Structured Review

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

Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1

Journal: Cancer Science

doi: 10.1111/cas.13754

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

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

2) Product Images from "Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation"

Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

Journal: Genome Medicine

doi: 10.1186/s13073-018-0589-3

T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control
Figure Legend Snippet: T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

Techniques Used: Activation Assay, Transformation Assay, Purification, Selection, RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Activity Assay, In Vitro, Expressing, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Staining, FACS

3) Product Images from "Effects of GADL1 overexpression on cell migration and the associated morphological changes"

Article Title: Effects of GADL1 overexpression on cell migration and the associated morphological changes

Journal: Scientific Reports

doi: 10.1038/s41598-019-41689-x

Genes downregulated upon GADL1 overexpression. ( a ) RNA expression array analyses were used to determine the levels of GADL1 , FN1 , ITGA2 , ITGAV and CCL2 mRNAs in the GADL1 -overexpressing cells (GADL1) relative to the parental cell line, SH-SY5Y (5Y). ( b ) Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for GADL1 , FN1 , ITGA2 , ITGAV and CCL2 . Data were normalized to that for ACTB in each sample, and the fold-change value for each gene is shown for GADL1 -overexpressing cells relative to SH-SY5Y cells. RNA samples for expression microarray analysis and RT-qPCR validation were prepared independently. ( c–g ) GADL1 -overexpressing cells were transfected with RISC-free negative control siRNA or siRNA targeting GADL1 (siGADL1) at 0.1 μM using DharmaFECT1 (FECT1) transfection reagent. Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for ( c ) GADL1 , ( d ) FN1 , ( e ) ITGA2 , ( f ) ITGAV and ( g ) CCL2 . The fold-change value for each gene was normalized to ACTB expression. ( b–g ) Data were combined from two independent experiments.
Figure Legend Snippet: Genes downregulated upon GADL1 overexpression. ( a ) RNA expression array analyses were used to determine the levels of GADL1 , FN1 , ITGA2 , ITGAV and CCL2 mRNAs in the GADL1 -overexpressing cells (GADL1) relative to the parental cell line, SH-SY5Y (5Y). ( b ) Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for GADL1 , FN1 , ITGA2 , ITGAV and CCL2 . Data were normalized to that for ACTB in each sample, and the fold-change value for each gene is shown for GADL1 -overexpressing cells relative to SH-SY5Y cells. RNA samples for expression microarray analysis and RT-qPCR validation were prepared independently. ( c–g ) GADL1 -overexpressing cells were transfected with RISC-free negative control siRNA or siRNA targeting GADL1 (siGADL1) at 0.1 μM using DharmaFECT1 (FECT1) transfection reagent. Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for ( c ) GADL1 , ( d ) FN1 , ( e ) ITGA2 , ( f ) ITGAV and ( g ) CCL2 . The fold-change value for each gene was normalized to ACTB expression. ( b–g ) Data were combined from two independent experiments.

Techniques Used: Over Expression, RNA Expression, Quantitative RT-PCR, Expressing, Microarray, Transfection, Negative Control

4) Product Images from "Depletion of the cdk Inhibitor p16INK4a Differentially Affects Proliferation of Established Cervical Carcinoma Cells"

Article Title: Depletion of the cdk Inhibitor p16INK4a Differentially Affects Proliferation of Established Cervical Carcinoma Cells

Journal: Journal of Virology

doi: 10.1128/JVI.03817-13

Knockdown of p16 INK4A induces senescence of HeLa cells. (A) HeLa cells were transfected with p16 INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7). p16 INK4A knockdown was verified by Western blotting (inset). Cells were counted, and a growth curve was established. Shown are the results (mean ± standard deviation [SD]) from three independent experiments. cPDL, cumulated population doublings. (B) At day 12, cells were assessed for presence of SA-β-gal activity. Bars represent the relative percentage of SA-β-gal-positive cells (mean ± SD) observed within three visual fields from three independent experiments. Two representative micrographs are also shown. (C) HeLa cells were treated as described for panel A and harvested at day 10 posttransfection. Left panel, extraction of total RNA was followed by subsequent cDNA synthesis, and mRNA levels of B2M and p21 WAF-1/Cip-1 were assessed by qRT-PCR. p21 WAF-1/Cip-1 mRNA levels were normalized to B2M mRNA. Shown are the results (mean ± SD) of three independent experiments. Right panel, cell extracts were subjected to SDS-PAGE and analyzed by Western blotting using antibodies for the detection of p21 WAF-1/Cip-1 , p53, and tubulin, as indicated. Extracts of cisplatin-treated human fibroblasts were added as a positive control for the expression of p21 WAF-1/Cip-1 and p53; as a further control, extracts of untreated HeLa cells were used. Three independent experiments were performed, and results of one representative experiment are shown. (D) Left panel, HeLa cells were treated as for panel A, stained with annexin V, and analyzed by flow cytometry, and the relative percentage of annexin V-positive cells was determined. Shown are the results (mean ± SD) of three independent experiments. Right panel, HeLa cells were treated as for panel A, and caspase-3/7 activity was determined and displayed as relative luminescence units (RLU). Staurosporine-treated cells were used as a positive control for the assay. Shown are the results (mean ± SD) of three independent experiments. n.s., not significant.
Figure Legend Snippet: Knockdown of p16 INK4A induces senescence of HeLa cells. (A) HeLa cells were transfected with p16 INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7). p16 INK4A knockdown was verified by Western blotting (inset). Cells were counted, and a growth curve was established. Shown are the results (mean ± standard deviation [SD]) from three independent experiments. cPDL, cumulated population doublings. (B) At day 12, cells were assessed for presence of SA-β-gal activity. Bars represent the relative percentage of SA-β-gal-positive cells (mean ± SD) observed within three visual fields from three independent experiments. Two representative micrographs are also shown. (C) HeLa cells were treated as described for panel A and harvested at day 10 posttransfection. Left panel, extraction of total RNA was followed by subsequent cDNA synthesis, and mRNA levels of B2M and p21 WAF-1/Cip-1 were assessed by qRT-PCR. p21 WAF-1/Cip-1 mRNA levels were normalized to B2M mRNA. Shown are the results (mean ± SD) of three independent experiments. Right panel, cell extracts were subjected to SDS-PAGE and analyzed by Western blotting using antibodies for the detection of p21 WAF-1/Cip-1 , p53, and tubulin, as indicated. Extracts of cisplatin-treated human fibroblasts were added as a positive control for the expression of p21 WAF-1/Cip-1 and p53; as a further control, extracts of untreated HeLa cells were used. Three independent experiments were performed, and results of one representative experiment are shown. (D) Left panel, HeLa cells were treated as for panel A, stained with annexin V, and analyzed by flow cytometry, and the relative percentage of annexin V-positive cells was determined. Shown are the results (mean ± SD) of three independent experiments. Right panel, HeLa cells were treated as for panel A, and caspase-3/7 activity was determined and displayed as relative luminescence units (RLU). Staurosporine-treated cells were used as a positive control for the assay. Shown are the results (mean ± SD) of three independent experiments. n.s., not significant.

