivity in DRG. ( B ) Lack of activation of off-target cytokines and innate immunity related genes in DRG after PEI-delivered plasmid transduction in DRG. Once again, T7-sip75 NTR showed significant upregulation of mRNA for all of these genes. ( C ) ED1 immunoreactivity was low in DC + PEI-nt3/ gfp -treated animals whilst abundant ED1 + cells were present within the lesion site (*) in DC + PEI-shAMIGO3/ nt3 -treated animals. ( D ) GFAP + astrocytes were restricted to the lesion edge in DC + PEI-nt3/ gfp -treated animals whilst GFAP + astrocytes invaded the lesion site in DC + PEI-shAMIGO3/ nt3 -treated animals. Scale bars in A = 100 µm; C and D = 500 µm.

    Techniques Used: Activation Assay, Plasmid Preparation, Transduction

    27) Product Images from "Non-viral-mediated suppression of AMIGO3 promotes disinhibited NT3-mediated regeneration of spinal cord dorsal column axons"

    Article Title: Non-viral-mediated suppression of AMIGO3 promotes disinhibited NT3-mediated regeneration of spinal cord dorsal column axons

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29124-z

    Knockdown of AMIGO3 and NT3 over-expression by PEI-delivered plasmid DNA disinhibited DRGN neurite outgrowth. ( A ) Increasing concentrations of plasmid DNA encoding shAMIGO3/ nt3 efficiently suppressed AMIGO3 mRNA in cultured DRGN. ( B ) Plasmids encoding nt3 significantly increased the titres of NT3 in DRGN culture media. ( C ) Representative images show that in the presence of CME, plasmid DNA encoding gfp , nt3 or shAMIGO3/ gfp did not, but that plasmids encoding shAMIGO3 and nt3 did promote DRGN neurite outgrowth. Note: DRGN do not have neurites due to the presence of inhibitory concentrations of CME, which does not affect their survival. ( D ) Quantification of the mean DRGN neurite length and ( E ) the proportion of DRGN with neurites showed that AMIGO3 suppression combined with nt3 overexpression promoted significant disinhibited DRGN neurite outgrowth. Scale bars in C = 50 μm. ***P
    Figure Legend Snippet: Knockdown of AMIGO3 and NT3 over-expression by PEI-delivered plasmid DNA disinhibited DRGN neurite outgrowth. ( A ) Increasing concentrations of plasmid DNA encoding shAMIGO3/ nt3 efficiently suppressed AMIGO3 mRNA in cultured DRGN. ( B ) Plasmids encoding nt3 significantly increased the titres of NT3 in DRGN culture media. ( C ) Representative images show that in the presence of CME, plasmid DNA encoding gfp , nt3 or shAMIGO3/ gfp did not, but that plasmids encoding shAMIGO3 and nt3 did promote DRGN neurite outgrowth. Note: DRGN do not have neurites due to the presence of inhibitory concentrations of CME, which does not affect their survival. ( D ) Quantification of the mean DRGN neurite length and ( E ) the proportion of DRGN with neurites showed that AMIGO3 suppression combined with nt3 overexpression promoted significant disinhibited DRGN neurite outgrowth. Scale bars in C = 50 μm. ***P

    Techniques Used: Over Expression, Plasmid Preparation, Cell Culture

    28) Product Images from "Secondary amplification of siRNA machinery limits the application of spray‐induced gene silencing"

    Article Title: Secondary amplification of siRNA machinery limits the application of spray‐induced gene silencing

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12728

    Conidiation, sexual reproduction, virulence and Myo5 gene expression of wild‐type (WT) and Myo5RNAi transformants. (A) Numbers of conidia produced in carboxymethylcellulose (CMC) medium after 5 days. Experiments were performed in triplicate.
    Figure Legend Snippet: Conidiation, sexual reproduction, virulence and Myo5 gene expression of wild‐type (WT) and Myo5RNAi transformants. (A) Numbers of conidia produced in carboxymethylcellulose (CMC) medium after 5 days. Experiments were performed in triplicate.

    Techniques Used: Expressing, Produced

    Sensitivity of wild‐type (WT) and Myo5RNAi [ Myo5 gene RNA interference (RNAi) transformants] to osmotic and cell wall stress. (A) Growth of different strains on potato dextrose agar (PDA) plates or PDA amended with various compounds that generate
    Figure Legend Snippet: Sensitivity of wild‐type (WT) and Myo5RNAi [ Myo5 gene RNA interference (RNAi) transformants] to osmotic and cell wall stress. (A) Growth of different strains on potato dextrose agar (PDA) plates or PDA amended with various compounds that generate

    Techniques Used:

    29) Product Images from "Leukemia Inhibitory Factor-Receptor is Dispensable for Prenatal Testis Development but is Required in Sertoli cells for Normal Spermatogenesis in Mice"

    Article Title: Leukemia Inhibitory Factor-Receptor is Dispensable for Prenatal Testis Development but is Required in Sertoli cells for Normal Spermatogenesis in Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30011-w

    Reduced Sertoli cell and spermatogonia volume in SC-KO testes. (A) qRT-PCR analysis of spermatogonia ( Stra8 ), spermatocyte ( Spo11 ) and spermatid ( Tpn1 ) specific mRNAs. Expression levels were similar between WT and SC-KO testes at d180 (unpaired t- test, n = 9–10). (B) Stereological analysis revealed a reduction in the absolute nuclear volume of Sertoli cells and spermatogonia in the SC-KO testis at d180 (unpaired t- test; p = 0.0172 and 0.0325 respectively; n = 7). Spermatogonia/Sertoli cell ratio is maintained in the SC-KO testis (unpaired t- test, n = 7). (C) Representative immunostaining for activated CASP3 (brown), as a marker of apoptosis, in WT and SC-KO testes at d180. No difference in the number of CASP3-positive tubules, or CASP3-positive cells lining the basal region of the tubules (black arrows, higher magnification insets) was observed between WT and SC-KO testes (unpaired t- test; n = 5–6). CASP3 immunoreactivity was occasionally observed throughout the seminiferous epithelium (open arrowheads) in SC-KO animals. Primary antibody negative control and experimentally-induced Sertoli cell death positive controls are included. Scale bars = 100 µm. (D) mRNA expression of Gdnf, Cyp26b1 and Kitl was similar between WT and SC-KO testes (unpaired t -tests, n = 9–10). All values are expressed as the mean ± S.E.M.
    Figure Legend Snippet: Reduced Sertoli cell and spermatogonia volume in SC-KO testes. (A) qRT-PCR analysis of spermatogonia ( Stra8 ), spermatocyte ( Spo11 ) and spermatid ( Tpn1 ) specific mRNAs. Expression levels were similar between WT and SC-KO testes at d180 (unpaired t- test, n = 9–10). (B) Stereological analysis revealed a reduction in the absolute nuclear volume of Sertoli cells and spermatogonia in the SC-KO testis at d180 (unpaired t- test; p = 0.0172 and 0.0325 respectively; n = 7). Spermatogonia/Sertoli cell ratio is maintained in the SC-KO testis (unpaired t- test, n = 7). (C) Representative immunostaining for activated CASP3 (brown), as a marker of apoptosis, in WT and SC-KO testes at d180. No difference in the number of CASP3-positive tubules, or CASP3-positive cells lining the basal region of the tubules (black arrows, higher magnification insets) was observed between WT and SC-KO testes (unpaired t- test; n = 5–6). CASP3 immunoreactivity was occasionally observed throughout the seminiferous epithelium (open arrowheads) in SC-KO animals. Primary antibody negative control and experimentally-induced Sertoli cell death positive controls are included. Scale bars = 100 µm. (D) mRNA expression of Gdnf, Cyp26b1 and Kitl was similar between WT and SC-KO testes (unpaired t -tests, n = 9–10). All values are expressed as the mean ± S.E.M.

    Techniques Used: Quantitative RT-PCR, Expressing, Immunostaining, Marker, Negative Control

    30) Product Images from "MicroRNAs as Predictor Markers for Response to Interferon Treatment of Chronic Hepatitis C Genotype-4 in Egyptian Patients"

    Article Title: MicroRNAs as Predictor Markers for Response to Interferon Treatment of Chronic Hepatitis C Genotype-4 in Egyptian Patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121524

    Correlation between log HCV PCR and miR-21 (a), miR-122 (b), and miR-221 (c) in patients with HCV-4. Points represent 2 −ΔΔt values for miRNAs normalised to normal controls. Difference was considered significant at P
    Figure Legend Snippet: Correlation between log HCV PCR and miR-21 (a), miR-122 (b), and miR-221 (c) in patients with HCV-4. Points represent 2 −ΔΔt values for miRNAs normalised to normal controls. Difference was considered significant at P

    Techniques Used: Polymerase Chain Reaction

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077306

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

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

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

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

    32) Product Images from "Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone"

    Article Title: Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/527561

    Effects of WIN-34B on chondrogenic differentiation of IL-1 β -stimulated progenitor cells from rabbit subchondral bone. (a) Histological analysis of WIN-34B by alcian blue staining of chondrogenic differentiation in IL-1 β -treated progenitor cells. Control, IL-1 β , WIN-34B 1 μ g/mL, WIN-34B 10 μ g/mL, and WIN-34B 20 μ g/mL after 7 days of culture in chondrogenic differentiation media. Magnified view (×100). (b) Dose response of WIN-34B on the mRNA expression of chondrogenic markers. Chondrogenic differentiation of subchondral progenitor cells that were incubated for seven days with 1, 10, and 20 μ g/mL of WIN-34B in the presence of IL-1 β . qRT-PCR was then performed for type II α 1 collagen, cartilage link protein, and aggrecan. (c) Inhibitory effects of WIN-34B on GAG and type II collagen degradation on chondrogenic differentiation of IL-1 β -stimulated subchondral progenitor cells. GAG and type II collagen degradation are shown as a cumulative release into the culture medium. Values are the mean ± SEM. ### P
    Figure Legend Snippet: Effects of WIN-34B on chondrogenic differentiation of IL-1 β -stimulated progenitor cells from rabbit subchondral bone. (a) Histological analysis of WIN-34B by alcian blue staining of chondrogenic differentiation in IL-1 β -treated progenitor cells. Control, IL-1 β , WIN-34B 1 μ g/mL, WIN-34B 10 μ g/mL, and WIN-34B 20 μ g/mL after 7 days of culture in chondrogenic differentiation media. Magnified view (×100). (b) Dose response of WIN-34B on the mRNA expression of chondrogenic markers. Chondrogenic differentiation of subchondral progenitor cells that were incubated for seven days with 1, 10, and 20 μ g/mL of WIN-34B in the presence of IL-1 β . qRT-PCR was then performed for type II α 1 collagen, cartilage link protein, and aggrecan. (c) Inhibitory effects of WIN-34B on GAG and type II collagen degradation on chondrogenic differentiation of IL-1 β -stimulated subchondral progenitor cells. GAG and type II collagen degradation are shown as a cumulative release into the culture medium. Values are the mean ± SEM. ### P

    Techniques Used: Staining, Expressing, Incubation, Quantitative RT-PCR

    33) Product Images from "E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif"

    Article Title: E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif

    Journal: Biology Open

    doi: 10.1242/bio.010405

    Ufd2 mediates Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). (B) Yap8 is destabilized in the ufd2 mutant. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2 U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2 U-boxΔ mutant strains carrying the GAL1 promoter MPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti- c -myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2 . Exponential phase BY4742 WT, yap8 , ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P
    Figure Legend Snippet: Ufd2 mediates Yap8 stabilization. (A) Yap8 levels are reduced in ufd2 mutant cells compared to the wild type strain. BY4742 wild type (WT) and ufd2 mutant strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). (B) Yap8 is destabilized in the ufd2 mutant. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting with the antibodies indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 98 min in the WT strain and 37 min in the ufd2 mutant. (C) Mps1 stability is increased in ufd2 and Ufd2 U-boxΔ mutant cells in comparison to WT strain. BY4741 WT, ufd2 and Ufd2 U-boxΔ mutant strains carrying the GAL1 promoter MPS1-c-myc construct were induced with galactose before being challenged with glucose and 0.1 mg/ml CHX. Cells were harvested at the indicated time-points and subjected to immunoblotting using anti- c -myc and anti-Pgk1 antibodies. The graph represents the percentage of remaining Mps1 protein after CHX addition. A representative experiment is shown. (D) Epistasis analyses of YAP8 and UFD2 . Exponential phase BY4742 WT, yap8 , ufd2 and yap8ufd2 cells were serially diluted and spotted onto MM media supplemented or not with increasing concentrations of As(V) (up to 2 mM; upper panel) or As(III) (up to 1.5 mM; lower panel). Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. (E) ACR3 expression is similar in the double yap8ufd2 and single yap8 mutants. The same strains referred in D were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P

    Techniques Used: Mutagenesis, Expressing, Incubation, Construct, Quantitative RT-PCR

    Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4 , rad23 and dsk2 mutant cells. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates. No significant statistical differences were observed.
    Figure Legend Snippet: Ubiquitin proteasome pathway (UPP) enzymes Ubc4, Rad23 and Dsk2 do not interfere with Yap8 stability in arsenic-exposed cells. BY4742 wild type (WT), ubc4 (A), rad23 (B) and dsk2 (C) mutant strains expressing Yap8-HA were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 120 min prior to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graphs represent the percentage of remaining Yap8 protein after CHX addition. Representative experiments are shown. (D) ACR3 mRNA levels remain unaltered in ubc4 , rad23 and dsk2 mutant cells. The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates. No significant statistical differences were observed.

    Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR

    Ufd2 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2 U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2 U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2 U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2 U-boxΔ . (C) ACR3 mRNA levels are unaltered in the Ufd2 U-boxΔ . The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P
    Figure Legend Snippet: Ufd2 U-box motif is not required for Yap8 stabilization. (A) Yap8 levels are unaffected in the Ufd2 U-boxΔ mutant strain compared to the wild type strain. BY4741 wild type (WT), ufd2 and Ufd2 U-boxΔ strains expressing Yap8-HA were incubated with 1.5 mM As(III), harvested at the indicated time-points and subjected to immunoblotting using anti-HA and anti-Pgk1 antibodies. The graph represents relative Yap8 levels (AU, Arbitrary Units). A representative experiment is shown; SD, control. (B) Yap8 stability is similar in WT and Ufd2 U-boxΔ mutant strains. The same strains were first exposed to 1.5 mM As(III) for 90 min, washed and subsequently treated with 0.1 mg/ml cycloheximide (CHX) up to 90 min prior to immunoblotting, as indicated above. The graph represents the percentage of remaining Yap8 protein after CHX addition. Estimated Yap8 half-life is 63 min in the WT strain, 25 min in ufd2 and 67 min in Ufd2 U-boxΔ . (C) ACR3 mRNA levels are unaltered in the Ufd2 U-boxΔ . The same strains were challenged with 1.5 mM As(III) for 90 min and ACR3 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as * P

    Techniques Used: Mutagenesis, Expressing, Incubation, Quantitative RT-PCR

    Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD 600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as *** P
    Figure Legend Snippet: Ufd2 mediates arsenic tolerance. (A) ufd2 cells are sensitive to arsenic stress. Exponential phase BY4742 wild type (WT) and the ufd2 mutant were serially diluted and spotted onto SC media supplemented or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III). SD, control. Growth was recorded after 2 days incubation at 30°C. A representative experiment is shown. Cell growth was also monitored by means of growth curves. Exponential phase BY4742 WT and ufd2 mutant cells were exposed or not to 2 mM As(V) or 1.5 mM As(III) for 22 h and OD 600 was monitored in intervals of 1 h. The curves represent the mean±s.d. of three biological replicates. (B) UFD2 is induced in cells injured with arsenic. BY4742 cells were challenged or not with 1.5 mM As(III) or 2 mM As(V) or 1.5 mM As(III) and UFD2 mRNA levels were determined by qRT-PCR (AU, Arbitrary Units). Values represent the mean±s.d. of three biological replicates and statistical differences denoted as *** P

    Techniques Used: Mutagenesis, Incubation, Quantitative RT-PCR

    34) Product Images from "MicroRNA-27a alleviates LPS-induced acute lung injury in mice via inhibiting inflammation and apoptosis through modulating TLR4/MyD88/NF-κB pathway"

    Article Title: MicroRNA-27a alleviates LPS-induced acute lung injury in mice via inhibiting inflammation and apoptosis through modulating TLR4/MyD88/NF-κB pathway

    Journal: Cell Cycle

    doi: 10.1080/15384101.2018.1509635

    Increased miR-27a ameliorated LPS induced ALI in mice. Groups of mice were given agomir-27a or agomir NC (2 mg/kg) by tail intravenous injection 24 h prior to 1 mg/kg LPS treatment. The mice were sacrificed after LPS administration for 24 h and then lung tissues were collected for analysis. (a) The miR-27a level in lung tissue samples were measured by qRT-PCR (n = 3/group). (b) Lung tissues from each experimental group were processed for histological evaluation (n = 3/group). (c, d) The Evans blue content and lung wet/dry assay (n = 3/group). (e) Oxygenation index (PaO 2 /FIO 2 ) were determined (n = 3/group). Data represent the mean ± SD of three independent experiments. *p
    Figure Legend Snippet: Increased miR-27a ameliorated LPS induced ALI in mice. Groups of mice were given agomir-27a or agomir NC (2 mg/kg) by tail intravenous injection 24 h prior to 1 mg/kg LPS treatment. The mice were sacrificed after LPS administration for 24 h and then lung tissues were collected for analysis. (a) The miR-27a level in lung tissue samples were measured by qRT-PCR (n = 3/group). (b) Lung tissues from each experimental group were processed for histological evaluation (n = 3/group). (c, d) The Evans blue content and lung wet/dry assay (n = 3/group). (e) Oxygenation index (PaO 2 /FIO 2 ) were determined (n = 3/group). Data represent the mean ± SD of three independent experiments. *p

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

    35) Product Images from "Invading Basement Membrane Matrix Is Sufficient for MDA-MB-231 Breast Cancer Cells to Develop a Stable In Vivo Metastatic Phenotype"

    Article Title: Invading Basement Membrane Matrix Is Sufficient for MDA-MB-231 Breast Cancer Cells to Develop a Stable In Vivo Metastatic Phenotype

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023334

    INV and REF breast cancer cell invasion and migration. Cells (2.5×10 5 ) were added to the upper side of each 8 µm insert of Boyden chambers coated (A) or not (B) with Matrigel for invasion or migration assays respectively; cells were then counted as described in Materials and Methods . MMP-9 activity secreted by REF (lane 1) and INV (lane 2) cells (C) was assessed by subjecting aliquots of lyophilized conditioned media normalized to the number of cells to 10% SDS-polyacrylamide gels containing 1 mg/mL gelatin. For QRT-PCR analysis (D), total RNA (1 µg) was reverse-transcribed using MMLV RT and subjected to qRT-PCR as described in Material and Methods . Representative data was normalised to PPIA is given. Each column represents a mean (± SD) of three independent experiments. * P
    Figure Legend Snippet: INV and REF breast cancer cell invasion and migration. Cells (2.5×10 5 ) were added to the upper side of each 8 µm insert of Boyden chambers coated (A) or not (B) with Matrigel for invasion or migration assays respectively; cells were then counted as described in Materials and Methods . MMP-9 activity secreted by REF (lane 1) and INV (lane 2) cells (C) was assessed by subjecting aliquots of lyophilized conditioned media normalized to the number of cells to 10% SDS-polyacrylamide gels containing 1 mg/mL gelatin. For QRT-PCR analysis (D), total RNA (1 µg) was reverse-transcribed using MMLV RT and subjected to qRT-PCR as described in Material and Methods . Representative data was normalised to PPIA is given. Each column represents a mean (± SD) of three independent experiments. * P