Techniques Used: Transfection, Western Blot, Standard Deviation, Activity Assay, Quantitative RT-PCR, SDS Page, Positive Control, Expressing, Staining, Flow Cytometry, Cytometry

Knockdown of p16 INK4A leads to reduced expression of the E7 oncogene in HeLa cells (A) Left panel, HeLa cells were transfected with p16 INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7) and harvested at different time points after initial transfection (24 h, 6 days, and 8 days). Extraction of total RNA was followed by subsequent cDNA synthesis, and HPV-18 E7 mRNA levels were assessed by qRT-PCR. Shown are the results (mean ± SD) of three independent experiments. Right panel, HeLa cells were transfected with p16 INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7) and harvested at different time points after initial transfection (24 h, 48 h, 6 days, and 8 days). Cell extracts were subjected to SDS-PAGE and analyzed by Western blotting using antibodies for the detection of p16 INK4A , HPV-18 E7 protein, and alpha-tubulin, as indicated. Three independent experiments were performed, and results from one representative experiment are shown. (B) HeLa cells were stably transfected with pLSXN and pLSXN-HPV-18 E6/E7, as indicated. p16 INK4A was silenced by siRNA as for panel A, and the percentage of senescent cells was determined after SA-β-gal staining. Shown are the results (mean ± SD) of three independent experiments. (C) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-18 E7 or the empty vector pCMV. HPV-18 E7 mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-18 E7 and cotransfected by p16 INK4A siRNA or control siRNA, as indicated. HPV-18 E7 mRNA was analyzed by qRT-PCR in both cases; shown are the results (mean ± SD) of three independent experiments. (D) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for p16 INK4A or the empty vector pCMV. p16 INK4A mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for p16 INK4A and cotransfected by p16 INK4A siRNA or control siRNA, as indicated. p16 INK4A mRNA was analyzed by qRT-PCR in both cases; shown are the results (mean ± SD) of three independent experiments.
Figure Legend Snippet: Knockdown of p16 INK4A leads to reduced expression of the E7 oncogene in HeLa cells (A) Left panel, HeLa cells were transfected with p16 INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7) and harvested at different time points after initial transfection (24 h, 6 days, and 8 days). Extraction of total RNA was followed by subsequent cDNA synthesis, and HPV-18 E7 mRNA levels were assessed by qRT-PCR. Shown are the results (mean ± SD) of three independent experiments. Right panel, HeLa cells were transfected with p16 INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7) and harvested at different time points after initial transfection (24 h, 48 h, 6 days, and 8 days). Cell extracts were subjected to SDS-PAGE and analyzed by Western blotting using antibodies for the detection of p16 INK4A , HPV-18 E7 protein, and alpha-tubulin, as indicated. Three independent experiments were performed, and results from one representative experiment are shown. (B) HeLa cells were stably transfected with pLSXN and pLSXN-HPV-18 E6/E7, as indicated. p16 INK4A was silenced by siRNA as for panel A, and the percentage of senescent cells was determined after SA-β-gal staining. Shown are the results (mean ± SD) of three independent experiments. (C) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-18 E7 or the empty vector pCMV. HPV-18 E7 mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for HPV-18 E7 and cotransfected by p16 INK4A siRNA or control siRNA, as indicated. HPV-18 E7 mRNA was analyzed by qRT-PCR in both cases; shown are the results (mean ± SD) of three independent experiments. (D) Left panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for p16 INK4A or the empty vector pCMV. p16 INK4A mRNA was analyzed by qRT-PCR in both cases; the fold difference of expression is shown on a logarithmic scale. Right panel, U2-OS cells were transiently transfected with a CMV-driven expression vector for p16 INK4A and cotransfected by p16 INK4A siRNA or control siRNA, as indicated. p16 INK4A mRNA was analyzed by qRT-PCR in both cases; shown are the results (mean ± SD) of three independent experiments.

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, SDS Page, Western Blot, Stable Transfection, Staining, Plasmid Preparation

5) Product Images from "Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact"

Article Title: Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036349

Correlations between different markers for viral productivity in liver and serum. A-C show strong correlation in 19 liver biopsies between cccDNA levels and pgRNA (A) and S-RNA (B), and significant correlation between cccDNA and pgRNA/S-RNA ratio (C). D shows strong correlation between pgRNA and serum levels of HBV DNA (but lack of correlation within the HBeAg-negative subgroup). E shows relatively strong correlation between S-RNA and HBsAg in serum. Filled dots HBeAg positive, open dots HBeAg negative.
Figure Legend Snippet: Correlations between different markers for viral productivity in liver and serum. A-C show strong correlation in 19 liver biopsies between cccDNA levels and pgRNA (A) and S-RNA (B), and significant correlation between cccDNA and pgRNA/S-RNA ratio (C). D shows strong correlation between pgRNA and serum levels of HBV DNA (but lack of correlation within the HBeAg-negative subgroup). E shows relatively strong correlation between S-RNA and HBsAg in serum. Filled dots HBeAg positive, open dots HBeAg negative.

Techniques Used:

Levels of cccDNA and HBV RNA in vivo and in vitro. The cccDNA levels (A) and pgRNA per cccDNA (B), as well as pgRNA/cccDNA ratios (C) were higher in liver tissue from HBeAg-positive as compared with HBeAg-negative patients. In PLC/PRF/5 cells, the cccDNA PCR amplifies integrated HBV DNA (a segment containing the promoter for pgRNA). In these cells which contain multiple integrations of the S region, the pgRNA/S-RNA ratio was low (C). In Huh7.5 cells, the cccDNA levels, pgRNA per cccDNA and ratio between pgRNA and S-RNA were similar in cells transfected with HBV without or with mutations in the core promoter region, indicating that these mutations have low impact on pgRNA transcription.
Figure Legend Snippet: Levels of cccDNA and HBV RNA in vivo and in vitro. The cccDNA levels (A) and pgRNA per cccDNA (B), as well as pgRNA/cccDNA ratios (C) were higher in liver tissue from HBeAg-positive as compared with HBeAg-negative patients. In PLC/PRF/5 cells, the cccDNA PCR amplifies integrated HBV DNA (a segment containing the promoter for pgRNA). In these cells which contain multiple integrations of the S region, the pgRNA/S-RNA ratio was low (C). In Huh7.5 cells, the cccDNA levels, pgRNA per cccDNA and ratio between pgRNA and S-RNA were similar in cells transfected with HBV without or with mutations in the core promoter region, indicating that these mutations have low impact on pgRNA transcription.

Techniques Used: In Vivo, In Vitro, Planar Chromatography, Polymerase Chain Reaction, Transfection

6) Product Images from "Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion"

Article Title: Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074465

Expression of meiotic and HR specific genes during serum starvation in E. histolytica and encystation in E. invadens . (A) Real Time PCR analysis in E. histolytica . Total RNA was extracted at different time points (0, 6, 12, 18 and 24 h) during serum starvation using TRIZOL reagent ( Invitro gen). cDNA was prepared by using Superscript III reverse transcriptase and qRT-PCR was performed with SYBR Green dye as a probe. 18S rDNA was used for normalization. The relative mRNA levels were expressed against those of trophozoites as 1. (B) Real Time PCR analysis in E. invadens . Total RNA was extracted at different time points (0, 8, 16, 24, 40, 48, and 72 h) during encystation and qRT-PCR was performed. mRNA levels of each gene were expressed relative to 0 h (beginning of encystation), shown in the bar graph. Size of all genes was checked by northern blotting (data not shown) by using appropriate probe, amplified by gene specific primers (Table S1in file S1 ). SPO11 , DMC1 and MND1 are meiotic specific genes.
Figure Legend Snippet: Expression of meiotic and HR specific genes during serum starvation in E. histolytica and encystation in E. invadens . (A) Real Time PCR analysis in E. histolytica . Total RNA was extracted at different time points (0, 6, 12, 18 and 24 h) during serum starvation using TRIZOL reagent ( Invitro gen). cDNA was prepared by using Superscript III reverse transcriptase and qRT-PCR was performed with SYBR Green dye as a probe. 18S rDNA was used for normalization. The relative mRNA levels were expressed against those of trophozoites as 1. (B) Real Time PCR analysis in E. invadens . Total RNA was extracted at different time points (0, 8, 16, 24, 40, 48, and 72 h) during encystation and qRT-PCR was performed. mRNA levels of each gene were expressed relative to 0 h (beginning of encystation), shown in the bar graph. Size of all genes was checked by northern blotting (data not shown) by using appropriate probe, amplified by gene specific primers (Table S1in file S1 ). SPO11 , DMC1 and MND1 are meiotic specific genes.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Northern Blot, Amplification

7) Product Images from "Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases"

Article Title: Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-5-14

Transgenic tomato plants that overexpress the ETO1 transgene did not show altered fruit ripening . A. Expression of the ETO1 transgene in leaves of T 1 individuals. Expression of ETO1 was analyzed by RT-PCR. GAPDH was used as an internal control. One microgram of total RNA was used for each reaction. WT: wild type ( Lycopersicon esculentum cv. Shu-gyoku); OE 9, OE 14-0, OE 14-1, OE 14-2 represent independent T 0 transformants. Numbers under the horizontal lines represent T 1 segregating individuals derived from the corresponding T 0 parents. Genotyping for the ETO1 transgene was also performed by PCR and shown under the photograph. B. Representative phenotype of two independent T 1 progenies of ETO1 transgenic tomato (line #14-1-5 and #9-2) and wild type (WT). Fruits were harvested at breaker stage and allowed to ripen for further days as indicated.
Figure Legend Snippet: Transgenic tomato plants that overexpress the ETO1 transgene did not show altered fruit ripening . A. Expression of the ETO1 transgene in leaves of T 1 individuals. Expression of ETO1 was analyzed by RT-PCR. GAPDH was used as an internal control. One microgram of total RNA was used for each reaction. WT: wild type ( Lycopersicon esculentum cv. Shu-gyoku); OE 9, OE 14-0, OE 14-1, OE 14-2 represent independent T 0 transformants. Numbers under the horizontal lines represent T 1 segregating individuals derived from the corresponding T 0 parents. Genotyping for the ETO1 transgene was also performed by PCR and shown under the photograph. B. Representative phenotype of two independent T 1 progenies of ETO1 transgenic tomato (line #14-1-5 and #9-2) and wild type (WT). Fruits were harvested at breaker stage and allowed to ripen for further days as indicated.

Techniques Used: Transgenic Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction

8) Product Images from "MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-? treatment"

Article Title: MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-? treatment

Journal: The Journal of General Virology

doi: 10.1099/vir.0.052910-0

Sensitivity of MERS-CoV and SARS-CoV to PEG-IFN. Vero cells were incubated with 0–1000 ng PEG-IFN ml −1 at t = −4, t = 0 and t = 4 h p.i. Cells were infected with 100 TCID 50 virus per well (a, b). At 2 days p.i., cells were examined for CPE. The effect of PEG-IFN treatment on CPE induced by SARS-CoV (a) or MERS-CoV (b) is shown. CPE was scored as none (0), mild (1), moderate (2), severe (3) or complete (4). (c, d) Viral genomes in the culture medium of virus-infected cells were determined by RT-PCR. The influence of PEG-IFN treatment on the viral RNA load [genome equivalents (gen. eq.) ml −1 ] in the supernatants of cells infected with SARS-CoV (c) or MERS-CoV (d) is shown.
Figure Legend Snippet: Sensitivity of MERS-CoV and SARS-CoV to PEG-IFN. Vero cells were incubated with 0–1000 ng PEG-IFN ml −1 at t = −4, t = 0 and t = 4 h p.i. Cells were infected with 100 TCID 50 virus per well (a, b). At 2 days p.i., cells were examined for CPE. The effect of PEG-IFN treatment on CPE induced by SARS-CoV (a) or MERS-CoV (b) is shown. CPE was scored as none (0), mild (1), moderate (2), severe (3) or complete (4). (c, d) Viral genomes in the culture medium of virus-infected cells were determined by RT-PCR. The influence of PEG-IFN treatment on the viral RNA load [genome equivalents (gen. eq.) ml −1 ] in the supernatants of cells infected with SARS-CoV (c) or MERS-CoV (d) is shown.