    Techniques Used: Migration, Activity Assay, Quantitative RT-PCR

    36) Product Images from "Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer, et al. Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer"

    Article Title: Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer, et al. Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14200

    SLC25A5‐AS1 modulated the miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway. (A, B) qRT‐PCR were used to detected the relative expression of potential mRNA targets, included KBTBD8, ZMYND11, CHIC1, MXD1, SOSC1 and PTEN, of miR‐19a‐3p in BGC‐823 and SGC‐7901 cells transfected with or without pcDNA‐SLC25A5‐AS1. C, MRNA expression level of PTEN in GC and normal tissues were detected by qRT‐PCR. D, The correlation analysis was performed between the expression level of SLC25A5‐AS1 and PTEN in GC tissues by qRT‐PCR. E, The correlation analysis between the expression level of miR‐19a‐3p and PTEN in GC tissues was detected by qRT‐PCR. F, After transfected for 72 h, cells were collected and total proteins were extracted for Western blot. The protein levels of PTEN in BGC‐823 and SGC‐7901 cells transfected with or without pcDNA‐SLC25A5‐AS1 were analysed by Western blot. G, Rescue experiments of Western blot analyses were used to analyse the protein levels of p27, cyclinD1, BCL‐2, BAX, PTEN, p‐PI3K, PI3K, p‐AKT and AKT in cells of each group. * P
    Figure Legend Snippet: SLC25A5‐AS1 modulated the miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway. (A, B) qRT‐PCR were used to detected the relative expression of potential mRNA targets, included KBTBD8, ZMYND11, CHIC1, MXD1, SOSC1 and PTEN, of miR‐19a‐3p in BGC‐823 and SGC‐7901 cells transfected with or without pcDNA‐SLC25A5‐AS1. C, MRNA expression level of PTEN in GC and normal tissues were detected by qRT‐PCR. D, The correlation analysis was performed between the expression level of SLC25A5‐AS1 and PTEN in GC tissues by qRT‐PCR. E, The correlation analysis between the expression level of miR‐19a‐3p and PTEN in GC tissues was detected by qRT‐PCR. F, After transfected for 72 h, cells were collected and total proteins were extracted for Western blot. The protein levels of PTEN in BGC‐823 and SGC‐7901 cells transfected with or without pcDNA‐SLC25A5‐AS1 were analysed by Western blot. G, Rescue experiments of Western blot analyses were used to analyse the protein levels of p27, cyclinD1, BCL‐2, BAX, PTEN, p‐PI3K, PI3K, p‐AKT and AKT in cells of each group. * P

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

    Expression of SLC25A5‐AS1 in GC cell lines and subcellar location. A, qRT‐PCR analyses of SLC25A5‐AS1 expression in GC cell lines (HGC‐27, BGC‐823, SGC‐7901 and AGS) and normal gastric mucosa epithelial cells (GES1). B, Relative nuclear and cytoplasmic levels of SLC25A5‐AS1 in GC cells assessed by qRT‐PCR with U6 and GAPDH as nuclear and cytoplasmic markers respectively. (C and D) Relative SLC25A5‐AS1 expression level in shRNA‐treated HGC‐27 cells, and pcDNA‐SLC25A5‐AS1‐treated BGC‐823 and SGC‐7901 cells. * P
    Figure Legend Snippet: Expression of SLC25A5‐AS1 in GC cell lines and subcellar location. A, qRT‐PCR analyses of SLC25A5‐AS1 expression in GC cell lines (HGC‐27, BGC‐823, SGC‐7901 and AGS) and normal gastric mucosa epithelial cells (GES1). B, Relative nuclear and cytoplasmic levels of SLC25A5‐AS1 in GC cells assessed by qRT‐PCR with U6 and GAPDH as nuclear and cytoplasmic markers respectively. (C and D) Relative SLC25A5‐AS1 expression level in shRNA‐treated HGC‐27 cells, and pcDNA‐SLC25A5‐AS1‐treated BGC‐823 and SGC‐7901 cells. * P

    Techniques Used: Expressing, Quantitative RT-PCR, shRNA

    SLC25A5‐AS1 directly interacted with miR‐19a‐3p. A, Bioinformatics software predicted miR‐19a‐3p has one binding site on the SLC25A5‐AS1 transcript. B, qRT‐PCR detected miR‐19a‐3p expression after pcDNA‐SLC25A5‐AS1 transfection in BGC‐823 and SGC‐7901 cells. C, Expression of miR‐19a‐3p after transfected with miR‐19a‐3p inhibitor and mimics in BGC‐823 and SGC‐7901 cells. D, qRT‐PCR analysed SLC25A5‐AS1 expression after miR‐19a‐3p knockdown or up‐regulation in BGC‐823 and SGC‐7901 cells. E, The miR‐19a‐3p binding site predicted in the sequence of SLC25A5‐AS1, red font as mutant locus. F, Relative firefly/renilla luminescence was analysed in BGC‐823 cells co‐transfected with miR‐19a‐3p mimics and wild‐type or mutant SLC25A5‐AS1 sequence constructed luciferase plasmid. (G and H) miR‐19a‐3p expression levels were analysed in GC tissues and GC cell lines by qRT‐PCR. I, Correlation analysis was performed between SLC25A5‐AS1 expression levels and miR‐19a‐3p expression levels in GC tissues, ** P
    Figure Legend Snippet: SLC25A5‐AS1 directly interacted with miR‐19a‐3p. A, Bioinformatics software predicted miR‐19a‐3p has one binding site on the SLC25A5‐AS1 transcript. B, qRT‐PCR detected miR‐19a‐3p expression after pcDNA‐SLC25A5‐AS1 transfection in BGC‐823 and SGC‐7901 cells. C, Expression of miR‐19a‐3p after transfected with miR‐19a‐3p inhibitor and mimics in BGC‐823 and SGC‐7901 cells. D, qRT‐PCR analysed SLC25A5‐AS1 expression after miR‐19a‐3p knockdown or up‐regulation in BGC‐823 and SGC‐7901 cells. E, The miR‐19a‐3p binding site predicted in the sequence of SLC25A5‐AS1, red font as mutant locus. F, Relative firefly/renilla luminescence was analysed in BGC‐823 cells co‐transfected with miR‐19a‐3p mimics and wild‐type or mutant SLC25A5‐AS1 sequence constructed luciferase plasmid. (G and H) miR‐19a‐3p expression levels were analysed in GC tissues and GC cell lines by qRT‐PCR. I, Correlation analysis was performed between SLC25A5‐AS1 expression levels and miR‐19a‐3p expression levels in GC tissues, ** P

    Techniques Used: Software, Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Sequencing, Mutagenesis, Construct, Luciferase, Plasmid Preparation

    37) Product Images from "Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension"

    Article Title: Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension

    Journal: American journal of physiology. Heart and circulatory physiology

    doi: 10.1152/ajpheart.00595.2010

    Effects of short-interfering RNA (siRNA) knockdown on ROCK isoform expression and apoptosis in primary cultured PASMCs. ROCK-I or -II isoform expression ( A ), quantified by quantitative PCR, and apoptosis in PASMCs cultured from days 1 to 14 neonatal rats electroporated with control (scrambled) siRNA (white bars; B ) or siRNAs designed to knockdown ROCK-I (gray bars) or ROCK-II (black bars) are shown. Values represent means ± SE for 4 samples or 6 wells per group. * P
    Figure Legend Snippet: Effects of short-interfering RNA (siRNA) knockdown on ROCK isoform expression and apoptosis in primary cultured PASMCs. ROCK-I or -II isoform expression ( A ), quantified by quantitative PCR, and apoptosis in PASMCs cultured from days 1 to 14 neonatal rats electroporated with control (scrambled) siRNA (white bars; B ) or siRNAs designed to knockdown ROCK-I (gray bars) or ROCK-II (black bars) are shown. Values represent means ± SE for 4 samples or 6 wells per group. * P

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

    38) Product Images from "Does p-coumaric acid improve cardiac injury following LPS-induced lung inflammation through miRNA-146a activity?"

    Article Title: Does p-coumaric acid improve cardiac injury following LPS-induced lung inflammation through miRNA-146a activity?

    Journal: Avicenna Journal of Phytomedicine

    doi:

    PCA pre-treatment effects on cardiac injury following LPS-induced lung inflammation. The relative expression of miR-146a in heart tissue of ALI rat was measured by qRT-PCR. Values are expressed as mean±SEM (n=8). **p
    Figure Legend Snippet: PCA pre-treatment effects on cardiac injury following LPS-induced lung inflammation. The relative expression of miR-146a in heart tissue of ALI rat was measured by qRT-PCR. Values are expressed as mean±SEM (n=8). **p

    Techniques Used: Expressing, Quantitative RT-PCR

    39) Product Images from "Epstein–Barr Virus BALF0 and BALF1 Modulate Autophagy"

    Article Title: Epstein–Barr Virus BALF0 and BALF1 Modulate Autophagy

    Journal: Viruses

    doi: 10.3390/v11121099

    Characterization of BALF0 and BALF1 expression. ( A ) Characterization of rabbit anti-sera against BALF0/1. HeLa cells were transfected with a plasmid encoding for BALF0/1 (pcDNA3.1-BALF0/1-HA) or corresponding negative control (empty vector, EV). BALF0/1 expression was analyzed at 48 h post-transfection (p.t.) by immunoblot (left panel) and immunofluorescence (right panel) using rabbit anti-sera directed against BALF0/1. Scale bar = 20 µm. Two polypeptides whose relative mobility following SDS-PAGE corresponded to the predicted size of BALF0 (26 kDa) and BALF1 (22 kDa) were detected by immunoblot. ( B ) Time-course accumulation of BALF0/1 mRNA in reactivated Akata cells. Akata cells were reactivated by cross-linking of surface immunoglobulin for 2 to 48 h. At the indicated time post-reactivation, mRNA encoding for BALF0/1 was quantified by qRT-PCR. ( C ) BALF0 and BALF1 protein expression in reactivated Akata cells. Total protein was extracted from Akata cells as described in ( B ) and analyzed by immunoblot (left panel) using rabbit anti-sera against BALF0/1. Immediate-early protein ZEBRA was used as a marker for viral reactivation. Relative expression of BALF0 and BALF1 was analyzed using ImageJ (right panel) and compared to β-actin loading control at each time point. ( D ) Schematic diagram of expression vectors encoding for BALF0/1 (pcDNA3.1-BALF0/1-HA), BALF0 (pcDNA3.1-BALF0-HA), and BALF1 (pcDNA3.1-BALF1-HA). BALF0 and BALF1 alone were obtained by replacing the methionine at amino acid 39 and 1 with glycine and isoleucine, respectively. ( E ) Immunofluorescence staining of HA-tagged BALF0/1, BALF0, and BALF1. HeLa cells were analyzed 24 h p.t. by immunofluorescence using an anti-HA antibody. Scale bar = 10 µm. ( F ) Immunoblot analysis of BALF0/1, BALF0, and BALF1. HeLa cells were co-transfected with 0.25 μg of each indicated plasmid for a total plasmid amount of 0.5 μg. Protein expression was analyzed 48 h p.t. by immunoblot using rabbit anti-sera against BALF0/1. ( G , H ) Cross-regulation of BALF0 and BALF1 expression. HeLa cells were transfected with a constant amount (0.25 μg) of BALF0 ( G ) or BALF1 ( H ) encoding plasmid in the presence of increasing amount of BALF1 ( G ) or BALF0 ( H ) encoding plasmid. EV plasmid was added to keep the total amount of transfected plasmids constant. Immunoblot analysis was carried out as described in ( F ). Relative expression of BALF0 and BALF1 was evaluated by densitometric analysis by ImageJ and compared to that of β-actin loading control. Values are mean ± SEM of three independent experiments. One representative set of immunoblotting results is shown. * P
    Figure Legend Snippet: Characterization of BALF0 and BALF1 expression. ( A ) Characterization of rabbit anti-sera against BALF0/1. HeLa cells were transfected with a plasmid encoding for BALF0/1 (pcDNA3.1-BALF0/1-HA) or corresponding negative control (empty vector, EV). BALF0/1 expression was analyzed at 48 h post-transfection (p.t.) by immunoblot (left panel) and immunofluorescence (right panel) using rabbit anti-sera directed against BALF0/1. Scale bar = 20 µm. Two polypeptides whose relative mobility following SDS-PAGE corresponded to the predicted size of BALF0 (26 kDa) and BALF1 (22 kDa) were detected by immunoblot. ( B ) Time-course accumulation of BALF0/1 mRNA in reactivated Akata cells. Akata cells were reactivated by cross-linking of surface immunoglobulin for 2 to 48 h. At the indicated time post-reactivation, mRNA encoding for BALF0/1 was quantified by qRT-PCR. ( C ) BALF0 and BALF1 protein expression in reactivated Akata cells. Total protein was extracted from Akata cells as described in ( B ) and analyzed by immunoblot (left panel) using rabbit anti-sera against BALF0/1. Immediate-early protein ZEBRA was used as a marker for viral reactivation. Relative expression of BALF0 and BALF1 was analyzed using ImageJ (right panel) and compared to β-actin loading control at each time point. ( D ) Schematic diagram of expression vectors encoding for BALF0/1 (pcDNA3.1-BALF0/1-HA), BALF0 (pcDNA3.1-BALF0-HA), and BALF1 (pcDNA3.1-BALF1-HA). BALF0 and BALF1 alone were obtained by replacing the methionine at amino acid 39 and 1 with glycine and isoleucine, respectively. ( E ) Immunofluorescence staining of HA-tagged BALF0/1, BALF0, and BALF1. HeLa cells were analyzed 24 h p.t. by immunofluorescence using an anti-HA antibody. Scale bar = 10 µm. ( F ) Immunoblot analysis of BALF0/1, BALF0, and BALF1. HeLa cells were co-transfected with 0.25 μg of each indicated plasmid for a total plasmid amount of 0.5 μg. Protein expression was analyzed 48 h p.t. by immunoblot using rabbit anti-sera against BALF0/1. ( G , H ) Cross-regulation of BALF0 and BALF1 expression. HeLa cells were transfected with a constant amount (0.25 μg) of BALF0 ( G ) or BALF1 ( H ) encoding plasmid in the presence of increasing amount of BALF1 ( G ) or BALF0 ( H ) encoding plasmid. EV plasmid was added to keep the total amount of transfected plasmids constant. Immunoblot analysis was carried out as described in ( F ). Relative expression of BALF0 and BALF1 was evaluated by densitometric analysis by ImageJ and compared to that of β-actin loading control. Values are mean ± SEM of three independent experiments. One representative set of immunoblotting results is shown. * P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Negative Control, Immunofluorescence, SDS Page, Quantitative RT-PCR, Marker, Staining

    40) Product Images from "Multi-Factor Regulation of the Master Modulator LeuO for the Cyclic-(Phe-Pro) Signaling Pathway in Vibrio vulnificus"

    Article Title: Multi-Factor Regulation of the Master Modulator LeuO for the Cyclic-(Phe-Pro) Signaling Pathway in Vibrio vulnificus

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-56855-4

    ChIP analysis showing that cFP enhances the ToxR binding affinity for the cis -acting elements in the DNA region upstream of leuO and ompU . The toxR null mutant cells expressing strep-tagged ToxR were cross-linked, washed, and sonicated as described in Materials and Methods. Lysates were then treated with either anti-Strep-tag II monoclonal antibody or normal mouse IgG as a control. Free DNA was purified and analyzed by quantitative real-time PCR (qRT-PCR) on a Light Cycler 480 II real-time PCR system. The relative enrichment was calculated as the amount of transcript compared to transcript from cells without cFP. Values are averages from biological experiments done in triplicate. Error bars indicate the standard deviations (**P
    Figure Legend Snippet: ChIP analysis showing that cFP enhances the ToxR binding affinity for the cis -acting elements in the DNA region upstream of leuO and ompU . The toxR null mutant cells expressing strep-tagged ToxR were cross-linked, washed, and sonicated as described in Materials and Methods. Lysates were then treated with either anti-Strep-tag II monoclonal antibody or normal mouse IgG as a control. Free DNA was purified and analyzed by quantitative real-time PCR (qRT-PCR) on a Light Cycler 480 II real-time PCR system. The relative enrichment was calculated as the amount of transcript compared to transcript from cells without cFP. Values are averages from biological experiments done in triplicate. Error bars indicate the standard deviations (**P

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Mutagenesis, Expressing, Sonication, Strep-tag, Purification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    41) Product Images from "Downregulation of SPARC Expression Decreases Cell Migration and Invasion Involving Epithelial-Mesenchymal Transition through the p-FAK/p-ERK Pathway in Esophageal Squamous Cell Carcinoma"

    Article Title: Downregulation of SPARC Expression Decreases Cell Migration and Invasion Involving Epithelial-Mesenchymal Transition through the p-FAK/p-ERK Pathway in Esophageal Squamous Cell Carcinoma

    Journal: Journal of Cancer

    doi: 10.7150/jca.31427

    Expression of secreted protein acidic and rich in cysteine (SPARC) in esophageal squamous cell carcinoma (ESCC) cell lines, control and SPARC siRNA transfected cells. (A) and (B) The relative mRNA and protein expression levels of SPARC were determined using real-time RT-PCR and western blot analysis for eight ESCC cell lines. GAPDH was used as an internal control. (C) Quantification of western blot analysis for SPARC expression in eight ESCC cell lines. (D) and (F) The relative mRNA and protein expression levels of SPARC in Eca109 and HKESC cells transfected with equimolar quantities of siRNA of a non-targeting control (CTRL) or SPARC (SP). β -actin was used as loading control. (E) Quantification of western blot analysis for SPARC expression in Eca109 and HKESC cells transfected with equimolar quantities of siRNA of a non-targeting control (CTRL) or SPARC (SP). All experiments were performed in triplicate for each condition. All error bars represent s.d., n=3. Student's t -test was used for statistical analysis. ** P
    Figure Legend Snippet: Expression of secreted protein acidic and rich in cysteine (SPARC) in esophageal squamous cell carcinoma (ESCC) cell lines, control and SPARC siRNA transfected cells. (A) and (B) The relative mRNA and protein expression levels of SPARC were determined using real-time RT-PCR and western blot analysis for eight ESCC cell lines. GAPDH was used as an internal control. (C) Quantification of western blot analysis for SPARC expression in eight ESCC cell lines. (D) and (F) The relative mRNA and protein expression levels of SPARC in Eca109 and HKESC cells transfected with equimolar quantities of siRNA of a non-targeting control (CTRL) or SPARC (SP). β -actin was used as loading control. (E) Quantification of western blot analysis for SPARC expression in Eca109 and HKESC cells transfected with equimolar quantities of siRNA of a non-targeting control (CTRL) or SPARC (SP). All experiments were performed in triplicate for each condition. All error bars represent s.d., n=3. Student's t -test was used for statistical analysis. ** P