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

Kinetics of MERS-CoV replication in Vero and Huh7 cells. Vero and Huh7 cells were infected with MERS-CoV (m.o.i. of 5). (a) Hybridization analysis of viral mRNAs isolated from MERS-CoV-infected cells using an oligonucleotide recognizing the viral genome and all sg mRNAs. Additional minor bands of ~3 and ~4 kb were observed (*) and may represent additional viral mRNA species that remain to be studied in more detail. However, the corresponding positions in the ORF4a/b and ORF5 coding regions do not contain a canonical core TRS sequence (AACGAA; van Boheemen et al. , 2012 ) that might provide a direct explanation for their synthesis. (b) Analysis of the relative molarities of viral genome and each of the sg mRNAs (% of total viral mRNA). mRNA sizes were calculated on the basis of the TRS positions in the viral genome sequence ( van Boheemen et al. , 2012 ). Phosphorimager quantification was performed on the gel lanes with the RNA samples isolated from Vero cells at 10, 13 and 24 h p.i. ( Fig. 1a ; lanes 3–5, respectively; mean± sd ). (c) Release of infectious MERS-CoV progeny into the medium of infected Vero or Huh7 cells at the indicated time points, as determined by plaque assay (mean± sd ; n = 4).
Figure Legend Snippet: Kinetics of MERS-CoV replication in Vero and Huh7 cells. Vero and Huh7 cells were infected with MERS-CoV (m.o.i. of 5). (a) Hybridization analysis of viral mRNAs isolated from MERS-CoV-infected cells using an oligonucleotide recognizing the viral genome and all sg mRNAs. Additional minor bands of ~3 and ~4 kb were observed (*) and may represent additional viral mRNA species that remain to be studied in more detail. However, the corresponding positions in the ORF4a/b and ORF5 coding regions do not contain a canonical core TRS sequence (AACGAA; van Boheemen et al. , 2012 ) that might provide a direct explanation for their synthesis. (b) Analysis of the relative molarities of viral genome and each of the sg mRNAs (% of total viral mRNA). mRNA sizes were calculated on the basis of the TRS positions in the viral genome sequence ( van Boheemen et al. , 2012 ). Phosphorimager quantification was performed on the gel lanes with the RNA samples isolated from Vero cells at 10, 13 and 24 h p.i. ( Fig. 1a ; lanes 3–5, respectively; mean± sd ). (c) Release of infectious MERS-CoV progeny into the medium of infected Vero or Huh7 cells at the indicated time points, as determined by plaque assay (mean± sd ; n = 4).

Techniques Used: Infection, Hybridization, Isolation, Sequencing, Plaque Assay

9) Product Images from "Ddx42p--a human DEAD box protein with RNA chaperone activities"

Article Title: Ddx42p--a human DEAD box protein with RNA chaperone activities

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj403

Characterization of ddx42 mRNA and protein. ( A ) Schematic representation of the structure of the ddx42 mRNA. The ddx42 cds is represented as a bar starting with ATG and ending with TAG. ATG n denotes the true translation initiation codon of Ddx42p (this study), while ATG o is the previously reported one, and the shaded bar area shows the 5′ ward extended cds. Thin lines left and right of the ddx42 cds symbolize the 5′- and 3′-UTRs, respectively, and gray parts are genomic sequences not found in ddx42 poly(A) + RNA (i1, i2, i3 are introns 1, 2 and 3). Exon 2 (e2) is absent in the shorter ddx42 poly(A) + RNA. PCR primers used for analysis are indicated as arrows, and those not leading to a PCR product are highlighted in gray. ( B ) ddx42 mRNA is present in similar low levels in different cell lines. A northern blot of poly(A) + RNA (100 ng each) was probed with a ddx42-specific antisense RNA probe. β-Actin RNA served as a loading control. The size of RNA marker bands (Fermentas RNA ladder, high range) is indicated on the right. ( C ) The protein Ddx42p is expressed differentially in different cell lines. Ddx42p was detected by western blotting in whole cell extracts (15 µg total protein each) with antiserum α-D42N. Actin was detected as a loading control. The molecular weight of marker bands (RPN800, Amersham Biosciences) is indicated on the right. ( D ) Ddx42p is found exclusively in nuclear extracts. The western blot was performed as above using nuclear extracts (ne) from HeLa and COS cells (lanes 1 and 2). Soluble cellular extracts (ce; lanes 3 and 5) and ne (lanes 4 and 6) from COS cells transfected with full-length ddx42 cDNA (pCddx42; lanes 5 and 6) or a C-terminally shortened form (pCddx42Δ; lanes 3 and 4), respectively, show that even overexpressed protein resides in the nuclei. The overexpressed full-length form is probably subject to proteolytic degradation.
Figure Legend Snippet: Characterization of ddx42 mRNA and protein. ( A ) Schematic representation of the structure of the ddx42 mRNA. The ddx42 cds is represented as a bar starting with ATG and ending with TAG. ATG n denotes the true translation initiation codon of Ddx42p (this study), while ATG o is the previously reported one, and the shaded bar area shows the 5′ ward extended cds. Thin lines left and right of the ddx42 cds symbolize the 5′- and 3′-UTRs, respectively, and gray parts are genomic sequences not found in ddx42 poly(A) + RNA (i1, i2, i3 are introns 1, 2 and 3). Exon 2 (e2) is absent in the shorter ddx42 poly(A) + RNA. PCR primers used for analysis are indicated as arrows, and those not leading to a PCR product are highlighted in gray. ( B ) ddx42 mRNA is present in similar low levels in different cell lines. A northern blot of poly(A) + RNA (100 ng each) was probed with a ddx42-specific antisense RNA probe. β-Actin RNA served as a loading control. The size of RNA marker bands (Fermentas RNA ladder, high range) is indicated on the right. ( C ) The protein Ddx42p is expressed differentially in different cell lines. Ddx42p was detected by western blotting in whole cell extracts (15 µg total protein each) with antiserum α-D42N. Actin was detected as a loading control. The molecular weight of marker bands (RPN800, Amersham Biosciences) is indicated on the right. ( D ) Ddx42p is found exclusively in nuclear extracts. The western blot was performed as above using nuclear extracts (ne) from HeLa and COS cells (lanes 1 and 2). Soluble cellular extracts (ce; lanes 3 and 5) and ne (lanes 4 and 6) from COS cells transfected with full-length ddx42 cDNA (pCddx42; lanes 5 and 6) or a C-terminally shortened form (pCddx42Δ; lanes 3 and 4), respectively, show that even overexpressed protein resides in the nuclei. The overexpressed full-length form is probably subject to proteolytic degradation.

Techniques Used: Genomic Sequencing, Polymerase Chain Reaction, Northern Blot, Marker, Western Blot, Molecular Weight, Transfection

10) Product Images from "Peroxisome proliferator-activated receptor (PPAR)? expression in T cells mediates gender differences in development of T cell-mediated autoimmunity"

Article Title: Peroxisome proliferator-activated receptor (PPAR)? expression in T cells mediates gender differences in development of T cell-mediated autoimmunity

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061839

PPARα mediates androgen sensitivity of Th responses. (A and B) Total RNA was obtained from naive CD4 + T cells that were pooled from the spleens of castrated- or sham-operated male ( n = 4 mice/group) (A) or placebo or α-DHT pellet-implanted female ( n = 4 mice/group) (B) mice. The expression of PPARα mRNA in these cells was measured using real-time RT-PCR, and abundance was expressed relative to β-actin mRNA. Values are means ± SEM of PCR product abundance in arbitrary units (AU). * indicates a significant difference (P
Figure Legend Snippet: PPARα mediates androgen sensitivity of Th responses. (A and B) Total RNA was obtained from naive CD4 + T cells that were pooled from the spleens of castrated- or sham-operated male ( n = 4 mice/group) (A) or placebo or α-DHT pellet-implanted female ( n = 4 mice/group) (B) mice. The expression of PPARα mRNA in these cells was measured using real-time RT-PCR, and abundance was expressed relative to β-actin mRNA. Values are means ± SEM of PCR product abundance in arbitrary units (AU). * indicates a significant difference (P

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

PPARα was more abundant in male as compared with female T cells and was associated with decreased NF-κB and c-jun activity and increased IFN-γ production. (A) Total RNA was obtained from naive CD4 + T cells that were pooled from the spleens of male and female SV.129 mice ( n = 4 mice/group). The expression of PPARα mRNA in these cells was measured using real-time RT-PCR, and abundance was expressed relative to β-actin mRNA. Values are means ± SEM of PPARα/β-actin product abundance in triplicate reactions expressed in arbitrary units (AU). (B) Western blot analysis of PPARα in nuclear extracts (250 μg) prepared from male and female T cells. Histone H3 was used as a loading control. (C) c-jun (left) and NF-κB (right) DNA binding was measured in nuclear extracts from male and female SV.129 WT or PPARα −/− CD3 + T cells using an ELISA-based assay. Nuclear extracts were prepared from T cells at 16 h after stimulation with 5 μg/ml anti-CD3 and 5 μg/ml anti-CD28. Values are means ± SEM of absorbance units (duplicate culture wells) of stimulated wells expressed relative to absorbance in nonstimulated control wells. (D) CD3 + T cells from male and female SV.129 WT or PPARα −/− mice were stimulated with 0–2 μg/ml anti-CD3 (left) and 0–5 μg/ml anti-CD28 (right). IFN-γ production in culture supernatants was measured by ELISA at 72 h after stimulation. Values are means ± SEM of triplicate culture wells. Note that anti-CD28 and anti-CD3 were held constant at 0.5 μg/ml in the left and right panels, respectively. Results in A–D are representative of two independent experiments.
Figure Legend Snippet: PPARα was more abundant in male as compared with female T cells and was associated with decreased NF-κB and c-jun activity and increased IFN-γ production. (A) Total RNA was obtained from naive CD4 + T cells that were pooled from the spleens of male and female SV.129 mice ( n = 4 mice/group). The expression of PPARα mRNA in these cells was measured using real-time RT-PCR, and abundance was expressed relative to β-actin mRNA. Values are means ± SEM of PPARα/β-actin product abundance in triplicate reactions expressed in arbitrary units (AU). (B) Western blot analysis of PPARα in nuclear extracts (250 μg) prepared from male and female T cells. Histone H3 was used as a loading control. (C) c-jun (left) and NF-κB (right) DNA binding was measured in nuclear extracts from male and female SV.129 WT or PPARα −/− CD3 + T cells using an ELISA-based assay. Nuclear extracts were prepared from T cells at 16 h after stimulation with 5 μg/ml anti-CD3 and 5 μg/ml anti-CD28. Values are means ± SEM of absorbance units (duplicate culture wells) of stimulated wells expressed relative to absorbance in nonstimulated control wells. (D) CD3 + T cells from male and female SV.129 WT or PPARα −/− mice were stimulated with 0–2 μg/ml anti-CD3 (left) and 0–5 μg/ml anti-CD28 (right). IFN-γ production in culture supernatants was measured by ELISA at 72 h after stimulation. Values are means ± SEM of triplicate culture wells. Note that anti-CD28 and anti-CD3 were held constant at 0.5 μg/ml in the left and right panels, respectively. Results in A–D are representative of two independent experiments.

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

11) Product Images from "Direct induction of ramified microglia-like cells from human monocytes: Dynamic microglial dysfunction in Nasu-Hakola disease"

Article Title: Direct induction of ramified microglia-like cells from human monocytes: Dynamic microglial dysfunction in Nasu-Hakola disease

Journal: Scientific Reports

doi: 10.1038/srep04957

The iMG cells show the character of human resident microglia. (A and B) The expression levels of surface markers on the iMG cells and induced macrophage were performed by flow cytometer. Peripheral monocytes were incubated with GM-CSF (macrophage) or cocktail of GM-CSF and IL-34 (iMG cells) for 14 days. The iMG cells showed the specific phenotypes of microglia compared to macrophage. (C to E) The expression pattern of CCR2 and CX3CR1 between monocytes and iMG cells were observed by immunocytochemistry. The monocytes and iMG cells were cultured for 14 days, and stained with specific antibodies. (C and D) The iMG cells were stained with bright green fluorescence (CX3CR1) bearing highly branched forms. Scale bar, 50 μm. (E) The expression ratio (CX3CR1/CCR2) of iMG cells was significantly higher than that of monocytes by flow cytometry (n = 3). The iMG cells were incubated with IL-4 (F) or dexamethasone (G) for 72 hours, and extracted RNA was analyzed by qRT-PCR (n = 6). Fold changes were depicted in mRNA levels after stimulation compared with unstimulated cells. * P
Figure Legend Snippet: The iMG cells show the character of human resident microglia. (A and B) The expression levels of surface markers on the iMG cells and induced macrophage were performed by flow cytometer. Peripheral monocytes were incubated with GM-CSF (macrophage) or cocktail of GM-CSF and IL-34 (iMG cells) for 14 days. The iMG cells showed the specific phenotypes of microglia compared to macrophage. (C to E) The expression pattern of CCR2 and CX3CR1 between monocytes and iMG cells were observed by immunocytochemistry. The monocytes and iMG cells were cultured for 14 days, and stained with specific antibodies. (C and D) The iMG cells were stained with bright green fluorescence (CX3CR1) bearing highly branched forms. Scale bar, 50 μm. (E) The expression ratio (CX3CR1/CCR2) of iMG cells was significantly higher than that of monocytes by flow cytometry (n = 3). The iMG cells were incubated with IL-4 (F) or dexamethasone (G) for 72 hours, and extracted RNA was analyzed by qRT-PCR (n = 6). Fold changes were depicted in mRNA levels after stimulation compared with unstimulated cells. * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation, Immunocytochemistry, Cell Culture, Staining, Fluorescence, Quantitative RT-PCR