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

    42) Product Images from "MicroRNA-937 is overexpressed and predicts poor prognosis in patients with colon cancer"

    Article Title: MicroRNA-937 is overexpressed and predicts poor prognosis in patients with colon cancer

    Journal: Diagnostic Pathology

    doi: 10.1186/s13000-019-0920-3

    Kaplan-Meier analysis showed that colon cancer patients with high miR-937 expression had shorter overall survival rate. The log-rank test was applied, P = 0.037
    Figure Legend Snippet: Kaplan-Meier analysis showed that colon cancer patients with high miR-937 expression had shorter overall survival rate. The log-rank test was applied, P = 0.037

    Techniques Used: Expressing

    Overexpression of miR-937 promotes the migratory and invasive capabilities, while the downregulation of miR-937 inhibits the migratory and invasive capabilities of colon cancer cells (LoVo and SW620), compared with untreated cells. a . Representative images of Transwell migration assay (magnification × 200). b . Transwell migration assay was applied to investigate migration ability. c . Representative images of Transwell invasion assay (magnification × 200). d . Transwell invasion assay was applied to investigate invasion ability. ** P
    Figure Legend Snippet: Overexpression of miR-937 promotes the migratory and invasive capabilities, while the downregulation of miR-937 inhibits the migratory and invasive capabilities of colon cancer cells (LoVo and SW620), compared with untreated cells. a . Representative images of Transwell migration assay (magnification × 200). b . Transwell migration assay was applied to investigate migration ability. c . Representative images of Transwell invasion assay (magnification × 200). d . Transwell invasion assay was applied to investigate invasion ability. ** P

    Techniques Used: Over Expression, Transwell Migration Assay, Migration, Transwell Invasion Assay

    Overexpression and downregulation of miR-937 promotes and inhibits the proliferation of colon cancer cells (LoVo and SW620), respectively, compared with untreated cells. a . The relative expression level of miR-937 was significantly increased by miR-937 mimic, while decreased by miR-937 inhibitor in LoVo and SW620 cells, compared with that in the untreated cell. b . Cell proliferation was detected by CCK-8 assay. * P
    Figure Legend Snippet: Overexpression and downregulation of miR-937 promotes and inhibits the proliferation of colon cancer cells (LoVo and SW620), respectively, compared with untreated cells. a . The relative expression level of miR-937 was significantly increased by miR-937 mimic, while decreased by miR-937 inhibitor in LoVo and SW620 cells, compared with that in the untreated cell. b . Cell proliferation was detected by CCK-8 assay. * P

    Techniques Used: Over Expression, Expressing, CCK-8 Assay

    The miR-937 expression is detected in colon cancer tissues and cell lines via qRT-PCR analysis. a . miR-937 expression is increased in colon cancer tissues compared with adjacent normal tissues. b . miR-937 expression is upregulated in colon cancer cells (LoVo, SW620, CW-2, HCT116) compared with a normal colonic epithelial cell line (NCM460). ** P
    Figure Legend Snippet: The miR-937 expression is detected in colon cancer tissues and cell lines via qRT-PCR analysis. a . miR-937 expression is increased in colon cancer tissues compared with adjacent normal tissues. b . miR-937 expression is upregulated in colon cancer cells (LoVo, SW620, CW-2, HCT116) compared with a normal colonic epithelial cell line (NCM460). ** P

    Techniques Used: Expressing, Quantitative RT-PCR

    43) Product Images from "MiR-215, an activator of the CTNNBIP1/β-catenin pathway, is a marker of poor prognosis in human glioma"

    Article Title: MiR-215, an activator of the CTNNBIP1/β-catenin pathway, is a marker of poor prognosis in human glioma

    Journal: Oncotarget

    doi:

    miR-215 overexpression in glioma tissues as detected by qRT-PCR assay A. The expression level of miR-215 was higher in tumor tissues than in non-tumor tissues. B. The expression levels of miR-215 in different stages of glioma. C. The expression level of miR-215 in WHO I+II and WHO III+IV stages of the glioma. The stages were graded with WHO classification criteria.
    Figure Legend Snippet: miR-215 overexpression in glioma tissues as detected by qRT-PCR assay A. The expression level of miR-215 was higher in tumor tissues than in non-tumor tissues. B. The expression levels of miR-215 in different stages of glioma. C. The expression level of miR-215 in WHO I+II and WHO III+IV stages of the glioma. The stages were graded with WHO classification criteria.

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

    miRNAs expression in glioma tissues The expression level of 15 miRNAs were examined by qRT-PCR assay.
    Figure Legend Snippet: miRNAs expression in glioma tissues The expression level of 15 miRNAs were examined by qRT-PCR assay.

    Techniques Used: Expressing, Quantitative RT-PCR

    44) Product Images from "Changes of Ammonia-Metabolizing Enzyme Activity and Gene Expression of Two Strains in Shrimp Litopenaeus vannamei Under Ammonia Stress"

    Article Title: Changes of Ammonia-Metabolizing Enzyme Activity and Gene Expression of Two Strains in Shrimp Litopenaeus vannamei Under Ammonia Stress

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00211

    The effect of ammonia-N on the expression of GDH -β, GS , and TG genes expression in hepatopancreas from strains A and B. Different letters indicated significant differences at the level of 0.05. (A) GDH -β gene relative expression level in the hepatopancreas of strain A and B. (B) GS gene relative expression level in the hepatopancreas of strain A and B. (C) TG gene relative expression level in the hepatopancreas of strain A and B. Keys: GDH , Glutamate dehydrogenase; TG , Transglutaminase; GS , Glutamine synthetase.
    Figure Legend Snippet: The effect of ammonia-N on the expression of GDH -β, GS , and TG genes expression in hepatopancreas from strains A and B. Different letters indicated significant differences at the level of 0.05. (A) GDH -β gene relative expression level in the hepatopancreas of strain A and B. (B) GS gene relative expression level in the hepatopancreas of strain A and B. (C) TG gene relative expression level in the hepatopancreas of strain A and B. Keys: GDH , Glutamate dehydrogenase; TG , Transglutaminase; GS , Glutamine synthetase.

    Techniques Used: Expressing

    The effect of ammonia-N on the expression of GDH -β, GS , and TG genes expression in muscles from strains A and B. Different letters indicated significant differences at the level of 0.05. (A) GDH -β gene relative expression level in the muscle of strain A and B. (B) GS gene relative expression level in the muscle of strain A and B. (C) TG gene relative expression level in the muscle of strain A and B. Keys: GDH , Glutamate dehydrogenase; TG , Transglutaminase; GS , Glutamine synthetase.
    Figure Legend Snippet: The effect of ammonia-N on the expression of GDH -β, GS , and TG genes expression in muscles from strains A and B. Different letters indicated significant differences at the level of 0.05. (A) GDH -β gene relative expression level in the muscle of strain A and B. (B) GS gene relative expression level in the muscle of strain A and B. (C) TG gene relative expression level in the muscle of strain A and B. Keys: GDH , Glutamate dehydrogenase; TG , Transglutaminase; GS , Glutamine synthetase.

    Techniques Used: Expressing

    45) Product Images from "Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models"

    Article Title: Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgs285

    Up-regulation of ILK and ILK-regulated proteins involved in EMT in fERG-PrECs. ( A ) qRT-PCR analysis of mRNA expression for ILK (upper left), E-cadherin (lower left), Snail (upper right) and LEF-1 (lower right) in Mock- (open bars) and fERG- (solid bars)
    Figure Legend Snippet: Up-regulation of ILK and ILK-regulated proteins involved in EMT in fERG-PrECs. ( A ) qRT-PCR analysis of mRNA expression for ILK (upper left), E-cadherin (lower left), Snail (upper right) and LEF-1 (lower right) in Mock- (open bars) and fERG- (solid bars)

    Techniques Used: Quantitative RT-PCR, Expressing

    46) Product Images from "The Gene ssl3076 Encodes a Protein Mediating the Salt-Induced Expression of ggpS for the Biosynthesis of the Compatible Solute Glucosylglycerol in Synechocystis sp. Strain PCC 6803 ▿ sp. Strain PCC 6803 ▿ §"

    Article Title: The Gene ssl3076 Encodes a Protein Mediating the Salt-Induced Expression of ggpS for the Biosynthesis of the Compatible Solute Glucosylglycerol in Synechocystis sp. Strain PCC 6803 ▿ sp. Strain PCC 6803 ▿ §

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00481-10

    Influence of ggpR mutation on the salt-inducible ggpS expression analyzed by semiquantitative RT-PCR (A) and real-time PCR (B). (A) Total RNA for cDNA synthesis was obtained from cells grown at control conditions as well as 2 or 96 h after addition of
    Figure Legend Snippet: Influence of ggpR mutation on the salt-inducible ggpS expression analyzed by semiquantitative RT-PCR (A) and real-time PCR (B). (A) Total RNA for cDNA synthesis was obtained from cells grown at control conditions as well as 2 or 96 h after addition of

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    47) Product Images from "Interleukin-37 is increased in adult-onset Still’s disease and associated with disease activity"

    Article Title: Interleukin-37 is increased in adult-onset Still’s disease and associated with disease activity

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1555-6

    Associations between serum interleukin (IL)-37 levels and inflammatory cytokines in patients with adult-onset Still’s disease. Serum IL-37 levels were positively correlated with IL-1β ( a ), IL-18 ( b ), and IL-10 ( c ) respectively except for IL-6 ( d ) and TNF-α ( e ). Each symbol represents an individual patient. The correlations were evaluated with Spearman’s nonparametric test. P
    Figure Legend Snippet: Associations between serum interleukin (IL)-37 levels and inflammatory cytokines in patients with adult-onset Still’s disease. Serum IL-37 levels were positively correlated with IL-1β ( a ), IL-18 ( b ), and IL-10 ( c ) respectively except for IL-6 ( d ) and TNF-α ( e ). Each symbol represents an individual patient. The correlations were evaluated with Spearman’s nonparametric test. P

    Techniques Used:

    48) Product Images from "Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I"

    Article Title: Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku485

    TAF-I negatively regulates ISG transcription. ( A ) Expression level of TAF-I in TAF-I KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or with TAF-I siRNA expression vector (siTAF-I, lane 4). Cell lysates were subjected to western blot analysis using anti-TAF-I and anti-β-actin antibodies. ( B ) and ( C ) Effects of TAF-I KD in ISG transcription. Total RNA was prepared from siCont- and siTAF-I-transfected cells treated with or without IFN-β for 3 h (B) or for indicated periods (C) and subjected to qRT-PCR using specific primer sets for ISG mRNAs, ISG15 pre-mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. The amounts of ISG15 mRNA and ISG15 pre-mRNA in TAF-I KD cells relative to those of control cells were shown in the right panel of (C). Error bars represent standard deviation ( n ≥ 3). * P
    Figure Legend Snippet: TAF-I negatively regulates ISG transcription. ( A ) Expression level of TAF-I in TAF-I KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or with TAF-I siRNA expression vector (siTAF-I, lane 4). Cell lysates were subjected to western blot analysis using anti-TAF-I and anti-β-actin antibodies. ( B ) and ( C ) Effects of TAF-I KD in ISG transcription. Total RNA was prepared from siCont- and siTAF-I-transfected cells treated with or without IFN-β for 3 h (B) or for indicated periods (C) and subjected to qRT-PCR using specific primer sets for ISG mRNAs, ISG15 pre-mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. The amounts of ISG15 mRNA and ISG15 pre-mRNA in TAF-I KD cells relative to those of control cells were shown in the right panel of (C). Error bars represent standard deviation ( n ≥ 3). * P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Standard Deviation

    Histone H1 negatively regulates ISG transcription. ( A ) Expression level of histone H1 in histone H1 KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or histone H1 siRNA expression vector (siH1, lane 4). Cell lysates were subjected to western blot analysis using anti-histone H1.2 (H1) and anti-β-actin antibodies. ( B ) Effects of histone H1 KD on ISG transcription. Total RNA was prepared from siCont- and siH1-transfected cells treated with or without IFN-β for 3 h and subjected to qRT-PCR using specific primer sets for each ISG mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as relative amounts of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P
    Figure Legend Snippet: Histone H1 negatively regulates ISG transcription. ( A ) Expression level of histone H1 in histone H1 KD cells. HeLa S3 cells were transfected with EGFP siRNA expression vector used as a control (siCont, lanes 1–3) or histone H1 siRNA expression vector (siH1, lane 4). Cell lysates were subjected to western blot analysis using anti-histone H1.2 (H1) and anti-β-actin antibodies. ( B ) Effects of histone H1 KD on ISG transcription. Total RNA was prepared from siCont- and siH1-transfected cells treated with or without IFN-β for 3 h and subjected to qRT-PCR using specific primer sets for each ISG mRNA and GAPDH mRNA. The amount of ISG mRNA was normalized as relative amounts of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Standard Deviation

    TAF-I and histone H1 regulate the chromatin structure of ISG promoters. ( A ) Nucleosome digestion by MNase. HeLa S3 cells were transfected with EGFP siRNA expression vector (siCont, lanes 1–4), TAF-I siRNA expression vector (siTAF-I, lanes 5–8) and histone H1 siRNA expression vector (siH1, lanes 9–12), and then treated with or without IFN-β for indicated periods followed by the fixation with formaldehyde. MNase digestion was carried out as described in the Materials and Methods section, and then genomic DNAs were purified, separated by electrophoresis in 1% agarose gel and visualized by ethidium bromide staining. Lane M shows DNA size markers. ( B ) Increase of the MNase sensitivity of the ISG15 promoter in TAF-I KD and H1 KD cells. MNase-digested DNA was prepared as shown in (A) and subjected to qRT-PCR using specific primer sets for the ISG15 and GAPDH promoters. The amount of the ISG15 promoter DNA was normalized by that of the GAPDH promoter DNA and is shown as a relative amount to that from IFN-untreated control cells. Error bars represent standard deviation ( n ≥ 3). * P
    Figure Legend Snippet: TAF-I and histone H1 regulate the chromatin structure of ISG promoters. ( A ) Nucleosome digestion by MNase. HeLa S3 cells were transfected with EGFP siRNA expression vector (siCont, lanes 1–4), TAF-I siRNA expression vector (siTAF-I, lanes 5–8) and histone H1 siRNA expression vector (siH1, lanes 9–12), and then treated with or without IFN-β for indicated periods followed by the fixation with formaldehyde. MNase digestion was carried out as described in the Materials and Methods section, and then genomic DNAs were purified, separated by electrophoresis in 1% agarose gel and visualized by ethidium bromide staining. Lane M shows DNA size markers. ( B ) Increase of the MNase sensitivity of the ISG15 promoter in TAF-I KD and H1 KD cells. MNase-digested DNA was prepared as shown in (A) and subjected to qRT-PCR using specific primer sets for the ISG15 and GAPDH promoters. The amount of the ISG15 promoter DNA was normalized by that of the GAPDH promoter DNA and is shown as a relative amount to that from IFN-untreated control cells. Error bars represent standard deviation ( n ≥ 3). * P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Purification, Electrophoresis, Agarose Gel Electrophoresis, Staining, Quantitative RT-PCR, Standard Deviation

    TAF-I regulates the amounts of the transcription factors and histone H1 on ISG promoters. ( A ) Promoter binding of transcription factors in TAF-I KD cells. siCont- and siTAF-I-transfected cells were treated with or without IFN-β for indicated periods, and cell lysates were subjected to ChIP assays using antibodies specific for STAT1 (left), STAT2 (middle) and Pol II (right) followed by qRT-PCR using a specific primer set for the ISG15 promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P
    Figure Legend Snippet: TAF-I regulates the amounts of the transcription factors and histone H1 on ISG promoters. ( A ) Promoter binding of transcription factors in TAF-I KD cells. siCont- and siTAF-I-transfected cells were treated with or without IFN-β for indicated periods, and cell lysates were subjected to ChIP assays using antibodies specific for STAT1 (left), STAT2 (middle) and Pol II (right) followed by qRT-PCR using a specific primer set for the ISG15 promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P

    Techniques Used: Binding Assay, Transfection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation, Standard Deviation

    The acidic domain of TAF-I is essential for ISG transcriptional regulation. ( A ) IFN-responsive STAT phosphorylation in TAF-I KD cells. Cell lysates were prepared from siCont- (lanes 1–4) and siTAF-I-transfected (lanes 5–8) cells treated with or without IFN-β for indicated periods, and subjected to western blot analysis using anti-tyrosine phosphorylated (pY)-STAT1, anti-pY-STAT2, anti-serine phosphorylated (pS)-STAT1, anti-STAT1, anti-STAT2, anti-IRF9, anti-TAF-I and anti-β-actin antibodies. ( B ) Dispensability of TAF-I in ISRE-dependent transcription. HEK293 cells were transfected with EGFP siRNA expression vector used as a control (siCont) or with TAF-I siRNA expression vector (siTAF-I) together with pISRE-TA- Luc and pSEAP-control, and then cells were treated with or without IFN-β for 6 h followed by the luciferase assay. The luciferase activity was normalized with the SEAP activity and represented as fold-induction relative to that from IFN-treated control cells. Error bars represent standard deviation ( n ≥ 3). ‘n/s’ indicates ‘not significant’. ( C ) Schematic representation of TAF-I proteins. The N-terminal regions specific for TAF-Iα (aa 1–37) and TAF-Iβ (aa 1–24) and the C-terminal acidic region for TAF-Iα (aa 239–290) and TAF-Iβ (aa 226–227) are indicated. ( D ) Expression level of Flag-TAF-Is in TAF-I KD cells. Cell lysates were prepared from HeLa S3 cells transfected with EGFP siRNA expression vector used as a control (lanes 1–3) or TAF-I siRNA expression vector (siTAF-I, lanes 4–8) together with expression vectors expressing Flag-tagged TAF-Iα (lane 5), Flag-tagged TAF-IαΔC (lane 6), Flag-tagged TAF-Iβ (lane 7), Flag-tagged TAF-IβΔC (lane 8) or empty vector (lane 4), and cell lysates were subjected to western blot analysis using anti-TAF-I, anti-Flag and anti-β-actin antibodies. ( E ) Transcription rescue experiments by Flag-TAF-I. Cells were prepared as shown in (D) and treated with or without IFN-β for 3 h. Total RNA was subjected to qRT-PCR using specific primer sets for ISG15 mRNA (left), ISG56 mRNA (right) and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P
    Figure Legend Snippet: The acidic domain of TAF-I is essential for ISG transcriptional regulation. ( A ) IFN-responsive STAT phosphorylation in TAF-I KD cells. Cell lysates were prepared from siCont- (lanes 1–4) and siTAF-I-transfected (lanes 5–8) cells treated with or without IFN-β for indicated periods, and subjected to western blot analysis using anti-tyrosine phosphorylated (pY)-STAT1, anti-pY-STAT2, anti-serine phosphorylated (pS)-STAT1, anti-STAT1, anti-STAT2, anti-IRF9, anti-TAF-I and anti-β-actin antibodies. ( B ) Dispensability of TAF-I in ISRE-dependent transcription. HEK293 cells were transfected with EGFP siRNA expression vector used as a control (siCont) or with TAF-I siRNA expression vector (siTAF-I) together with pISRE-TA- Luc and pSEAP-control, and then cells were treated with or without IFN-β for 6 h followed by the luciferase assay. The luciferase activity was normalized with the SEAP activity and represented as fold-induction relative to that from IFN-treated control cells. Error bars represent standard deviation ( n ≥ 3). ‘n/s’ indicates ‘not significant’. ( C ) Schematic representation of TAF-I proteins. The N-terminal regions specific for TAF-Iα (aa 1–37) and TAF-Iβ (aa 1–24) and the C-terminal acidic region for TAF-Iα (aa 239–290) and TAF-Iβ (aa 226–227) are indicated. ( D ) Expression level of Flag-TAF-Is in TAF-I KD cells. Cell lysates were prepared from HeLa S3 cells transfected with EGFP siRNA expression vector used as a control (lanes 1–3) or TAF-I siRNA expression vector (siTAF-I, lanes 4–8) together with expression vectors expressing Flag-tagged TAF-Iα (lane 5), Flag-tagged TAF-IαΔC (lane 6), Flag-tagged TAF-Iβ (lane 7), Flag-tagged TAF-IβΔC (lane 8) or empty vector (lane 4), and cell lysates were subjected to western blot analysis using anti-TAF-I, anti-Flag and anti-β-actin antibodies. ( E ) Transcription rescue experiments by Flag-TAF-I. Cells were prepared as shown in (D) and treated with or without IFN-β for 3 h. Total RNA was subjected to qRT-PCR using specific primer sets for ISG15 mRNA (left), ISG56 mRNA (right) and GAPDH mRNA. The amount of ISG mRNA was normalized as a relative amount of GAPDH mRNA. Error bars represent standard deviation ( n ≥ 3). * P