Dynamic functional analysis of the iMG cells. (A) The iMG cells were incubated with FITC-conjugated latex beads for 24 hours, and phagocytic activity was observed by fluorescent microscopy. The iMG cells showed the ability of phagocytosis with morphological changes into an ameboid form (arrow head). Scale bar, 50 μm. (B and C) The ability of TNF-α production during phagocytosis was measured on the iMG cells. The iMG cells were incubated with latex beads for 72 hours. The extracted RNA and culture supernatant were analyzed by qRT-PCR and Cytometric Beads Array System (CBA), respectively. The mRNA expression (B) and protein level of TNF-α (C) on the iMG cells were significantly higher compared to controls (B, n = 4; C, n = 6). * P
Figure Legend Snippet: Dynamic functional analysis of the iMG cells. (A) The iMG cells were incubated with FITC-conjugated latex beads for 24 hours, and phagocytic activity was observed by fluorescent microscopy. The iMG cells showed the ability of phagocytosis with morphological changes into an ameboid form (arrow head). Scale bar, 50 μm. (B and C) The ability of TNF-α production during phagocytosis was measured on the iMG cells. The iMG cells were incubated with latex beads for 72 hours. The extracted RNA and culture supernatant were analyzed by qRT-PCR and Cytometric Beads Array System (CBA), respectively. The mRNA expression (B) and protein level of TNF-α (C) on the iMG cells were significantly higher compared to controls (B, n = 4; C, n = 6). * P

Techniques Used: Functional Assay, Incubation, Activity Assay, Microscopy, Quantitative RT-PCR, Crocin Bleaching Assay, Expressing

12) Product Images from "High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus"

Article Title: High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-9-96

Correlation between P19 protein accumulation and levels of Nef specific siRNAs . (A) ELISA of leaf extracts from Nicotiana benthamiana plants agroinfiltrated with Nef, Nef/TBSV-P19 and Nef/AMCV-P19 was performed using a rabbit polyclonal antibody against TBSV-P19. Leaves were collected at 9 d.p.i. and plant extracts were normalized for total soluble protein (TSP). Fifty micrograms of TSP were loaded in each ELISA plate well. Values are means of triplicate determinations ± standard errors of the means. C-: leaf extract of plants infiltrated with infiltration buffer. (B) Detection of Nef specific siRNAs. Total RNA (15 μg) extracted from leaves agroinfiltrated with Nef, Nef/AMCV-P19, Nef/TBSV-P19, and from mock infiltrated plant used as a control (WT), was separated on denaturing 15% (w/v) polyacrylamide gel with 8 M Urea, stained with ethidium bromide to display relative amounts of rRNA and transferred to a positively charged nylon membrane. Nef specific digoxigenin-labelled RNA (+) (618 nt) was used as probe. M: si RNA low molecular weight RNA marker (synthetic siRNA duplexes 17, 21 and 25 bp).
Figure Legend Snippet: Correlation between P19 protein accumulation and levels of Nef specific siRNAs . (A) ELISA of leaf extracts from Nicotiana benthamiana plants agroinfiltrated with Nef, Nef/TBSV-P19 and Nef/AMCV-P19 was performed using a rabbit polyclonal antibody against TBSV-P19. Leaves were collected at 9 d.p.i. and plant extracts were normalized for total soluble protein (TSP). Fifty micrograms of TSP were loaded in each ELISA plate well. Values are means of triplicate determinations ± standard errors of the means. C-: leaf extract of plants infiltrated with infiltration buffer. (B) Detection of Nef specific siRNAs. Total RNA (15 μg) extracted from leaves agroinfiltrated with Nef, Nef/AMCV-P19, Nef/TBSV-P19, and from mock infiltrated plant used as a control (WT), was separated on denaturing 15% (w/v) polyacrylamide gel with 8 M Urea, stained with ethidium bromide to display relative amounts of rRNA and transferred to a positively charged nylon membrane. Nef specific digoxigenin-labelled RNA (+) (618 nt) was used as probe. M: si RNA low molecular weight RNA marker (synthetic siRNA duplexes 17, 21 and 25 bp).

Techniques Used: Enzyme-linked Immunosorbent Assay, Staining, Molecular Weight, Marker

13) Product Images from "Predictable suppression of gene expression by 5?-UTR-based RNA quadruplexes"

Article Title: Predictable suppression of gene expression by 5?-UTR-based RNA quadruplexes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp696

In vitro stability and in vivo abundancy of the quadruplex motifs. The CD scans (black) show a positive band around 264 nm and negative maxima around 240 nm for 4G 3 U ( A ), 4G 3 U 2 ( B ) and 4G 3 U 3 ( C ), characteristic for a parallel stranded quadruplex motif, while the respective control sequences; con4G 3 U ( A ), con4G 3 U 2 ( B ) and con4G 3 U 3 ( C ) does not show any quadruplex characteristic peaks (grey). In CD melting experiments ( D ), denaturing (hollow) and annealing (solid) curves are almost identical and show a T m of > 95, 73 and 61°C for 4G 3 U (dark grey), 4G 3 U 2 (grey) and 4G 3 U 3 (light grey) RNA-quadruplex motifs, respectively. ( E ) Relative hRluc mRNA levels for different constructs determined by real-time PCR assays [ hRluc ( C T )] is normalized to hluc mRNA levels [ hluc ( C T )], as reported earlier ( 35 ). Error bars represent standard deviation of three independent experiments.
Figure Legend Snippet: In vitro stability and in vivo abundancy of the quadruplex motifs. The CD scans (black) show a positive band around 264 nm and negative maxima around 240 nm for 4G 3 U ( A ), 4G 3 U 2 ( B ) and 4G 3 U 3 ( C ), characteristic for a parallel stranded quadruplex motif, while the respective control sequences; con4G 3 U ( A ), con4G 3 U 2 ( B ) and con4G 3 U 3 ( C ) does not show any quadruplex characteristic peaks (grey). In CD melting experiments ( D ), denaturing (hollow) and annealing (solid) curves are almost identical and show a T m of > 95, 73 and 61°C for 4G 3 U (dark grey), 4G 3 U 2 (grey) and 4G 3 U 3 (light grey) RNA-quadruplex motifs, respectively. ( E ) Relative hRluc mRNA levels for different constructs determined by real-time PCR assays [ hRluc ( C T )] is normalized to hluc mRNA levels [ hluc ( C T )], as reported earlier ( 35 ). Error bars represent standard deviation of three independent experiments.

Techniques Used: In Vitro, In Vivo, Construct, Real-time Polymerase Chain Reaction, Standard Deviation

14) Product Images from "BDCA-2, a Novel Plasmacytoid Dendritic Cell-specific Type II C-type Lectin, Mediates Antigen Capture and Is a Potent Inhibitor of Interferon ?/? Induction"

Article Title: BDCA-2, a Novel Plasmacytoid Dendritic Cell-specific Type II C-type Lectin, Mediates Antigen Capture and Is a Potent Inhibitor of Interferon ?/? Induction

Journal: The Journal of Experimental Medicine

doi:

(A) Identification of putative alternative splice forms of BDCA-2 mRNA. Poly(A) + RNA was isolated from purified PDCs and analyzed for the presence of BDCA-2 mRNA by RT-PCR amplification (lane 2, 20 PCR cycles; lane 3, 25 PCR cycles; lane 4, 30 PCR cycles). The PCR products were size-fractionated by 4–12% TBE PAGE. A further PCR amplification of individual excised bands enabled the cloning and sequencing of individual splice variants. (B) Schematic drawing of the coding region of full-length BDCA-2 mRNA. The structural domains are indicated by different colors (violet, cytoplasmic domain; red, transmembrane domain; green, neck domain; and blue, CRD) and the individual exons (exons 2–7) of the coding region are represented as boxes. Below the full-length BDCA-2 mRNA, BDCA-2 splice variants are shown with the missing exons indicated by gaps.
Figure Legend Snippet: (A) Identification of putative alternative splice forms of BDCA-2 mRNA. Poly(A) + RNA was isolated from purified PDCs and analyzed for the presence of BDCA-2 mRNA by RT-PCR amplification (lane 2, 20 PCR cycles; lane 3, 25 PCR cycles; lane 4, 30 PCR cycles). The PCR products were size-fractionated by 4–12% TBE PAGE. A further PCR amplification of individual excised bands enabled the cloning and sequencing of individual splice variants. (B) Schematic drawing of the coding region of full-length BDCA-2 mRNA. The structural domains are indicated by different colors (violet, cytoplasmic domain; red, transmembrane domain; green, neck domain; and blue, CRD) and the individual exons (exons 2–7) of the coding region are represented as boxes. Below the full-length BDCA-2 mRNA, BDCA-2 splice variants are shown with the missing exons indicated by gaps.

Techniques Used: Isolation, Purification, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Clone Assay, Sequencing

15) Product Images from "Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations *"

Article Title: Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M110.003129

NE composition changes during muscle differentiation. Expression levels of several muscle NETs were tested in both C2C12 (mouse) and RD (human) myoblast to myotube differentiation systems. RNA extracted from both untreated myoblast and chemically differentiated myotube populations were subjected to RT-PCR for each NET, and expression levels were quantified. The relative change between myoblast and myotube populations was determined after first normalizing values to the control peptidylprolyl isomerase A ( PPIA ). Relative transcript levels for both human and mouse systems are shown, and error bars indicate standard deviations between three replicates for each differentiation system. Four NETs were induced similarly to positive differentiation controls, MYOG and MYH1 .
Figure Legend Snippet: NE composition changes during muscle differentiation. Expression levels of several muscle NETs were tested in both C2C12 (mouse) and RD (human) myoblast to myotube differentiation systems. RNA extracted from both untreated myoblast and chemically differentiated myotube populations were subjected to RT-PCR for each NET, and expression levels were quantified. The relative change between myoblast and myotube populations was determined after first normalizing values to the control peptidylprolyl isomerase A ( PPIA ). Relative transcript levels for both human and mouse systems are shown, and error bars indicate standard deviations between three replicates for each differentiation system. Four NETs were induced similarly to positive differentiation controls, MYOG and MYH1 .

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

16) Product Images from "Time course analysis of RNA stability in human placenta"

Article Title: Time course analysis of RNA stability in human placenta

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-10-21

Analysis of mRNA integrity . Comparison of FASN (a) and GAPDH (b) mRNA 5'/3' ratios in placental samples stored at + 4°C for 0 to 96 h, as determined by qRT-PCR assays targeting sequences close to the 5' and 3' ends of the transcripts. RNA samples prepared with protocol A were compared to protocol B (n = 14 in each group). Results are expressed as mean +/- SEM. Each experiment was performed in duplicate. p values were determined by ANOVA. p
Figure Legend Snippet: Analysis of mRNA integrity . Comparison of FASN (a) and GAPDH (b) mRNA 5'/3' ratios in placental samples stored at + 4°C for 0 to 96 h, as determined by qRT-PCR assays targeting sequences close to the 5' and 3' ends of the transcripts. RNA samples prepared with protocol A were compared to protocol B (n = 14 in each group). Results are expressed as mean +/- SEM. Each experiment was performed in duplicate. p values were determined by ANOVA. p

Techniques Used: Quantitative RT-PCR

Time course analysis of housekeeping genes . Effect of storage time and handling conditions on housekeeping gene expression. Relative qRT-PCR amount synthetised on 1 μg of total RNA from placental tissues stored at +4°C from 0 h to 96 h according to protocol A or protocol B (n = 14 for each group). Reactions were normalised to contain equivalent amounts of total RNA. (a): ALAS (b): B2M, (c): Cyclophilin. Data are plotted as mean +/- SEM. (n = 14). p values were determined by ANOVA. p
Figure Legend Snippet: Time course analysis of housekeeping genes . Effect of storage time and handling conditions on housekeeping gene expression. Relative qRT-PCR amount synthetised on 1 μg of total RNA from placental tissues stored at +4°C from 0 h to 96 h according to protocol A or protocol B (n = 14 for each group). Reactions were normalised to contain equivalent amounts of total RNA. (a): ALAS (b): B2M, (c): Cyclophilin. Data are plotted as mean +/- SEM. (n = 14). p values were determined by ANOVA. p

Techniques Used: Expressing, Quantitative RT-PCR

17) Product Images from "Erythroid-Specific Expression of ?-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells"

Article Title: Erythroid-Specific Expression of ?-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029110

Increased β-globin transcript is detectable in non-erythroid progeny of IHK-transduced CD34 + cells. Twenty ng of total RNA was used for RT-PCR specific for spliced β-globin transcript and spliced β-actin. RNA from adult RBCs and erythrocyte-depleted adult WBCs was used as control.
Figure Legend Snippet: Increased β-globin transcript is detectable in non-erythroid progeny of IHK-transduced CD34 + cells. Twenty ng of total RNA was used for RT-PCR specific for spliced β-globin transcript and spliced β-actin. RNA from adult RBCs and erythrocyte-depleted adult WBCs was used as control.