    Techniques Used: Transfection, Western Blot, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Quantitative RT-PCR

    Dissociation of TAF-I from ISG promoters. ( A ) Dynamics of TAF-I at ISG promoters in an IFN-dependent manner. HeLa S3 cells were transfected with TAF-I siRNA expression vector (lanes 1–4) together with empty vector (lane 4) or Flag-tagged TAF-Iα expression vector (lanes 1–3) and then treated with or without IFN-β for indicated periods. Cells were, then, subjected to ChIP assays using the agarose-conjugated antibody against Flag followed by qRT-PCR using specific primer sets for each ISG promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P
    Figure Legend Snippet: Dissociation of TAF-I from ISG promoters. ( A ) Dynamics of TAF-I at ISG promoters in an IFN-dependent manner. HeLa S3 cells were transfected with TAF-I siRNA expression vector (lanes 1–4) together with empty vector (lane 4) or Flag-tagged TAF-Iα expression vector (lanes 1–3) and then treated with or without IFN-β for indicated periods. Cells were, then, subjected to ChIP assays using the agarose-conjugated antibody against Flag followed by qRT-PCR using specific primer sets for each ISG promoter. The amount of DNA co-immunoprecipitated with each antibody was shown as % of input. Error bars represent standard deviation ( n ≥ 3). * P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation, Standard Deviation

    49) Product Images from "Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models"

    Article Title: Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models

    Journal: BMC Systems Biology

    doi: 10.1186/1752-0509-4-159

    qRT-PCR validation of the differential expression of six candidate genes at different time points . Filled circles located on solid lines represent differential expression values from the microarray whereas filled squares on dashed lines show qRT-PCR expression values of AtT-20 cells independently treated with CRH related to their untreated controls and normalized to the house keeping gene Hprt. p-values were evaluated by ANOVA analyses.
    Figure Legend Snippet: qRT-PCR validation of the differential expression of six candidate genes at different time points . Filled circles located on solid lines represent differential expression values from the microarray whereas filled squares on dashed lines show qRT-PCR expression values of AtT-20 cells independently treated with CRH related to their untreated controls and normalized to the house keeping gene Hprt. p-values were evaluated by ANOVA analyses.

    Techniques Used: Quantitative RT-PCR, Expressing, Microarray

    Result of shortest path searches between all candidate genes investigated by GeneNet . After manual curation of each interaction the resulting pathways were combined in this picture. Experimentally with qRT-PCR validated genes are drawn by rectangles and intermediates are indicated by circles. Lines with an arrowhead reflect positive regulation, other lines indicate inhibition.
    Figure Legend Snippet: Result of shortest path searches between all candidate genes investigated by GeneNet . After manual curation of each interaction the resulting pathways were combined in this picture. Experimentally with qRT-PCR validated genes are drawn by rectangles and intermediates are indicated by circles. Lines with an arrowhead reflect positive regulation, other lines indicate inhibition.

    Techniques Used: Quantitative RT-PCR, Inhibition

    50) Product Images from "High-Resolution Recording of the Circadian Oscillator in Primary Mouse α- and β-Cell Culture"

    Article Title: High-Resolution Recording of the Circadian Oscillator in Primary Mouse α- and β-Cell Culture

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2017.00068

    Isolated around-the-clock α- and β-cells express in vivo circadian rhythm for selected core clock genes after fluorescence-activated cell sorting (FACS) separation . Triple transgenic mice (ProGcg-Venus/RIP-Cherry/Per2:Luc) were kept at standard 12 h/12 h light–dark cycles (ZT0 (Zeitgeber) corresponds to 07:00 a.m., and ZT12 corresponds to 07:00 p.m.), with constant access to the drinking water and night-restricted feeding regime for 2 weeks prior to islet collection. Pancreatic islets were isolated around-the-clock every 4 h and subjected to the FACS separation procedure. Temporal profiles of selected core clock transcripts ( Clock, Per1 , and Per2 ) were assessed in freshly extracted mRNA at each time point from α- and β-cell populations by quantitative RT-PCR analysis. Transcript levels were normalized to Hprt expression. Data expressed as mean ± SD ( N = 2 biological replicates of 6 mice at each time point).
    Figure Legend Snippet: Isolated around-the-clock α- and β-cells express in vivo circadian rhythm for selected core clock genes after fluorescence-activated cell sorting (FACS) separation . Triple transgenic mice (ProGcg-Venus/RIP-Cherry/Per2:Luc) were kept at standard 12 h/12 h light–dark cycles (ZT0 (Zeitgeber) corresponds to 07:00 a.m., and ZT12 corresponds to 07:00 p.m.), with constant access to the drinking water and night-restricted feeding regime for 2 weeks prior to islet collection. Pancreatic islets were isolated around-the-clock every 4 h and subjected to the FACS separation procedure. Temporal profiles of selected core clock transcripts ( Clock, Per1 , and Per2 ) were assessed in freshly extracted mRNA at each time point from α- and β-cell populations by quantitative RT-PCR analysis. Transcript levels were normalized to Hprt expression. Data expressed as mean ± SD ( N = 2 biological replicates of 6 mice at each time point).

    Techniques Used: Isolation, In Vivo, Fluorescence, FACS, Transgenic Assay, Mouse Assay, Quantitative RT-PCR, Expressing

    51) Product Images from "Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus"

    Article Title: Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-1097-0

    Validation of the interaction between ANXA2 and NS1. a Co-IP assay confirming the interaction between NS1 and ANXA2. A549 cells were transfected with HA-ANXA2 plasmid and infected with GD1322 24 h later. Cell lysates, harvested 36–48 h after infection, were subjected to IP and western blotting with anti-HA or anti-NS1 D7 antibodies. b Co-IP assay detecting the association between NS1 and endogenous ANXA2. A549 cells were infected with GD1322 and collected after 12 h. Next, cell lysates from infected or uninfected A549 cells were immunoprecipitated with an anti-ANXA2 rabbit antibody or the anti-NS1 D7 mouse antibody. After incubation with protein A/G-agarose beads, the Immunoprecipitation pellets were immunoblotted with the anti-NS1 D7 mouse antibody or the anti-ANXA2 rabbit antibody. c Pull down assay to verify that the ED of NS1 binds with ANXA2. Full-length NS1 and each functional domain were fused with GST and then incubated with uninfected A549 lysate. GST and ANXA2 were detected by western blotting. d Colocalization of ANXA2 and NS1 in A549 cells. HA-ANXA2 and FLAG-NS1 plasmids were co-transfected into A549 or 293 T cells. After incubation for 24 h, the cells were double-immunostained for FLAG-NS1 (green) and HA-ANXA2 (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue)
    Figure Legend Snippet: Validation of the interaction between ANXA2 and NS1. a Co-IP assay confirming the interaction between NS1 and ANXA2. A549 cells were transfected with HA-ANXA2 plasmid and infected with GD1322 24 h later. Cell lysates, harvested 36–48 h after infection, were subjected to IP and western blotting with anti-HA or anti-NS1 D7 antibodies. b Co-IP assay detecting the association between NS1 and endogenous ANXA2. A549 cells were infected with GD1322 and collected after 12 h. Next, cell lysates from infected or uninfected A549 cells were immunoprecipitated with an anti-ANXA2 rabbit antibody or the anti-NS1 D7 mouse antibody. After incubation with protein A/G-agarose beads, the Immunoprecipitation pellets were immunoblotted with the anti-NS1 D7 mouse antibody or the anti-ANXA2 rabbit antibody. c Pull down assay to verify that the ED of NS1 binds with ANXA2. Full-length NS1 and each functional domain were fused with GST and then incubated with uninfected A549 lysate. GST and ANXA2 were detected by western blotting. d Colocalization of ANXA2 and NS1 in A549 cells. HA-ANXA2 and FLAG-NS1 plasmids were co-transfected into A549 or 293 T cells. After incubation for 24 h, the cells were double-immunostained for FLAG-NS1 (green) and HA-ANXA2 (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue)

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Infection, Western Blot, Immunoprecipitation, Incubation, Pull Down Assay, Functional Assay

    ANXA2 influences the replication of H5 subtype AIV. A549 cells were infected with GD1322 (MOI = 0.1) after transfection with siNC or siANXA2 for 24 h. The cells were then collected at 4, 12, 24 and 48 hpi to extract RNA or protein ( a , b ). A549 cells were also collected at 0, 4, 12, 24, and 48 hpi to extract protein ( c ). a qRT-PCR detection of gene synthesis of viral genes after transfection with siRNA. Total RNA was extracted from A549 cells, and ANXA2 mRNA, M vRNA and HA vRNA were used for relative quantification. GAPDH was used as a control. b Western blotting detection of viral protein expression after transfection of siRNA. After extracting total proteins from A549 cells, viral protein expression was determined using H5-specific antiserum from our lab. ANXA2 and tubulin were detected using commercial MAbs. The results are presented as the mean and SD from three independent experiments. c Western blotting detection of ANXA2 and HA expression after infection with H5N1 AIV. The same antibodies described above were used
    Figure Legend Snippet: ANXA2 influences the replication of H5 subtype AIV. A549 cells were infected with GD1322 (MOI = 0.1) after transfection with siNC or siANXA2 for 24 h. The cells were then collected at 4, 12, 24 and 48 hpi to extract RNA or protein ( a , b ). A549 cells were also collected at 0, 4, 12, 24, and 48 hpi to extract protein ( c ). a qRT-PCR detection of gene synthesis of viral genes after transfection with siRNA. Total RNA was extracted from A549 cells, and ANXA2 mRNA, M vRNA and HA vRNA were used for relative quantification. GAPDH was used as a control. b Western blotting detection of viral protein expression after transfection of siRNA. After extracting total proteins from A549 cells, viral protein expression was determined using H5-specific antiserum from our lab. ANXA2 and tubulin were detected using commercial MAbs. The results are presented as the mean and SD from three independent experiments. c Western blotting detection of ANXA2 and HA expression after infection with H5N1 AIV. The same antibodies described above were used

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

    ANXA2 influences AIV replication in A549 cells. a Overexpression of ANXA2 in A549 cells. A549 cells were transfected with HA-ANXA2 plasmid or an empty vector (Vec) and then infected with GD1322 (MOI = 0.1). After 24 h, HA-ANXA2 and NS1 proteins were detected by western blotting. b Progeny virus titers increased significantly in A549 cells overexpressing ANXA2. Viral supernatants were collected at 12 h and 24 h after infection. Viral titers were assayed based on hemagglutination, and the results are expressed as TCID 50 per mL. c Knockdown efficiency of ANXA2. A549 cells were transfected with siNC or siANXA2. The results shown are from qPCR and western blotting analyses performed 24 h after infection. d Progeny virus titers decreased significantly after transfection with siANXA2 at 24 hpi. The results are presented as the mean and SD from three independent experiments. *, p
    Figure Legend Snippet: ANXA2 influences AIV replication in A549 cells. a Overexpression of ANXA2 in A549 cells. A549 cells were transfected with HA-ANXA2 plasmid or an empty vector (Vec) and then infected with GD1322 (MOI = 0.1). After 24 h, HA-ANXA2 and NS1 proteins were detected by western blotting. b Progeny virus titers increased significantly in A549 cells overexpressing ANXA2. Viral supernatants were collected at 12 h and 24 h after infection. Viral titers were assayed based on hemagglutination, and the results are expressed as TCID 50 per mL. c Knockdown efficiency of ANXA2. A549 cells were transfected with siNC or siANXA2. The results shown are from qPCR and western blotting analyses performed 24 h after infection. d Progeny virus titers decreased significantly after transfection with siANXA2 at 24 hpi. The results are presented as the mean and SD from three independent experiments. *, p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Infection, Western Blot, Real-time Polymerase Chain Reaction

    ANXA2 was confirmed as a novel host protein that binds to NS1. NS1-associated proteins from infected (InfA) or uninfected (Mock) A549 cell lysates were immunoprecipitated using the anti-NS1 D7 antibody, separated by SDS-PAGE (8%), and visualized by silver staining. The InfA group-specific band (indicated by an asterisk) was identified as ANXA2 by LTQ-MS
    Figure Legend Snippet: ANXA2 was confirmed as a novel host protein that binds to NS1. NS1-associated proteins from infected (InfA) or uninfected (Mock) A549 cell lysates were immunoprecipitated using the anti-NS1 D7 antibody, separated by SDS-PAGE (8%), and visualized by silver staining. The InfA group-specific band (indicated by an asterisk) was identified as ANXA2 by LTQ-MS

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry

    52) Product Images from "Follistatin like-1 (Fstl1) is required for the normal formation of lung airway and vascular smooth muscle at birth"

    Article Title: Follistatin like-1 (Fstl1) is required for the normal formation of lung airway and vascular smooth muscle at birth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177899

    Loss of Fstl1 led to abnormal tracheal and bronchial SM formation in E18.5 embryos. ( A , B ) α-SMA whole-mount staining revealed an extremely attenuated α-SMA signal in Fstl1 −/− trachea. Trachea ( C , D ), proximal bronchi ( E , F , the sections at the points where the tracheas split into the left and right main bronchi) and distal bronchi ( G , H ) sections stained for α-SMA confirmed reduced α-SMA expression (arrows). ( I , J ) Trachea sections of similar planes, as indicated by the common carotid artery and thymus, stained for SM22α revealed reduced SM cells in Fstl1 −/− trachea. ( K , L ) Stitched images showed airway SM defects from proximal bronchi to distal bronchi in Fstl1 −/− lung. ( M ) qRT-PCR analysis of the expression of Fstl1 and α-SMA in E18.5 tracheas and lungs (n = 5 per group). aa, arch of the aorta, th, thymus, ca, common carotid artery, lb, left main bronchus, rb, right main bronchus. *, P
    Figure Legend Snippet: Loss of Fstl1 led to abnormal tracheal and bronchial SM formation in E18.5 embryos. ( A , B ) α-SMA whole-mount staining revealed an extremely attenuated α-SMA signal in Fstl1 −/− trachea. Trachea ( C , D ), proximal bronchi ( E , F , the sections at the points where the tracheas split into the left and right main bronchi) and distal bronchi ( G , H ) sections stained for α-SMA confirmed reduced α-SMA expression (arrows). ( I , J ) Trachea sections of similar planes, as indicated by the common carotid artery and thymus, stained for SM22α revealed reduced SM cells in Fstl1 −/− trachea. ( K , L ) Stitched images showed airway SM defects from proximal bronchi to distal bronchi in Fstl1 −/− lung. ( M ) qRT-PCR analysis of the expression of Fstl1 and α-SMA in E18.5 tracheas and lungs (n = 5 per group). aa, arch of the aorta, th, thymus, ca, common carotid artery, lb, left main bronchus, rb, right main bronchus. *, P

    Techniques Used: Staining, Expressing, Quantitative RT-PCR

    ASM differentiation was significantly reduced in Fstl1 −/− lungs. ( A , B ) α-SMA immunostaining of E10.5 trachea sections revealed rare SM cell differentiation in both WT and Fstl1 −/− (arrows). Immunofluorescence staining for α-SMA of E11.5 ( C , D ), E12.5 ( E , F ), E13.5 ( G , H ), E15.5 ( I , J ) tracheas showed less SM formation and expansion in Fstl1 −/− tracheas during the early development (arrows). ( K ) qRT-PCR of Fstl1 , α-SMA , myocardin and SRF expression demonstrated a significant reduction in E11.5 Fstl1 −/− lungs compared to control WT lungs (WT, n = 5, Fstl1 −/− , n = 6). ( L ) Loss of Fstl1 inhibited the TGF-β1-induced α-SMA , SRF , and myocardin expression in MEFs. *, P
    Figure Legend Snippet: ASM differentiation was significantly reduced in Fstl1 −/− lungs. ( A , B ) α-SMA immunostaining of E10.5 trachea sections revealed rare SM cell differentiation in both WT and Fstl1 −/− (arrows). Immunofluorescence staining for α-SMA of E11.5 ( C , D ), E12.5 ( E , F ), E13.5 ( G , H ), E15.5 ( I , J ) tracheas showed less SM formation and expansion in Fstl1 −/− tracheas during the early development (arrows). ( K ) qRT-PCR of Fstl1 , α-SMA , myocardin and SRF expression demonstrated a significant reduction in E11.5 Fstl1 −/− lungs compared to control WT lungs (WT, n = 5, Fstl1 −/− , n = 6). ( L ) Loss of Fstl1 inhibited the TGF-β1-induced α-SMA , SRF , and myocardin expression in MEFs. *, P

    Techniques Used: Immunostaining, Cell Differentiation, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

    53) Product Images from "Nitric oxide blocks the development of the human parasite Schistosoma japonicum"

    Article Title: Nitric oxide blocks the development of the human parasite Schistosoma japonicum

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

    doi: 10.1073/pnas.1708578114

    Relative expression of the mitochondrial respiratory chain enzyme CcO I and NADH dehydrogenase in WT and iNOS −/− rats. The relative expression of each gene was normalized to the expression of β-actin. Data are shown as the mean ± SEM from two biological repeats ( n = 10). Significant differences have been noted. * P
    Figure Legend Snippet: Relative expression of the mitochondrial respiratory chain enzyme CcO I and NADH dehydrogenase in WT and iNOS −/− rats. The relative expression of each gene was normalized to the expression of β-actin. Data are shown as the mean ± SEM from two biological repeats ( n = 10). Significant differences have been noted. * P

    Techniques Used: Expressing

    54) Product Images from "Influence of CCND1 G870A polymorphism on the risk of HBV-related HCC and cyclin D1 splicing variant expression in Chinese population"

    Article Title: Influence of CCND1 G870A polymorphism on the risk of HBV-related HCC and cyclin D1 splicing variant expression in Chinese population

    Journal: Tumour Biology

    doi: 10.1007/s13277-015-3401-7

    Expression levels of both cyclin D1 variants in paired HCC tissues. Total RNA were prepared from 45 paired HCC tissues and 11 normal liver tissues. The expression of both cyclin D1 variants was quantitated by qRT-PCR and normalized to CTBP1. Each sample was tested in triplicate in two separate experiments. a The comparison of cyclin D1a expression levels among the HCC tumor, adjacent nontumor and the normal liver tissues. ANOVA test was used to analyze the difference of cyclin D1a expression levels among these groups. b The expression of cyclin D1b was compared as in ( a ). c Ratio of cyclin D1b versus cyclin D1a was compared as in ( a ). d The difference between cyclin D1a and cyclin D1b expression in the HCC tissues, adjacent nontumor tissues or the normal liver tissues. ANOVA test was used to analyze the difference of cyclin D1 expression levels among these groups. e The expression of cyclin D1a protein in 14 paired HCC tissues was detected by Western blot assay. Tubulin was used as an internal loading control in each lane. T tumor tissue, NT nontumor tissue
    Figure Legend Snippet: Expression levels of both cyclin D1 variants in paired HCC tissues. Total RNA were prepared from 45 paired HCC tissues and 11 normal liver tissues. The expression of both cyclin D1 variants was quantitated by qRT-PCR and normalized to CTBP1. Each sample was tested in triplicate in two separate experiments. a The comparison of cyclin D1a expression levels among the HCC tumor, adjacent nontumor and the normal liver tissues. ANOVA test was used to analyze the difference of cyclin D1a expression levels among these groups. b The expression of cyclin D1b was compared as in ( a ). c Ratio of cyclin D1b versus cyclin D1a was compared as in ( a ). d The difference between cyclin D1a and cyclin D1b expression in the HCC tissues, adjacent nontumor tissues or the normal liver tissues. ANOVA test was used to analyze the difference of cyclin D1 expression levels among these groups. e The expression of cyclin D1a protein in 14 paired HCC tissues was detected by Western blot assay. Tubulin was used as an internal loading control in each lane. T tumor tissue, NT nontumor tissue

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

    Epitopic expression of cyclin D1a and cyclin D1b promote the cell proliferation in Huh-7 and LO2 cells. Huh-7 and LO2 cells were transiently transfected with pFlex-cyclin D1a, pFlex-cyclin D1b, and pFlex vector. Ectopic expression of cyclin D1 variants was detected by Western blot analysis using Flag-tagged antibody ( a ). b The CCK-8 assay was performed to measure the growth curve of Huh-7 and LO2 cells transfected with plasmids as ( a ). All measurements were done in triplicate and data are shown as the means ± SD. P values
    Figure Legend Snippet: Epitopic expression of cyclin D1a and cyclin D1b promote the cell proliferation in Huh-7 and LO2 cells. Huh-7 and LO2 cells were transiently transfected with pFlex-cyclin D1a, pFlex-cyclin D1b, and pFlex vector. Ectopic expression of cyclin D1 variants was detected by Western blot analysis using Flag-tagged antibody ( a ). b The CCK-8 assay was performed to measure the growth curve of Huh-7 and LO2 cells transfected with plasmids as ( a ). All measurements were done in triplicate and data are shown as the means ± SD. P values

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, CCK-8 Assay

    Relationship between cyclin D1a and cyclin D1b expression in HCC tissues. a The correlation of cyclin D1a and cyclin D1b expression level in 45 cases tumor tissues was analyzed by liner correction. b The correlation of cyclin D1a and cyclin D1b expression level in nontumor tissues was analyzed as in ( a )
    Figure Legend Snippet: Relationship between cyclin D1a and cyclin D1b expression in HCC tissues. a The correlation of cyclin D1a and cyclin D1b expression level in 45 cases tumor tissues was analyzed by liner correction. b The correlation of cyclin D1a and cyclin D1b expression level in nontumor tissues was analyzed as in ( a )

    Techniques Used: Expressing

    The effect of G870A genotype on the expression of cyclin D1 variants in HCC tissues. The expression of cyclin D1 variants in 17 genotyped HCC tissues was detected by real-time PCR. The expression of cyclin D1a ( a ) or D1b ( b ) in HCC tissues with AA genotype ( n = 9) was compared to GG genotype ( n = 8). The housekeeping gene CTBP1 was used to normalize the expression levels of cyclin D1a and cyclin D1b. Each sample was tested in triplicate in two separate experiments
    Figure Legend Snippet: The effect of G870A genotype on the expression of cyclin D1 variants in HCC tissues. The expression of cyclin D1 variants in 17 genotyped HCC tissues was detected by real-time PCR. The expression of cyclin D1a ( a ) or D1b ( b ) in HCC tissues with AA genotype ( n = 9) was compared to GG genotype ( n = 8). The housekeeping gene CTBP1 was used to normalize the expression levels of cyclin D1a and cyclin D1b. Each sample was tested in triplicate in two separate experiments

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    55) Product Images from "Fiber2 and hexon genes are closely associated with the virulence of the emerging and highly pathogenic fowl adenovirus 4"

    Article Title: Fiber2 and hexon genes are closely associated with the virulence of the emerging and highly pathogenic fowl adenovirus 4

    Journal: Emerging Microbes & Infections

    doi: 10.1038/s41426-018-0203-1

    Viral loads in different tissues of chickens inoculated with rescued FAdV-4. Heart, liver, kidney, spleen, lung, proventriculus, duodenum, bursa of Fabricius, and cecal tonsil tissue samples of chickens in each group were collected from dead chickens during the experiment or euthanized chickens at the end of experiment. Viral loads in different tissues were determined by a SYBR Green I quantitative real-time PCR using FAdV-4 ORF14 gene as an indicator for the presence of viral DNA. The final concentration was calculated as copy numbers per milligram of tissue sample. Results are presented at the means ± standard error of mean. Asterisks (*) mark the viral loads that were significantly different between the groups ( p
    Figure Legend Snippet: Viral loads in different tissues of chickens inoculated with rescued FAdV-4. Heart, liver, kidney, spleen, lung, proventriculus, duodenum, bursa of Fabricius, and cecal tonsil tissue samples of chickens in each group were collected from dead chickens during the experiment or euthanized chickens at the end of experiment. Viral loads in different tissues were determined by a SYBR Green I quantitative real-time PCR using FAdV-4 ORF14 gene as an indicator for the presence of viral DNA. The final concentration was calculated as copy numbers per milligram of tissue sample. Results are presented at the means ± standard error of mean. Asterisks (*) mark the viral loads that were significantly different between the groups ( p

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, Concentration Assay

    Viral shedding in chickens of different groups inoculated with rescued FAdV-4. Cloacal swabs were collected at 24, 48, 72, 96, and 120 h postinfection (h.p.i.) and their virus loads were determined by a SYBR Green I quantitative real-time PCR assay using the FAdV-4 ORF14 gene as an indicator for the presence of viral DNA. The final concentration was calculated as copy numbers per microliter of extracted DNA from cloacal swabs. The results are presented at the means ± standard error of mean
    Figure Legend Snippet: Viral shedding in chickens of different groups inoculated with rescued FAdV-4. Cloacal swabs were collected at 24, 48, 72, 96, and 120 h postinfection (h.p.i.) and their virus loads were determined by a SYBR Green I quantitative real-time PCR assay using the FAdV-4 ORF14 gene as an indicator for the presence of viral DNA. The final concentration was calculated as copy numbers per microliter of extracted DNA from cloacal swabs. The results are presented at the means ± standard error of mean

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, Concentration Assay

    56) Product Images from "STEROID RECEPTOR COACTIVATOR 2 (SRC-2) MODULATES STEROID-DEPENDENT MALE SEXUAL BEHAVIOR AND NEUROPLASTICITY IN JAPANESE QUAIL (COTURNIX JAPONICA)"

    Article Title: STEROID RECEPTOR COACTIVATOR 2 (SRC-2) MODULATES STEROID-DEPENDENT MALE SEXUAL BEHAVIOR AND NEUROPLASTICITY IN JAPANESE QUAIL (COTURNIX JAPONICA)

    Journal: Journal of neurochemistry

    doi: 10.1111/j.1471-4159.2011.07438.x

    Quantification of SRC-2 in the quail brain by quantitative PCR using the FastStart Universal SYBR Green Master (ROX) Mix. Bar graphs illustrate the SRC-2/GAPDH ratio in the medial preoptic nucleus (POM), cerebellum and optic lobes of male (white) and
    Figure Legend Snippet: Quantification of SRC-2 in the quail brain by quantitative PCR using the FastStart Universal SYBR Green Master (ROX) Mix. Bar graphs illustrate the SRC-2/GAPDH ratio in the medial preoptic nucleus (POM), cerebellum and optic lobes of male (white) and

    Techniques Used: Real-time Polymerase Chain Reaction, SYBR Green Assay

    57) Product Images from "Stanniocalcin-1 Controls Ion Regulation Functions of Ion-transporting Epithelium Other than Calcium Balance"

    Article Title: Stanniocalcin-1 Controls Ion Regulation Functions of Ion-transporting Epithelium Other than Calcium Balance

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.10773

    Different environments affect expression of STC genes in zebrafish embryos. Zebrafish embryos were incubated with high-Ca 2+ or acidic (pH=4) media for 4 d or double-deionized water for 1 d, and the mRNA levels of four STC genes were then measured by qRT-PCR. (A) stc-1 (STC1) and stc-2 (STC2) mRNAs were significantly decreased by low-Ca 2+ (A), acidic (B) and double-deionized water (DDW)(C) treatments, while that of stc-1 like (STC1L) and stc-2 like (STC2L) were unaffected. Low-Ca 2+ (A), acidic (B) and double-deionized water (C) treatments caused greater reductions of stc-1 mRNA (87%, 87% and 60%, respectively) than stc-2 mRNA (17%, 32% and 17%, respectively). qRT-PCR values were normalized to that of rpl13a . Mean ± SEM ( n = 4-5). * ( p
    Figure Legend Snippet: Different environments affect expression of STC genes in zebrafish embryos. Zebrafish embryos were incubated with high-Ca 2+ or acidic (pH=4) media for 4 d or double-deionized water for 1 d, and the mRNA levels of four STC genes were then measured by qRT-PCR. (A) stc-1 (STC1) and stc-2 (STC2) mRNAs were significantly decreased by low-Ca 2+ (A), acidic (B) and double-deionized water (DDW)(C) treatments, while that of stc-1 like (STC1L) and stc-2 like (STC2L) were unaffected. Low-Ca 2+ (A), acidic (B) and double-deionized water (C) treatments caused greater reductions of stc-1 mRNA (87%, 87% and 60%, respectively) than stc-2 mRNA (17%, 32% and 17%, respectively). qRT-PCR values were normalized to that of rpl13a . Mean ± SEM ( n = 4-5). * ( p

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR

    58) Product Images from "Retention of hexanucleotide repeat-containing intron in C9orf72 mRNA: implications for the pathogenesis of ALS/FTD"

    Article Title: Retention of hexanucleotide repeat-containing intron in C9orf72 mRNA: implications for the pathogenesis of ALS/FTD

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-016-0289-4

    Intron 1 retention in C9orf72 transcripts in the brain of C9orf72 expansion carriers. C9orf72 transcripts were analyzed in the frontal cortex from frontotemporal lobar degeneration (FTLD) or frontotemporal lobar degeneration with motor neuron disease (FTLD-MND) cases with confirmed C9orf72 hexanucleotide expansions and from control individuals. a RNA was analyzed by RT-PCR as in Fig. 1 . GAPDH was used as a loading control. The level of C9orf72 transcripts unspliced at the 5’ and 3’ ends in an FTLD case homozygous for the C9orf72 G 4 C 2 repeat expansion was markedly higher than in heterozygous cases (C9 (+/+) , far right lane). b Quantitative analysis of intron retention by real-time PCR. Levels of C9orf72 transcripts spliced or unspliced at the 5’ and 3’ end of intron 1 were determined by real-time qRT-PCR in heterozygous expansion carriers ( n = 11) and control cases ( n = 10). Data are shown as means ± SEM. Each data point represents an individual case, *** P
    Figure Legend Snippet: Intron 1 retention in C9orf72 transcripts in the brain of C9orf72 expansion carriers. C9orf72 transcripts were analyzed in the frontal cortex from frontotemporal lobar degeneration (FTLD) or frontotemporal lobar degeneration with motor neuron disease (FTLD-MND) cases with confirmed C9orf72 hexanucleotide expansions and from control individuals. a RNA was analyzed by RT-PCR as in Fig. 1 . GAPDH was used as a loading control. The level of C9orf72 transcripts unspliced at the 5’ and 3’ ends in an FTLD case homozygous for the C9orf72 G 4 C 2 repeat expansion was markedly higher than in heterozygous cases (C9 (+/+) , far right lane). b Quantitative analysis of intron retention by real-time PCR. Levels of C9orf72 transcripts spliced or unspliced at the 5’ and 3’ end of intron 1 were determined by real-time qRT-PCR in heterozygous expansion carriers ( n = 11) and control cases ( n = 10). Data are shown as means ± SEM. Each data point represents an individual case, *** P

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

    Intron 1 retention in C9orf72 transcripts in lymphoblasts. C9orf72 transcripts were analyzed in lymphoblasts from C9orf72 G 4 C 2 expansion carriers and control individuals. a RT-PCR analysis of poly(A) + RNA using primers spanning the 5’ splice site (left) or the 3’ splice site (right) of intron 1 demonstrating retention of intron 1in polyadenylated RNA. The position of the primers is indicated on the diagram above the gels. GAPDH was used as a loading control. b Correctly spliced transcripts detected in controls and expansion carrier cells using primers in exons 1a and 2. c Quantitative analysis of intron retention by real-time PCR. Levels of C9orf72 transcripts spliced or unspliced at the 5’ and 3’ end of intron 1 were determined by real-time qRT-PCR. Data are shown as means ± SEM. Each data point represents an individual case, n =15 (C9 - ); 15 (C9 + ). No significant differences were observed between the C9 − and C9 + groups. d Sequencing of the 3’ PCR product demonstrates an exact intron 1-exon 2 boundary. C9 − , controls; C9 + , expansion carriers
    Figure Legend Snippet: Intron 1 retention in C9orf72 transcripts in lymphoblasts. C9orf72 transcripts were analyzed in lymphoblasts from C9orf72 G 4 C 2 expansion carriers and control individuals. a RT-PCR analysis of poly(A) + RNA using primers spanning the 5’ splice site (left) or the 3’ splice site (right) of intron 1 demonstrating retention of intron 1in polyadenylated RNA. The position of the primers is indicated on the diagram above the gels. GAPDH was used as a loading control. b Correctly spliced transcripts detected in controls and expansion carrier cells using primers in exons 1a and 2. c Quantitative analysis of intron retention by real-time PCR. Levels of C9orf72 transcripts spliced or unspliced at the 5’ and 3’ end of intron 1 were determined by real-time qRT-PCR. Data are shown as means ± SEM. Each data point represents an individual case, n =15 (C9 - ); 15 (C9 + ). No significant differences were observed between the C9 − and C9 + groups. d Sequencing of the 3’ PCR product demonstrates an exact intron 1-exon 2 boundary. C9 − , controls; C9 + , expansion carriers

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

    59) Product Images from "Shorter Telomeres in Peripheral Blood Mononuclear Cells from Older Persons with Sarcopenia: Results from an Exploratory Study"

    Article Title: Shorter Telomeres in Peripheral Blood Mononuclear Cells from Older Persons with Sarcopenia: Results from an Exploratory Study

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2014.00233

    Scatter plot of telomere/single-copy gene ratio ( T/S ) and the skeletal muscle index ( n = 142) .
    Figure Legend Snippet: Scatter plot of telomere/single-copy gene ratio ( T/S ) and the skeletal muscle index ( n = 142) .

    Techniques Used:

    60) Product Images from "Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer"

    Article Title: Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083187

    Evaluation of LASP-1 expression in OSCC-derived cell lines. (A) Quantification of LASP-1 mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. mRNA expression levels are normalized to GAPDH. Significant up-regulation of LASP-1 mRNA is seen in seven OSCC-derived cell lines compared with that in HNOKs. Data are expressed as the means ± SEM of triplicate results (* P
    Figure Legend Snippet: Evaluation of LASP-1 expression in OSCC-derived cell lines. (A) Quantification of LASP-1 mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. mRNA expression levels are normalized to GAPDH. Significant up-regulation of LASP-1 mRNA is seen in seven OSCC-derived cell lines compared with that in HNOKs. Data are expressed as the means ± SEM of triplicate results (* P

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

    61) Product Images from "p53-Independent Cell Cycle and Erythroid Differentiation Defects in Murine Embryonic Stem Cells Haploinsufficient for Diamond Blackfan Anemia-Proteins: RPS19 versus RPL5"

    Article Title: p53-Independent Cell Cycle and Erythroid Differentiation Defects in Murine Embryonic Stem Cells Haploinsufficient for Diamond Blackfan Anemia-Proteins: RPS19 versus RPL5

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089098

    The differentiation defects observed in Rps19 and Rpl5 mutants are nonspecifically rescued by p53 inhibition. (A) Western blot analyses were performed from mutant ES cells with antibodies against p53, using β-Actin as a loading control. ES cells from the Rps19 mutant cells showed an increase in p53 expression. In contrast, the Rpl5 mutant expressed no increase in p53, compared with the parental line. Image J quantification of western blots from 3 independent experiments demonstrated that the Rps19 mutant ES cells had approximately a 4-fold increase in p53 protein compared to the wild type cells. (B) qRT-PCR performed on these ES cells showed an increase in p21 mRNA only in the Rps19 mutant ES cells (3 independent experiments) while there was no similar increase in the Rpl5 mutant ES cells (4 independent experiments). siRNA targeting p53 was used to transiently transfect ES cells 24 hours prior to primary differentiation, obtaining > 90% p53 knockdown by qRT-PCR. Both mutants (C) showed a significant increase in EB formation with p53 knockdown (4 independent pooled experiments). This effect was nonspecific, as p53 knockdown of parental cells also increased EB formation (D). The primitive erythroid colony defect was partially compensated in the Rps19 mutant after p53 inhibition and overcompensated in the Rpl5 mutant (E) (3 independent pooled experiments). This augmentation of colony formation was again nonspecific, as there was an increase in primitive colony formation with p53 knockdown in both parental ES cells when compared with the control siRNA (3 independent pooled experiments for Rpl5 parent and 4 independent experiments for Rps19 parent) (F).
    Figure Legend Snippet: The differentiation defects observed in Rps19 and Rpl5 mutants are nonspecifically rescued by p53 inhibition. (A) Western blot analyses were performed from mutant ES cells with antibodies against p53, using β-Actin as a loading control. ES cells from the Rps19 mutant cells showed an increase in p53 expression. In contrast, the Rpl5 mutant expressed no increase in p53, compared with the parental line. Image J quantification of western blots from 3 independent experiments demonstrated that the Rps19 mutant ES cells had approximately a 4-fold increase in p53 protein compared to the wild type cells. (B) qRT-PCR performed on these ES cells showed an increase in p21 mRNA only in the Rps19 mutant ES cells (3 independent experiments) while there was no similar increase in the Rpl5 mutant ES cells (4 independent experiments). siRNA targeting p53 was used to transiently transfect ES cells 24 hours prior to primary differentiation, obtaining > 90% p53 knockdown by qRT-PCR. Both mutants (C) showed a significant increase in EB formation with p53 knockdown (4 independent pooled experiments). This effect was nonspecific, as p53 knockdown of parental cells also increased EB formation (D). The primitive erythroid colony defect was partially compensated in the Rps19 mutant after p53 inhibition and overcompensated in the Rpl5 mutant (E) (3 independent pooled experiments). This augmentation of colony formation was again nonspecific, as there was an increase in primitive colony formation with p53 knockdown in both parental ES cells when compared with the control siRNA (3 independent pooled experiments for Rpl5 parent and 4 independent experiments for Rps19 parent) (F).

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

    62) Product Images from "Unaltered Prion Pathogenesis in a Mouse Model of High-Fat Diet-Induced Insulin Resistance"

    Article Title: Unaltered Prion Pathogenesis in a Mouse Model of High-Fat Diet-Induced Insulin Resistance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0144983

    Dysregulated insulin signaling but unchanged PrP C expression in HFD-fed mouse brains. ( A ) Left: Western blot of p Ser473 -Akt and total Akt in ND- or HFD-fed mouse brains. Right: densitometric quantification of the Western blot showed significantly lower p Ser473 -Akt/Akt ratio in HFD-fed mouse brains (n = 3, *, p = 0.0280) ( B ) Left: Western blot of p Ser9 -GSK3β and total GSK3β in ND- or HFD-fed mouse brains. Right: densitometric quantification of the Western blot revealed a trend of reduced lower p Ser9 -GSK3β/ GSK3β ratio in HFD-fed mouse brains (n = 3, n.s p = 0.0890). ( C ) qRT-PCR of Prnp expression in HFD-fed and ND-fed mouse brains showed similar levels of Prnp mRNA (n = 3, n.s p > 0.05). ( D ) Left: Western blot of PrP C in HFD-fed and ND-fed mouse brains. Right: densitometric quantification of the Western blot demonstrated a similar level of PrP C expression (n = 3, n.s p > 0.05). (E) ELISA of PrP C showed similar level of PrP C expression in HFD-fed and ND-fed mouse brains (n = 3, n.s p > 0.05).
    Figure Legend Snippet: Dysregulated insulin signaling but unchanged PrP C expression in HFD-fed mouse brains. ( A ) Left: Western blot of p Ser473 -Akt and total Akt in ND- or HFD-fed mouse brains. Right: densitometric quantification of the Western blot showed significantly lower p Ser473 -Akt/Akt ratio in HFD-fed mouse brains (n = 3, *, p = 0.0280) ( B ) Left: Western blot of p Ser9 -GSK3β and total GSK3β in ND- or HFD-fed mouse brains. Right: densitometric quantification of the Western blot revealed a trend of reduced lower p Ser9 -GSK3β/ GSK3β ratio in HFD-fed mouse brains (n = 3, n.s p = 0.0890). ( C ) qRT-PCR of Prnp expression in HFD-fed and ND-fed mouse brains showed similar levels of Prnp mRNA (n = 3, n.s p > 0.05). ( D ) Left: Western blot of PrP C in HFD-fed and ND-fed mouse brains. Right: densitometric quantification of the Western blot demonstrated a similar level of PrP C expression (n = 3, n.s p > 0.05). (E) ELISA of PrP C showed similar level of PrP C expression in HFD-fed and ND-fed mouse brains (n = 3, n.s p > 0.05).

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Similar level of astrogliosis and microglial activation in terminally sick HFD- and ND-fed tg a 20 mice. ( A ) Representative immunohistochemical staining for GFAP and Iba1 in brains of terminally sick HFD-fed and ND-fed tg a 20 mice. There was no detectable difference between the two groups in astrogliosis or microglial activation. Scale bar = 100μm. ( B ) Left: Western blot for Iba1 in terminally sick mouse brains. Right: densitometric quantification of the Western blot revealed no significant difference of Iba1 levels between HFD-fed and ND-fed tg a 20 mouse brains (n = 3; n.s.: p > 0.05). ( C ) Left: Western blot for GFAP in terminally sick mouse brains. Right: densitometric quantification of the Western blot showed no significant difference of GFAP levels between HFD-fed and ND-fed tg a 20 mouse brains (n = 3; n.s.: p > 0.05). ( D ) qRT-PCR of cytokines TNFα, IL-6 and IL-1β expression revealed similar expression levels of these cytokines in terminally sick HFD-fed and ND-fed mouse brains (n = 3; n.s.: p > 0.05).
    Figure Legend Snippet: Similar level of astrogliosis and microglial activation in terminally sick HFD- and ND-fed tg a 20 mice. ( A ) Representative immunohistochemical staining for GFAP and Iba1 in brains of terminally sick HFD-fed and ND-fed tg a 20 mice. There was no detectable difference between the two groups in astrogliosis or microglial activation. Scale bar = 100μm. ( B ) Left: Western blot for Iba1 in terminally sick mouse brains. Right: densitometric quantification of the Western blot revealed no significant difference of Iba1 levels between HFD-fed and ND-fed tg a 20 mouse brains (n = 3; n.s.: p > 0.05). ( C ) Left: Western blot for GFAP in terminally sick mouse brains. Right: densitometric quantification of the Western blot showed no significant difference of GFAP levels between HFD-fed and ND-fed tg a 20 mouse brains (n = 3; n.s.: p > 0.05). ( D ) qRT-PCR of cytokines TNFα, IL-6 and IL-1β expression revealed similar expression levels of these cytokines in terminally sick HFD-fed and ND-fed mouse brains (n = 3; n.s.: p > 0.05).

    Techniques Used: Activation Assay, Mouse Assay, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR, Expressing

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

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

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery432

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

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

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

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

    64) Product Images from "Collagen triple helix repeat containing-1 negatively regulated by microRNA-30c promotes cell proliferation and metastasis and indicates poor prognosis in breast cancer"

    Article Title: Collagen triple helix repeat containing-1 negatively regulated by microRNA-30c promotes cell proliferation and metastasis and indicates poor prognosis in breast cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0564-7

    CTHRC1 is frequently up-regulated in human breast cancer cells and tissues. a and b CTHRC1 was up-regulated in human breast cancer cells analyzed by qRT-PCR and western blot. * P
    Figure Legend Snippet: CTHRC1 is frequently up-regulated in human breast cancer cells and tissues. a and b CTHRC1 was up-regulated in human breast cancer cells analyzed by qRT-PCR and western blot. * P

    Techniques Used: Quantitative RT-PCR, Western Blot

    CTHRC1 is a direct target of miR-30c. a qRT-PCR analysis of CTHRC1 mRNA expression in indicated cells 24 h post-transfection. ** P
    Figure Legend Snippet: CTHRC1 is a direct target of miR-30c. a qRT-PCR analysis of CTHRC1 mRNA expression in indicated cells 24 h post-transfection. ** P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection

    CTHRC1 and miR-30c expression are inversely correlated in human breast cancer cells and tissues. a The relative expression level of miR-134, miR-155, miR-30c and miR-630 in breast cancer cells respectively was detected by qRT-PCR. * P
    Figure Legend Snippet: CTHRC1 and miR-30c expression are inversely correlated in human breast cancer cells and tissues. a The relative expression level of miR-134, miR-155, miR-30c and miR-630 in breast cancer cells respectively was detected by qRT-PCR. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    65) Product Images from "Synthetic regulatory RNAs selectively suppress the progression of bladder cancer"

    Article Title: Synthetic regulatory RNAs selectively suppress the progression of bladder cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0626-x

    The expression of MYC was upregulated in bladder cancer tissues and cells, and siRNA was able to knockdown the expression of this gene in cells. ( a ) qRT-PCR was used to detect the expression of MYC in bladder cancer tissues. ΔCT N means comparative CT in normal tissue. ΔCT C represents comparative CT in tumor tissue. ( b, c ) Compared with the negative control, the expression of MYC mRNA was significantly increased in bladder cancer tissues ( b , P
    Figure Legend Snippet: The expression of MYC was upregulated in bladder cancer tissues and cells, and siRNA was able to knockdown the expression of this gene in cells. ( a ) qRT-PCR was used to detect the expression of MYC in bladder cancer tissues. ΔCT N means comparative CT in normal tissue. ΔCT C represents comparative CT in tumor tissue. ( b, c ) Compared with the negative control, the expression of MYC mRNA was significantly increased in bladder cancer tissues ( b , P

    Techniques Used: Expressing, Quantitative RT-PCR, Negative Control

    Effects of synthetic regulatory RNAs on luciferase activity, and MYC mRNA and protein levels. ( a, b and c ) When aRNA was not expressed in the three cell lines, the luciferase activity was significantly decreased between the NC iRNA group and iRNA group in T24, 5637 and HFF. However, the luciferase activity was still significantly decreased in HFF ( a ) after addition of aRNA in cells between these two groups. But the luciferase activity was no statistical difference in T24 ( b ) and 5637 ( c ). ( d, e, f, g, h and i ) Without aRNA, there was no difference between column 1 and 2 in all three cell lines and therefore the transcriptional and translational levels of MYC was not inhibited. After the cells were transfected with aRNA as shown in T24 and 5637, the mRNA and protein levels of MYC was largely reduced by expression of MYC amiRNA (column 4 in E, F, H and I) compared with the negative group (column 3 in e, f, h and i ). However, there was no significant difference between column 3 and 4 ( d and g ) in HFF. All data are presented as the mean ± SD (* p
    Figure Legend Snippet: Effects of synthetic regulatory RNAs on luciferase activity, and MYC mRNA and protein levels. ( a, b and c ) When aRNA was not expressed in the three cell lines, the luciferase activity was significantly decreased between the NC iRNA group and iRNA group in T24, 5637 and HFF. However, the luciferase activity was still significantly decreased in HFF ( a ) after addition of aRNA in cells between these two groups. But the luciferase activity was no statistical difference in T24 ( b ) and 5637 ( c ). ( d, e, f, g, h and i ) Without aRNA, there was no difference between column 1 and 2 in all three cell lines and therefore the transcriptional and translational levels of MYC was not inhibited. After the cells were transfected with aRNA as shown in T24 and 5637, the mRNA and protein levels of MYC was largely reduced by expression of MYC amiRNA (column 4 in E, F, H and I) compared with the negative group (column 3 in e, f, h and i ). However, there was no significant difference between column 3 and 4 ( d and g ) in HFF. All data are presented as the mean ± SD (* p

    Techniques Used: Luciferase, Activity Assay, Transfection, Expressing

    A schematic of the mechanism of synthetic regulatory RNAs. The mutant hTERT promoter drives the expression of aRNA. The amiRNA targeting MYC is driven by UAS. In HFF cells, aRNA cannot be expressed. The iRNA interacts with UAS so GAL4-VP64 cannot bind UAS to activate the expression of amiRNA. Artificial miRNAs can generate miRNAs that interact with the 3′ UTR of MYC mRNA completely and causes MYC mRNA degradation. However, aRNA is overexpressed in bladder cancer cells. The aRNA can interact with iRNA so UAS is exposed to GAL4-VP64 again. Thus, MYC amiRNA will be driven by the UAS and GAL4-VP64 complex
    Figure Legend Snippet: A schematic of the mechanism of synthetic regulatory RNAs. The mutant hTERT promoter drives the expression of aRNA. The amiRNA targeting MYC is driven by UAS. In HFF cells, aRNA cannot be expressed. The iRNA interacts with UAS so GAL4-VP64 cannot bind UAS to activate the expression of amiRNA. Artificial miRNAs can generate miRNAs that interact with the 3′ UTR of MYC mRNA completely and causes MYC mRNA degradation. However, aRNA is overexpressed in bladder cancer cells. The aRNA can interact with iRNA so UAS is exposed to GAL4-VP64 again. Thus, MYC amiRNA will be driven by the UAS and GAL4-VP64 complex

    Techniques Used: Mutagenesis, Expressing

    66) Product Images from "STAT3-Mediated Transcriptional Regulation of Osteopontin in STAT3 Loss-of-Function Related Hyper IgE Syndrome"

    Article Title: STAT3-Mediated Transcriptional Regulation of Osteopontin in STAT3 Loss-of-Function Related Hyper IgE Syndrome

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01080

    (A) Representative flow cytometry histogram of STAT3 protein expression in PC-3 and LNCaP cells. PC-3 cells had no expression of STAT3 protein. (B) STAT3 mRNA expression in PC3, LNCaP, hyper-IgE syndrome (HIES) subjects and healthy controls (HCs) by conventional PCR. PC-3 cells showed no expression of STAT3 mRNA. (C) Agarose gel pictures showing either absence or non-specific/extremely faint bands of STAT3 amplification (exons 9–22) in PC-3 cells. Gel 1 (Exons 9–10/12–24/15 and 16–17), gel 2 (exon 11), and gel 3 (exons 18-20/21/22-23) have been grouped together for representation purpose. (D) Scatter plot showing per cent STAT3 phosphorylation in HCs, HIES subjects, LNCaP, and PC3 cells; mean values are shown as horizontal bars. Bar graphs showing quantitative real time PCR (qRT-PCR) of SOCS3 mRNA expression in HC peripheral blood mononuclear cells stimulated with IL-6 in a (E) time-dependent manner at 30 ng/ml and (F) dose-dependent manner for 2 h. Data are shown as mean ± SD for three independent experiments. (G) Bar graphs showing qRT-PCR results of SOCS3 expression from HCs, HIES subjects, LNCaP, and PC-3 cells stimulated with IL-6. The primary transcript for each gene were assayed at least in duplicate and normalized to β-actin expression. (Data are represented as mean ± SD, * p
    Figure Legend Snippet: (A) Representative flow cytometry histogram of STAT3 protein expression in PC-3 and LNCaP cells. PC-3 cells had no expression of STAT3 protein. (B) STAT3 mRNA expression in PC3, LNCaP, hyper-IgE syndrome (HIES) subjects and healthy controls (HCs) by conventional PCR. PC-3 cells showed no expression of STAT3 mRNA. (C) Agarose gel pictures showing either absence or non-specific/extremely faint bands of STAT3 amplification (exons 9–22) in PC-3 cells. Gel 1 (Exons 9–10/12–24/15 and 16–17), gel 2 (exon 11), and gel 3 (exons 18-20/21/22-23) have been grouped together for representation purpose. (D) Scatter plot showing per cent STAT3 phosphorylation in HCs, HIES subjects, LNCaP, and PC3 cells; mean values are shown as horizontal bars. Bar graphs showing quantitative real time PCR (qRT-PCR) of SOCS3 mRNA expression in HC peripheral blood mononuclear cells stimulated with IL-6 in a (E) time-dependent manner at 30 ng/ml and (F) dose-dependent manner for 2 h. Data are shown as mean ± SD for three independent experiments. (G) Bar graphs showing qRT-PCR results of SOCS3 expression from HCs, HIES subjects, LNCaP, and PC-3 cells stimulated with IL-6. The primary transcript for each gene were assayed at least in duplicate and normalized to β-actin expression. (Data are represented as mean ± SD, * p

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    67) Product Images from "Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids"

    Article Title: Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids

    Journal: Scientific Reports

    doi: 10.1038/srep09378

    TFEB and MTOR expression analysis in HDFa and MPS cells via real-time qRT-PCR. Data represent averaged values ± SD from n = 3, and mean significant differences for samples treated for 24 h with various flavonoids (100 µ M genistein, 100 µ M kaempferol, and mixtures of them of 30 µ M each) against non-treated, with respect to endogenous reference gene GAPDH (in HDFa) and RPLPO (in MPS II), with p
    Figure Legend Snippet: TFEB and MTOR expression analysis in HDFa and MPS cells via real-time qRT-PCR. Data represent averaged values ± SD from n = 3, and mean significant differences for samples treated for 24 h with various flavonoids (100 µ M genistein, 100 µ M kaempferol, and mixtures of them of 30 µ M each) against non-treated, with respect to endogenous reference gene GAPDH (in HDFa) and RPLPO (in MPS II), with p

    Techniques Used: Expressing, Quantitative RT-PCR

    68) Product Images from "Gene Silencing of TGFβRII Can Inhibit Glioblastoma Cell Growth"

    Article Title: Gene Silencing of TGFβRII Can Inhibit Glioblastoma Cell Growth

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    doi: 10.22034/APJCP.2018.19.9.2681

    The U-373 MG Tumor Cells Transfected with siRNA (40, 60, and 80 pmol). Total RNA was extracted and mRNA was analyzed via qRT-PCR at 48 hours after transfection. The relative expression of mRNA was determined with 2-(ΔΔCT) formula (β-actin, internal control) (data presented as mean±SD; *P
    Figure Legend Snippet: The U-373 MG Tumor Cells Transfected with siRNA (40, 60, and 80 pmol). Total RNA was extracted and mRNA was analyzed via qRT-PCR at 48 hours after transfection. The relative expression of mRNA was determined with 2-(ΔΔCT) formula (β-actin, internal control) (data presented as mean±SD; *P

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing

    The U-373 MG Tumor Cells Transfected with TGFβ RII siRNA. Total RNA was extracted and mRNA was analyzed via qRT-PCR assay at 24, 48, and 72 hours following transfection. The relative mRNA expression was quantified by 2-(ΔΔCT) formula (β-actin, internal control) (data presented as mean±SD; *P
    Figure Legend Snippet: The U-373 MG Tumor Cells Transfected with TGFβ RII siRNA. Total RNA was extracted and mRNA was analyzed via qRT-PCR assay at 24, 48, and 72 hours following transfection. The relative mRNA expression was quantified by 2-(ΔΔCT) formula (β-actin, internal control) (data presented as mean±SD; *P

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing

    69) Product Images from "Lowly Expressed Ribosomal Protein S19 in the Feces of Patients with Colorectal Cancer"

    Article Title: Lowly Expressed Ribosomal Protein S19 in the Feces of Patients with Colorectal Cancer

    Journal: ISRN Gastroenterology

    doi: 10.5402/2012/394545

    Efficiency of RPS19 silence in colonic cells by RNA interference. RPS19 silence is achieved by the lentivirus-mediated RNAi experiment. Relative mRNA levels of RPS19 are quantified by qRT-PCR with TaqMan probes and normalized by individual level of 18 s rRNA. The relative expression level of shLuc-infected cells is considered as 1. Results are representative of those obtained in two-to-three separate experiments with error bars showing standard error. Changes of protein levels are immunoblotted with antibodies against RPS19 and β -actin (in black square).
    Figure Legend Snippet: Efficiency of RPS19 silence in colonic cells by RNA interference. RPS19 silence is achieved by the lentivirus-mediated RNAi experiment. Relative mRNA levels of RPS19 are quantified by qRT-PCR with TaqMan probes and normalized by individual level of 18 s rRNA. The relative expression level of shLuc-infected cells is considered as 1. Results are representative of those obtained in two-to-three separate experiments with error bars showing standard error. Changes of protein levels are immunoblotted with antibodies against RPS19 and β -actin (in black square).

    Techniques Used: Quantitative RT-PCR, Expressing, Infection

    Changes of BAX expression in RPS19-silent colonic cells. Relative mRNA levels of BAX are quantified by qRT-PCR with TaqMan probes and normalized by individual level of 18 s rRNA. The relative expression level of shLuc-infected cells is considered as 1. Results are representative of those obtained in one experiment.
    Figure Legend Snippet: Changes of BAX expression in RPS19-silent colonic cells. Relative mRNA levels of BAX are quantified by qRT-PCR with TaqMan probes and normalized by individual level of 18 s rRNA. The relative expression level of shLuc-infected cells is considered as 1. Results are representative of those obtained in one experiment.

    Techniques Used: Expressing, Quantitative RT-PCR, Infection

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

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

    Journal: Nature

    doi: 10.1038/nature13322

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

    Techniques Used: Expressing, Activation Assay, Activity Assay

    71) Product Images from "Ezrin Mediates Tethering of the γ-Aminobutyric Acid Transporter GAT1 to Actin Filaments Via a C-Terminal PDZ-Interacting Domain"

    Article Title: Ezrin Mediates Tethering of the γ-Aminobutyric Acid Transporter GAT1 to Actin Filaments Via a C-Terminal PDZ-Interacting Domain

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2008.11.070

    Schematic of GAT1 interactions with Pals1, ezrin, and actin. ( A ) Schematic of GAT1-YFP8 interaction with actin via ezrin and Pals1. The additional PDZ domain after YFP moiety restores the interaction between GAT1 and the PDZ protein. ( B ) The addition of 5 μ M latrunculin B disrupts actin, and reduces the interaction between ezrin and GAT1 either by ( B1 ) pulling away the GAT1-PDZ interaction, or ( B2 ) reducing the interaction between ezrin and the PDZ protein. Altogether, this increases the mobility of GAT1 on the membrane. ( C ) GAT1 0 -GFP, a GAT1 with a disrupted PDZ domain, is more mobile on the membrane, indicating that this domain may also stabilize the GAT1-ezrin-actin interactions.
    Figure Legend Snippet: Schematic of GAT1 interactions with Pals1, ezrin, and actin. ( A ) Schematic of GAT1-YFP8 interaction with actin via ezrin and Pals1. The additional PDZ domain after YFP moiety restores the interaction between GAT1 and the PDZ protein. ( B ) The addition of 5 μ M latrunculin B disrupts actin, and reduces the interaction between ezrin and GAT1 either by ( B1 ) pulling away the GAT1-PDZ interaction, or ( B2 ) reducing the interaction between ezrin and the PDZ protein. Altogether, this increases the mobility of GAT1 on the membrane. ( C ) GAT1 0 -GFP, a GAT1 with a disrupted PDZ domain, is more mobile on the membrane, indicating that this domain may also stabilize the GAT1-ezrin-actin interactions.

    Techniques Used:

    Determination of expression of ezrin and Pals1 in N2a cells by qRT-PCR, and an interaction between ezrin and GAT1-YFP8 by FRET. ( A ) mRNA levels of β -actin, γ -actin, ezrin, and Pals1, normalized to β -actin expression. One-step qRT-PCR shows that ezrin is expressed in N2a cells at levels similar to those of PDZ protein Pals1. ( B – D ) FRET results. Prebleaching and postbleaching images of respective CFP and YFP fused proteins. Images are processed using Image J software with the enhance-image plugin (Rasband, W.S.; US National Institutes of Health, Bethesda, MD). Scale bar, 5 μ m. ( E ) GAT1-YFP8 and ezrin-CFP interact, as represented by the 25% ± 3% increase in ezrin-CFP fluorescence that accompanies photodestruction of GAT1-YFP8. The disruption of actin through the addition of 5 μ m latrunculin B significantly decreased FRET between ezrin-CFP and GAT1-YFP8. As a positive control, FRET between YFP-ezrin and ezrin-CFP was performed, resulting in a 26% ± 3% increase in ezrin-CFP fluorescence that accompanied the photodestruction of YFP-ezrin. ( F ) FRET efficiency for ezrin-CFP/GAT1-YFP8 is 19% ± 2%, for ezrin-CFP/GAT1-YFP8 + latrunculin B it is 7% ± 5%, and for ezrin-CFP/YFP-ezrin it is 20% ± 2%. Values are represented as the mean ± SE of 23 replicates. Significance was determined by one-way analysis of variance with Tukey's post hoc test ( p
    Figure Legend Snippet: Determination of expression of ezrin and Pals1 in N2a cells by qRT-PCR, and an interaction between ezrin and GAT1-YFP8 by FRET. ( A ) mRNA levels of β -actin, γ -actin, ezrin, and Pals1, normalized to β -actin expression. One-step qRT-PCR shows that ezrin is expressed in N2a cells at levels similar to those of PDZ protein Pals1. ( B – D ) FRET results. Prebleaching and postbleaching images of respective CFP and YFP fused proteins. Images are processed using Image J software with the enhance-image plugin (Rasband, W.S.; US National Institutes of Health, Bethesda, MD). Scale bar, 5 μ m. ( E ) GAT1-YFP8 and ezrin-CFP interact, as represented by the 25% ± 3% increase in ezrin-CFP fluorescence that accompanies photodestruction of GAT1-YFP8. The disruption of actin through the addition of 5 μ m latrunculin B significantly decreased FRET between ezrin-CFP and GAT1-YFP8. As a positive control, FRET between YFP-ezrin and ezrin-CFP was performed, resulting in a 26% ± 3% increase in ezrin-CFP fluorescence that accompanied the photodestruction of YFP-ezrin. ( F ) FRET efficiency for ezrin-CFP/GAT1-YFP8 is 19% ± 2%, for ezrin-CFP/GAT1-YFP8 + latrunculin B it is 7% ± 5%, and for ezrin-CFP/YFP-ezrin it is 20% ± 2%. Values are represented as the mean ± SE of 23 replicates. Significance was determined by one-way analysis of variance with Tukey's post hoc test ( p

    Techniques Used: Expressing, Quantitative RT-PCR, Software, Fluorescence, Positive Control

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077306

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

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

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

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

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

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

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

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

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

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

    73) Product Images from "Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes"

    Article Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes

    Journal: Biochemistry

    doi: 10.1021/bi501388y

    Functional implications from d -Ala- d -Ala TRAP SBPs. (A) Genome environment of the three TRAP SBPs that had DSF hits on the dipeptide d -Ala- d -Ala. Genes putatively assigned for the transport and catabolic degradation of d -Ala- d -Ala are shown in color. (B) Interactions of Csal_0660 with d -Ala- d -Ala. Hydrogen bonds are shown as dashed lines, and hydrophobic contacts are represented by an arc with spokes radiating toward the ligand atoms that they contact. (C) 1 H NMR verification of CsVanX (Csal_0663) dipeptisase activity on d -Ala- d -Ala. The control spectrum is show on the bottom, and the reaction is shown on top (glycerol from the enzyme prep is present between 3.6 and 3.4 ppm). Insets show magnifications of the control and reaction peaks. (D) Fold change in transcript measured by qRT-PCR for Csal_0660 genome neighborhood related genes when C. salexigens is grown on d -Ala- d -Ala versus d -glucose as a carbon source. (E) Growth curves of wild-type C. salexigens versus a deletion mutant of the d -Ala- d -Ala TRAP SBP (ΔCsDctP) or deletion mutant of the d -Ala- d -Ala dipeptidase (ΔCsVanX).
    Figure Legend Snippet: Functional implications from d -Ala- d -Ala TRAP SBPs. (A) Genome environment of the three TRAP SBPs that had DSF hits on the dipeptide d -Ala- d -Ala. Genes putatively assigned for the transport and catabolic degradation of d -Ala- d -Ala are shown in color. (B) Interactions of Csal_0660 with d -Ala- d -Ala. Hydrogen bonds are shown as dashed lines, and hydrophobic contacts are represented by an arc with spokes radiating toward the ligand atoms that they contact. (C) 1 H NMR verification of CsVanX (Csal_0663) dipeptisase activity on d -Ala- d -Ala. The control spectrum is show on the bottom, and the reaction is shown on top (glycerol from the enzyme prep is present between 3.6 and 3.4 ppm). Insets show magnifications of the control and reaction peaks. (D) Fold change in transcript measured by qRT-PCR for Csal_0660 genome neighborhood related genes when C. salexigens is grown on d -Ala- d -Ala versus d -glucose as a carbon source. (E) Growth curves of wild-type C. salexigens versus a deletion mutant of the d -Ala- d -Ala TRAP SBP (ΔCsDctP) or deletion mutant of the d -Ala- d -Ala dipeptidase (ΔCsVanX).

    Techniques Used: Functional Assay, Nuclear Magnetic Resonance, Activity Assay, Quantitative RT-PCR, Mutagenesis

    74) Product Images from "FAS system deregulation in T-cell lymphoblastic lymphoma"

    Article Title: FAS system deregulation in T-cell lymphoblastic lymphoma

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.83

    FAS mRNA expression level in RNA samples. qRT-PCR determining FAS mRNA expression levels in human T-LBL samples where only RNA was available. The mean values were normalized to that of JK. Error bars represent S.D. Data represent two independent experiments. *Denotes statistical significance compared with the JK control, with P
    Figure Legend Snippet: FAS mRNA expression level in RNA samples. qRT-PCR determining FAS mRNA expression levels in human T-LBL samples where only RNA was available. The mean values were normalized to that of JK. Error bars represent S.D. Data represent two independent experiments. *Denotes statistical significance compared with the JK control, with P

    Techniques Used: Expressing, Quantitative RT-PCR

    75) Product Images from "Transcriptome Analysis of ABA/JA-Dual Responsive Genes in Rice Shoot and Root"

    Article Title: Transcriptome Analysis of ABA/JA-Dual Responsive Genes in Rice Shoot and Root

    Journal: Current Genomics

    doi: 10.2174/1389202918666170228134205

    Validation of ABA- and JA-regulated genes using qRT-PCR. The ΔΔCT value method was used to determine the relative fold changes. All data were normalized to the expression level of Ubi5 . Error bars represent standard error of three replicates.
    Figure Legend Snippet: Validation of ABA- and JA-regulated genes using qRT-PCR. The ΔΔCT value method was used to determine the relative fold changes. All data were normalized to the expression level of Ubi5 . Error bars represent standard error of three replicates.

    Techniques Used: Quantitative RT-PCR, Expressing

    Validation of ABA regulation of genes expressed in root and shoot using qRT-PCR. The ΔΔCT method was used to determine the relative fold change. All data were normalized to the expression level of Ubi5 . Error bars represent standard error of three replicates.
    Figure Legend Snippet: Validation of ABA regulation of genes expressed in root and shoot using qRT-PCR. The ΔΔCT method was used to determine the relative fold change. All data were normalized to the expression level of Ubi5 . Error bars represent standard error of three replicates.

    Techniques Used: Quantitative RT-PCR, Expressing

    Related Articles

    Clone Assay:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: Site-directed mutagenesis (QuickChange, Stratagene) was utilized so that the DNA sequence of wild-type SP-A1 and SP-A2 clones exactly matched and , respectively. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    Centrifugation:

    Article Title: Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
    Article Snippet: To monitor the association between SERCA and Bcl-2, 150 μg of SR in lysis buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM EDTA, 1% Nonidet P40, 1 mM PMSF and protease inhibitors (Roche Diagnostics) was incubated with 3 μg of GST–Bcl-2 or GST alone for 2 h at 4 °C. .. After the incubation with 10 μl (packed volume) of glutathione–Sepharose beads for 2 h, the beads were separated by centrifugation at 7500 g for 10 min and washed six times with the lysis buffer.

    Amplification:

    Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
    Article Snippet: RT-qPCR was utilized to evaluate the expression of lncRNA, miRNA, and mRNA in the serum samples or cells on the Roche LightCycler 480 (Roche, Switzerland). .. The amplification of the appropriate product was confirmed by melting curve analysis.

    Article Title: Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression
    Article Snippet: Real-time quantitative RT-PCR The angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF cDNA were analyzed using a fluorescein quantitative real-time PCR detection system (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). .. For Amplification was followed by melting curve analysis to verify the correctness of the amplicon.

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: Full-length human SFTPA1 cDNA was PCR amplified from clone LIFESEQ90096303 (Open Biosystems, Waltham, MA) and subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) downstream of the CMV promoter. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    Cell Cycle Assay:

    Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
    Article Snippet: Paragraph title: Cell-cycle analysis ... MM cells were exposed to DMSO or 0.5 to 1μM of MLN8237 for 24 to 72 hours, permeabilized by 70% ethanol at −20°C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A (Roche Diagnostics).

    Cytometry:

    Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
    Article Snippet: MM cells were exposed to DMSO or 0.5 to 1μM of MLN8237 for 24 to 72 hours, permeabilized by 70% ethanol at −20°C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A (Roche Diagnostics). .. DNA content was analyzed by flow cytometry using BDFACS-Canto II (BD Biosciences) and FlowJo software.

    Quantitative RT-PCR:

    Article Title: Prospective Evaluation of Whole Genome MicroRNA Expression Profiling in Childhood Acute Lymphoblastic Leukemia
    Article Snippet: Quantitative Real Time Polymerase Chain Reaction and Validation Significantly dysregulated miRNAs were validated by Quantitative PCR (LightCycler 480, Roche Applied Science, Mannheim, Germany) after comparing the BM microarray miRNA expressions of patients with the controls. .. A master mix was designed for each primer set in accordance with the recommendations of the real time RT-PCR setup for “individual assays,” suggested in the kit.

    Article Title: Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer
    Article Snippet: .. Real-time quantitative polymerase chain reaction (QRT-PCR) All reagents and equipment for mRNA and cDNA preparation were purchased from Roche (Roche Applied Science, Mannheim, Germany). mRNA was prepared by automated isolation using the MagNA Pure LC instrument and isolation Kit I (for cells) and Kit II (for tissues). .. RNA was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) according to the manufacturer's instructions.

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

    Article Title: Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2
    Article Snippet: .. Quantitative PCR Since in silico analysis showed HOXA13 and HOXA9 homeodomains were very similar (75,4% of similarity; 57,9% of identity, score: 274; analysis performed with EMBOSS Matcher program 6.6.0 http://www.ebi.ac.uk/Tools/psa/emboss_matcher/ , matrix: BLOSUM80, gap penality: 14, extended penality: 4), we hypothesized NUP98/HOXA13 could bind MSI2 promoter and tested whether HOXA9 was involved in the present patient. qRT-PCR (LightCycler480, Roche Diagnostics, Germany) was performed using TaqMan assay probes (Applied Biosystems, Foster City, CA) Hs00292670_m for MSI2 gene, Hs00365956_m1 for HOXA9 and Hs00426284_m1 for HOXA13 . ..

    Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
    Article Snippet: .. RT-qPCR was utilized to evaluate the expression of lncRNA, miRNA, and mRNA in the serum samples or cells on the Roche LightCycler 480 (Roche, Switzerland). .. The amplification of the appropriate product was confirmed by melting curve analysis.

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice
    Article Snippet: .. RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands). .. Quantitative real-time PCR (qRT-PCR) was performed with SYBR green (Promega) on a CFX96 PCR Machine (Bio-Rad, Veenendaal, The Netherlands). mRNA expression levels were normalized to β2-microglobulin or glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) and hypoxanthine guanine phosphoribosyl transferase (Hprt ) as reference genes.

    Article Title: Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression
    Article Snippet: .. Real-time quantitative RT-PCR The angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF cDNA were analyzed using a fluorescein quantitative real-time PCR detection system (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). ..

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells
    Article Snippet: .. Real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cultured cells using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and DNase I (Roche Diagnostics). .. Real-time qRT-PCR was performed using a QuantiTect SYBR Green Reverse Transcription-PCR Kit (Qiagen, Tokyo, Japan) and a Chromo4 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).

    SYBR Green Assay:

    Article Title: Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer
    Article Snippet: Real-time quantitative polymerase chain reaction (QRT-PCR) All reagents and equipment for mRNA and cDNA preparation were purchased from Roche (Roche Applied Science, Mannheim, Germany). mRNA was prepared by automated isolation using the MagNA Pure LC instrument and isolation Kit I (for cells) and Kit II (for tissues). .. QRT-PCR was performed with the Light Cycler Fast Start DNA SYBR Green kit as described previously [ ].

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

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice
    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands). .. Quantitative real-time PCR (qRT-PCR) was performed with SYBR green (Promega) on a CFX96 PCR Machine (Bio-Rad, Veenendaal, The Netherlands). mRNA expression levels were normalized to β2-microglobulin or glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) and hypoxanthine guanine phosphoribosyl transferase (Hprt ) as reference genes.

    Article Title: Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression
    Article Snippet: .. Real-time quantitative RT-PCR The angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF cDNA were analyzed using a fluorescein quantitative real-time PCR detection system (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). ..

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells
    Article Snippet: Real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cultured cells using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and DNase I (Roche Diagnostics). .. Real-time qRT-PCR was performed using a QuantiTect SYBR Green Reverse Transcription-PCR Kit (Qiagen, Tokyo, Japan) and a Chromo4 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).

    Microarray:

    Article Title: Prospective Evaluation of Whole Genome MicroRNA Expression Profiling in Childhood Acute Lymphoblastic Leukemia
    Article Snippet: .. Quantitative Real Time Polymerase Chain Reaction and Validation Significantly dysregulated miRNAs were validated by Quantitative PCR (LightCycler 480, Roche Applied Science, Mannheim, Germany) after comparing the BM microarray miRNA expressions of patients with the controls. .. RNA was reverse-transcribed to cDNA by using a cDNA synthesis kit (Exiqon).

    Incubation:

    Article Title: Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
    Article Snippet: .. To monitor the association between SERCA and Bcl-2, 150 μg of SR in lysis buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM EDTA, 1% Nonidet P40, 1 mM PMSF and protease inhibitors (Roche Diagnostics) was incubated with 3 μg of GST–Bcl-2 or GST alone for 2 h at 4 °C. .. After the incubation with 10 μl (packed volume) of glutathione–Sepharose beads for 2 h, the beads were separated by centrifugation at 7500 g for 10 min and washed six times with the lysis buffer.

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits
    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States). .. The qRT-PCR conditions were an initial incubation at 95°C for 10 min followed by 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 10 s. The studied genes we identified from the publicly available Vaccinium transcriptome databases.

    Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
    Article Snippet: .. MM cells were exposed to DMSO or 0.5 to 1μM of MLN8237 for 24 to 72 hours, permeabilized by 70% ethanol at −20°C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A (Roche Diagnostics). .. DNA content was analyzed by flow cytometry using BDFACS-Canto II (BD Biosciences) and FlowJo software.

    In Silico:

    Article Title: Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2
    Article Snippet: .. Quantitative PCR Since in silico analysis showed HOXA13 and HOXA9 homeodomains were very similar (75,4% of similarity; 57,9% of identity, score: 274; analysis performed with EMBOSS Matcher program 6.6.0 http://www.ebi.ac.uk/Tools/psa/emboss_matcher/ , matrix: BLOSUM80, gap penality: 14, extended penality: 4), we hypothesized NUP98/HOXA13 could bind MSI2 promoter and tested whether HOXA9 was involved in the present patient. qRT-PCR (LightCycler480, Roche Diagnostics, Germany) was performed using TaqMan assay probes (Applied Biosystems, Foster City, CA) Hs00292670_m for MSI2 gene, Hs00365956_m1 for HOXA9 and Hs00426284_m1 for HOXA13 . ..

    Expressing:

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

    Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
    Article Snippet: .. RT-qPCR was utilized to evaluate the expression of lncRNA, miRNA, and mRNA in the serum samples or cells on the Roche LightCycler 480 (Roche, Switzerland). .. The amplification of the appropriate product was confirmed by melting curve analysis.

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice
    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands). .. Quantitative real-time PCR (qRT-PCR) was performed with SYBR green (Promega) on a CFX96 PCR Machine (Bio-Rad, Veenendaal, The Netherlands). mRNA expression levels were normalized to β2-microglobulin or glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) and hypoxanthine guanine phosphoribosyl transferase (Hprt ) as reference genes.

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol. .. Seventy-two hours after transfection, we examined the cell lysate and medium by immunoblotting to assay for the presence of SP-A.

    BIA-KA:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    Transfection:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol. .. Seventy-two hours after transfection, we examined the cell lysate and medium by immunoblotting to assay for the presence of SP-A.

    Immunoprecipitation:

    Article Title: Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
    Article Snippet: Paragraph title: Immunoprecipitation and solution-binding assay ... To monitor the association between SERCA and Bcl-2, 150 μg of SR in lysis buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM EDTA, 1% Nonidet P40, 1 mM PMSF and protease inhibitors (Roche Diagnostics) was incubated with 3 μg of GST–Bcl-2 or GST alone for 2 h at 4 °C.

    Cell Culture:

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells
    Article Snippet: .. Real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cultured cells using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and DNase I (Roche Diagnostics). .. Real-time qRT-PCR was performed using a QuantiTect SYBR Green Reverse Transcription-PCR Kit (Qiagen, Tokyo, Japan) and a Chromo4 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).

    Generated:

    Article Title: Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential
    Article Snippet: PCR, RT-PCR and 3′ RACE For the mouse, in general, 500 ng of mRNA were converted into cDNA with expand RT (Roche) and random nonamers in a 20 μl reaction volume. .. Less mRNA was used for the XE Sxrb O cDNAs, because RNA was extracted from testis fragments and yields were < 500 ng, but in all these cases, the corresponding XY control cDNA was generated from an equivalent amount of RNA.

    Polymerase Chain Reaction:

    Article Title: Prospective Evaluation of Whole Genome MicroRNA Expression Profiling in Childhood Acute Lymphoblastic Leukemia
    Article Snippet: Quantitative Real Time Polymerase Chain Reaction and Validation Significantly dysregulated miRNAs were validated by Quantitative PCR (LightCycler 480, Roche Applied Science, Mannheim, Germany) after comparing the BM microarray miRNA expressions of patients with the controls. .. For miRNA quantification, the miRCURY LNA Universal RT microRNA PCR system (Exiqon) was used in combination with the predesigned primers (Exiqon).

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

    Article Title: Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2
    Article Snippet: Quantitative PCR Since in silico analysis showed HOXA13 and HOXA9 homeodomains were very similar (75,4% of similarity; 57,9% of identity, score: 274; analysis performed with EMBOSS Matcher program 6.6.0 http://www.ebi.ac.uk/Tools/psa/emboss_matcher/ , matrix: BLOSUM80, gap penality: 14, extended penality: 4), we hypothesized NUP98/HOXA13 could bind MSI2 promoter and tested whether HOXA9 was involved in the present patient. qRT-PCR (LightCycler480, Roche Diagnostics, Germany) was performed using TaqMan assay probes (Applied Biosystems, Foster City, CA) Hs00292670_m for MSI2 gene, Hs00365956_m1 for HOXA9 and Hs00426284_m1 for HOXA13 . .. Reaction mixtures, of 25 μl each, contained 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems), 1.25 μl of the TaqMan assay probe and 5 μl of cDNA (1/10 of RT product).

    Article Title: Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential
    Article Snippet: .. PCR, RT-PCR and 3′ RACE For the mouse, in general, 500 ng of mRNA were converted into cDNA with expand RT (Roche) and random nonamers in a 20 μl reaction volume. .. Less mRNA was used for the XE Sxrb O cDNAs, because RNA was extracted from testis fragments and yields were < 500 ng, but in all these cases, the corresponding XY control cDNA was generated from an equivalent amount of RNA.

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice
    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands). .. Quantitative real-time PCR (qRT-PCR) was performed with SYBR green (Promega) on a CFX96 PCR Machine (Bio-Rad, Veenendaal, The Netherlands). mRNA expression levels were normalized to β2-microglobulin or glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) and hypoxanthine guanine phosphoribosyl transferase (Hprt ) as reference genes.

    Article Title: Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression
    Article Snippet: Real-time quantitative RT-PCR The angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF cDNA were analyzed using a fluorescein quantitative real-time PCR detection system (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). .. A negative control without cDNA was run with every PCR to assess the specificity of the reaction.

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: In a parallel set of experiments, we used expression constructs containing an in-frame 14 amino acid V5-tag immediately after the glutamic acid at position 21 by primer extension mutagenesis and zipper PCR. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    Protein Concentration:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer
    Article Snippet: Real-time quantitative polymerase chain reaction (QRT-PCR) All reagents and equipment for mRNA and cDNA preparation were purchased from Roche (Roche Applied Science, Mannheim, Germany). mRNA was prepared by automated isolation using the MagNA Pure LC instrument and isolation Kit I (for cells) and Kit II (for tissues). .. RNA was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) according to the manufacturer's instructions.

    Article Title: Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential
    Article Snippet: .. PCR, RT-PCR and 3′ RACE For the mouse, in general, 500 ng of mRNA were converted into cDNA with expand RT (Roche) and random nonamers in a 20 μl reaction volume. .. Less mRNA was used for the XE Sxrb O cDNAs, because RNA was extracted from testis fragments and yields were < 500 ng, but in all these cases, the corresponding XY control cDNA was generated from an equivalent amount of RNA.

    Sonication:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol. .. The cells were washed once with 2 ml ice-cold PBS, harvested in 300 μl of RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 1% NP-40, 0.1% SDS, and 0.1% deoxycholate with 1 tablet of protease cocktail [Roche] per 10 ml buffer), sonicated for 10 s., and centrifuged at 16,000 × g at 4°C for 10 min.

    TaqMan Assay:

    Article Title: Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2
    Article Snippet: .. Quantitative PCR Since in silico analysis showed HOXA13 and HOXA9 homeodomains were very similar (75,4% of similarity; 57,9% of identity, score: 274; analysis performed with EMBOSS Matcher program 6.6.0 http://www.ebi.ac.uk/Tools/psa/emboss_matcher/ , matrix: BLOSUM80, gap penality: 14, extended penality: 4), we hypothesized NUP98/HOXA13 could bind MSI2 promoter and tested whether HOXA9 was involved in the present patient. qRT-PCR (LightCycler480, Roche Diagnostics, Germany) was performed using TaqMan assay probes (Applied Biosystems, Foster City, CA) Hs00292670_m for MSI2 gene, Hs00365956_m1 for HOXA9 and Hs00426284_m1 for HOXA13 . ..

    Mutagenesis:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: In a parallel set of experiments, we used expression constructs containing an in-frame 14 amino acid V5-tag immediately after the glutamic acid at position 21 by primer extension mutagenesis and zipper PCR. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    Isolation:

    Article Title: Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer
    Article Snippet: .. Real-time quantitative polymerase chain reaction (QRT-PCR) All reagents and equipment for mRNA and cDNA preparation were purchased from Roche (Roche Applied Science, Mannheim, Germany). mRNA was prepared by automated isolation using the MagNA Pure LC instrument and isolation Kit I (for cells) and Kit II (for tissues). .. RNA was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) according to the manufacturer's instructions.

    Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
    Article Snippet: Total RNA of MM cells was isolated using TRIzol reagent (Invitrogen) and quantified with a nanophotometer. .. RT-qPCR was utilized to evaluate the expression of lncRNA, miRNA, and mRNA in the serum samples or cells on the Roche LightCycler 480 (Roche, Switzerland).

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice
    Article Snippet: .. RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands). .. Quantitative real-time PCR (qRT-PCR) was performed with SYBR green (Promega) on a CFX96 PCR Machine (Bio-Rad, Veenendaal, The Netherlands). mRNA expression levels were normalized to β2-microglobulin or glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) and hypoxanthine guanine phosphoribosyl transferase (Hprt ) as reference genes.

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells
    Article Snippet: .. Real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cultured cells using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and DNase I (Roche Diagnostics). .. Real-time qRT-PCR was performed using a QuantiTect SYBR Green Reverse Transcription-PCR Kit (Qiagen, Tokyo, Japan) and a Chromo4 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).

    Flow Cytometry:

    Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
    Article Snippet: MM cells were exposed to DMSO or 0.5 to 1μM of MLN8237 for 24 to 72 hours, permeabilized by 70% ethanol at −20°C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A (Roche Diagnostics). .. DNA content was analyzed by flow cytometry using BDFACS-Canto II (BD Biosciences) and FlowJo software.

    Sequencing:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: Site-directed mutagenesis (QuickChange, Stratagene) was utilized so that the DNA sequence of wild-type SP-A1 and SP-A2 clones exactly matched and , respectively. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    Construct:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: In a parallel set of experiments, we used expression constructs containing an in-frame 14 amino acid V5-tag immediately after the glutamic acid at position 21 by primer extension mutagenesis and zipper PCR. .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol.

    RNA Extraction:

    Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR (qPCR) ... RT-qPCR was utilized to evaluate the expression of lncRNA, miRNA, and mRNA in the serum samples or cells on the Roche LightCycler 480 (Roche, Switzerland).

    Software:

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

    Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
    Article Snippet: MM cells were exposed to DMSO or 0.5 to 1μM of MLN8237 for 24 to 72 hours, permeabilized by 70% ethanol at −20°C, and incubated with 50 μg/mL PI and 20 units/mL RNase-A (Roche Diagnostics). .. DNA content was analyzed by flow cytometry using BDFACS-Canto II (BD Biosciences) and FlowJo software.

    Real-time Polymerase Chain Reaction:

    Article Title: Prospective Evaluation of Whole Genome MicroRNA Expression Profiling in Childhood Acute Lymphoblastic Leukemia
    Article Snippet: .. Quantitative Real Time Polymerase Chain Reaction and Validation Significantly dysregulated miRNAs were validated by Quantitative PCR (LightCycler 480, Roche Applied Science, Mannheim, Germany) after comparing the BM microarray miRNA expressions of patients with the controls. .. RNA was reverse-transcribed to cDNA by using a cDNA synthesis kit (Exiqon).

    Article Title: Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer
    Article Snippet: .. Real-time quantitative polymerase chain reaction (QRT-PCR) All reagents and equipment for mRNA and cDNA preparation were purchased from Roche (Roche Applied Science, Mannheim, Germany). mRNA was prepared by automated isolation using the MagNA Pure LC instrument and isolation Kit I (for cells) and Kit II (for tissues). .. RNA was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) according to the manufacturer's instructions.

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

    Article Title: Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2
    Article Snippet: .. Quantitative PCR Since in silico analysis showed HOXA13 and HOXA9 homeodomains were very similar (75,4% of similarity; 57,9% of identity, score: 274; analysis performed with EMBOSS Matcher program 6.6.0 http://www.ebi.ac.uk/Tools/psa/emboss_matcher/ , matrix: BLOSUM80, gap penality: 14, extended penality: 4), we hypothesized NUP98/HOXA13 could bind MSI2 promoter and tested whether HOXA9 was involved in the present patient. qRT-PCR (LightCycler480, Roche Diagnostics, Germany) was performed using TaqMan assay probes (Applied Biosystems, Foster City, CA) Hs00292670_m for MSI2 gene, Hs00365956_m1 for HOXA9 and Hs00426284_m1 for HOXA13 . ..

    Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR (qPCR) ... RT-qPCR was utilized to evaluate the expression of lncRNA, miRNA, and mRNA in the serum samples or cells on the Roche LightCycler 480 (Roche, Switzerland).

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice
    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands). .. Quantitative real-time PCR (qRT-PCR) was performed with SYBR green (Promega) on a CFX96 PCR Machine (Bio-Rad, Veenendaal, The Netherlands). mRNA expression levels were normalized to β2-microglobulin or glyceraldehyde-3-phosphate dehydrogenase (Gapdh ) and hypoxanthine guanine phosphoribosyl transferase (Hprt ) as reference genes.

    Article Title: Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression
    Article Snippet: .. Real-time quantitative RT-PCR The angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF cDNA were analyzed using a fluorescein quantitative real-time PCR detection system (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). ..

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells
    Article Snippet: Real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cultured cells using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and DNase I (Roche Diagnostics). .. Real-time qRT-PCR was performed using a QuantiTect SYBR Green Reverse Transcription-PCR Kit (Qiagen, Tokyo, Japan) and a Chromo4 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).

    Negative Control:

    Article Title: Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression
    Article Snippet: Real-time quantitative RT-PCR The angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF cDNA were analyzed using a fluorescein quantitative real-time PCR detection system (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). .. A negative control without cDNA was run with every PCR to assess the specificity of the reaction.

    Activation Assay:

    Article Title: Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2
    Article Snippet: Quantitative PCR Since in silico analysis showed HOXA13 and HOXA9 homeodomains were very similar (75,4% of similarity; 57,9% of identity, score: 274; analysis performed with EMBOSS Matcher program 6.6.0 http://www.ebi.ac.uk/Tools/psa/emboss_matcher/ , matrix: BLOSUM80, gap penality: 14, extended penality: 4), we hypothesized NUP98/HOXA13 could bind MSI2 promoter and tested whether HOXA9 was involved in the present patient. qRT-PCR (LightCycler480, Roche Diagnostics, Germany) was performed using TaqMan assay probes (Applied Biosystems, Foster City, CA) Hs00292670_m for MSI2 gene, Hs00365956_m1 for HOXA9 and Hs00426284_m1 for HOXA13 . .. Protocol consisted of 2 minutes at 50°C for activation of AmpliTaq Gold and 10 minutes at 95°C for DNA denaturation.

    Lysis:

    Article Title: Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
    Article Snippet: .. To monitor the association between SERCA and Bcl-2, 150 μg of SR in lysis buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM EDTA, 1% Nonidet P40, 1 mM PMSF and protease inhibitors (Roche Diagnostics) was incubated with 3 μg of GST–Bcl-2 or GST alone for 2 h at 4 °C. .. After the incubation with 10 μl (packed volume) of glutathione–Sepharose beads for 2 h, the beads were separated by centrifugation at 7500 g for 10 min and washed six times with the lysis buffer.

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol. .. The cells were washed once with 2 ml ice-cold PBS, harvested in 300 μl of RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 1% NP-40, 0.1% SDS, and 0.1% deoxycholate with 1 tablet of protease cocktail [Roche] per 10 ml buffer), sonicated for 10 s., and centrifuged at 16,000 × g at 4°C for 10 min.

    Solution Binding Assay:

    Article Title: Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
    Article Snippet: Paragraph title: Immunoprecipitation and solution-binding assay ... To monitor the association between SERCA and Bcl-2, 150 μg of SR in lysis buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM EDTA, 1% Nonidet P40, 1 mM PMSF and protease inhibitors (Roche Diagnostics) was incubated with 3 μg of GST–Bcl-2 or GST alone for 2 h at 4 °C.

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    Roche gene expression real time quantitative reverse transcription pcr qrt pcr analyses
    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by <t>qRT-PCR</t> and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.
    Gene Expression Real Time Quantitative Reverse Transcription Pcr Qrt Pcr Analyses, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene expression real time quantitative reverse transcription pcr qrt pcr analyses/product/Roche
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    86
    Roche real time quantitative rt pcr qrt pcr total rna
    CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time <t>qRT-PCR</t> analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p
    Real Time Quantitative Rt Pcr Qrt Pcr Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche pectin methylesterase inhibitor
    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 <t>methylesterase</t> 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 .
    Pectin Methylesterase Inhibitor, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche qrt pcr amplification protocol
    NBAT1 inhibits invasion of breast cancer cells by activating DKK1 expression a. , b . <t>qRT-PCR</t> 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
    Qrt Pcr Amplification Protocol, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

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

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

    CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time qRT-PCR analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p

    Journal: PLoS ONE

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

    doi: 10.1371/journal.pone.0052682

    Figure Lengend Snippet: CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time qRT-PCR analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p

    Article Snippet: Real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cultured cells using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and DNase I (Roche Diagnostics).

    Techniques: Expressing, Quantitative RT-PCR

    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 .

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0126973

    Figure Lengend 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 .

    Article Snippet: The 16 genes selected include 11 up-regulated genes (two RPM1 interacting protein 4 (RIN4 ), invertase , Phloem protein 2-B15 (PP2-B15 ), NPR1 regulatory protein (NPR1) , trehalose-phosphate phosphatase-like (TPP) , act repeat 4 (ACR4), disease resistant protein ribosomal protein S2 (RPS2 ), disease resistance protein TIR-NBS-LRR class (DPI) , cysteine-rich protein kinase (CRPK ), xyloglucan endotransglucosylase hydrolase (XTR6 )) and 5 down-regulated genes (bri1 kinase inhibitor 1 (BRI1), bzip transcription protein factor-like (BZIP), 3-ketoacyl-CoA synthase 6 (KCS6 ), β-amylase (BAM) and pectin methylesterase inhibitor (PMEI )) identified by RNA-seq. qRT-PCR was carried out in Lightcycler 480II (Roche, Switzerland) using SYBR Green real time PCR Master Mix (Toyobo, Osaka, Japan) according to Cheng et al .

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

    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

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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

    Article Snippet: The qRT-PCR amplification protocol was conducted according to the user guide of the SYBR® Green master mixes on the Roche LightCycler® 480 Real-Time PCR System.

    Techniques: 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

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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

    Article Snippet: The qRT-PCR amplification protocol was conducted according to the user guide of the SYBR® Green master mixes on the Roche LightCycler® 480 Real-Time PCR System.

    Techniques: 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

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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

    Article Snippet: The qRT-PCR amplification protocol was conducted according to the user guide of the SYBR® Green master mixes on the Roche LightCycler® 480 Real-Time PCR System.

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