Techniques Used: Reverse Transcription Polymerase Chain Reaction

18) Product Images from "Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis"

Article Title: Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-7-38

Validation and extension of microarray analysis by quantitative/real-time PCR. Primer sets (see Additional File 1 ) were designed to target the Affymetrix probe set region of the selected genes and to amplify the region across exon-exon junctions whenever possible. Quantitative PCR was performed as described in Materials and Methods. Shown is the mean of all signal values obtained from 6 independent RNA samples. (A) novel NETs; (B) NE markers proteins; (C) muscle differentiation markers. The error bars indicate the standard deviation. Gray columns indicate the fold changes obtained in the microarray analysis, taken from Fig. 1.
Figure Legend Snippet: Validation and extension of microarray analysis by quantitative/real-time PCR. Primer sets (see Additional File 1 ) were designed to target the Affymetrix probe set region of the selected genes and to amplify the region across exon-exon junctions whenever possible. Quantitative PCR was performed as described in Materials and Methods. Shown is the mean of all signal values obtained from 6 independent RNA samples. (A) novel NETs; (B) NE markers proteins; (C) muscle differentiation markers. The error bars indicate the standard deviation. Gray columns indicate the fold changes obtained in the microarray analysis, taken from Fig. 1.

Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Standard Deviation

19) Product Images from "The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression"

Article Title: The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

Journal: bioRxiv

doi: 10.1101/2020.01.19.911503

Abrogating pUS28 expression does not impact US27 or US29 transcription. NuFF-1 fibroblasts were infected (moi = 0.5) with (A) BFX wt -based or (B) TB40/E mCherry -based viruses. Total RNA was harvested 96 hpi and US27 , US28 , US29 , UL123 , and UL99 mRNA levels were quantified by RTqPCR. Viral gene expression is plotted relative to cellular GAPDH . Each data point (circles) is the mean of three technical replicates (e.g. one biological replicate). Error bars indicate standard deviation of three biological replicates, and statistical significance was calculated using two-way ANOVA analyses followed by Tukey’s post-hoc analyses. * p
Figure Legend Snippet: Abrogating pUS28 expression does not impact US27 or US29 transcription. NuFF-1 fibroblasts were infected (moi = 0.5) with (A) BFX wt -based or (B) TB40/E mCherry -based viruses. Total RNA was harvested 96 hpi and US27 , US28 , US29 , UL123 , and UL99 mRNA levels were quantified by RTqPCR. Viral gene expression is plotted relative to cellular GAPDH . Each data point (circles) is the mean of three technical replicates (e.g. one biological replicate). Error bars indicate standard deviation of three biological replicates, and statistical significance was calculated using two-way ANOVA analyses followed by Tukey’s post-hoc analyses. * p

Techniques Used: Expressing, Infection, Standard Deviation

20) Product Images from "The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression"

Article Title: The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

Journal: bioRxiv

doi: 10.1101/2020.01.19.911503

Abrogating pUS28 expression does not impact US27 or US29 transcription. NuFF-1 fibroblasts were infected (moi = 0.5) with (A) BFX wt -based or (B) TB40/E mCherry -based viruses. Total RNA was harvested 96 hpi and US27 , US28 , US29 , UL123 , and UL99 mRNA levels were quantified by RTqPCR. Viral gene expression is plotted relative to cellular GAPDH . Each data point (circles) is the mean of three technical replicates (e.g. one biological replicate). Error bars indicate standard deviation of three biological replicates, and statistical significance was calculated using two-way ANOVA analyses followed by Tukey’s post-hoc analyses. * p
Figure Legend Snippet: Abrogating pUS28 expression does not impact US27 or US29 transcription. NuFF-1 fibroblasts were infected (moi = 0.5) with (A) BFX wt -based or (B) TB40/E mCherry -based viruses. Total RNA was harvested 96 hpi and US27 , US28 , US29 , UL123 , and UL99 mRNA levels were quantified by RTqPCR. Viral gene expression is plotted relative to cellular GAPDH . Each data point (circles) is the mean of three technical replicates (e.g. one biological replicate). Error bars indicate standard deviation of three biological replicates, and statistical significance was calculated using two-way ANOVA analyses followed by Tukey’s post-hoc analyses. * p

Techniques Used: Expressing, Infection, Standard Deviation

21) Product Images from "Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web"

Article Title: Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

Journal: PLoS ONE

doi: 10.1371/journal.pone.0114629

Effect of different nuclear transport signals bearing peptides on HCV infection. Cells were infected by HCV and treated with the indicated peptides. (A) Following 4 days of infection, virus titer was estimated by monitoring the levels of intracellular and extracellular viral RNA, using real time PCR. Significant differences between the intracellular and extracellular viral RNA levels are indicated by: * p
Figure Legend Snippet: Effect of different nuclear transport signals bearing peptides on HCV infection. Cells were infected by HCV and treated with the indicated peptides. (A) Following 4 days of infection, virus titer was estimated by monitoring the levels of intracellular and extracellular viral RNA, using real time PCR. Significant differences between the intracellular and extracellular viral RNA levels are indicated by: * p

Techniques Used: Infection, Real-time Polymerase Chain Reaction

Nuclear transport signal-bearing peptides affect specific steps of infection: Time of addition experiments. In order to elucidate the time points in infection when the nuclear transport signals bearing peptides are functional, cells were synchronously infected and treated with the indicated peptides. Each time point (2 h intervals) represents a culture treated with the indicated reagent at the specified time post temperature shift (PTS) and initiation of HCV infection. All samples were fixed and probed with anti-NS5A antibodies to determine the percentage of infected cells relative to untreated cells at 72 h PTS. (A) Known inhibitors of HCV production (Ezetimibe (30 µM), LMB (18 nM), BMS-986094 (10 nM) and DGAT 1 inhibitor (10 nM)) and control (Pen peptide 100 µM, DMSO 2%, no treatment, and no infection) were examined. Groups of NLS and NES peptides appeared to yield similar inhibitory effects and are grouped in panels B-D (B) a group of NLS peptides (core NLS 1-4, NS2 NLS and SV40 NLS) inhibiting intracellular and extracellular viral RNA to approximately the same level, (C) the NLS peptides (NS3 NLS 1-2, NS5A NLS and HIV-1 Rev NLS) which further inhibited the extracellular virus and (D) the NES peptides (core NES 1-2, NS2 NES and HIV-1 Rev NES). Error bars represent ± standard deviation, n = 3.
Figure Legend Snippet: Nuclear transport signal-bearing peptides affect specific steps of infection: Time of addition experiments. In order to elucidate the time points in infection when the nuclear transport signals bearing peptides are functional, cells were synchronously infected and treated with the indicated peptides. Each time point (2 h intervals) represents a culture treated with the indicated reagent at the specified time post temperature shift (PTS) and initiation of HCV infection. All samples were fixed and probed with anti-NS5A antibodies to determine the percentage of infected cells relative to untreated cells at 72 h PTS. (A) Known inhibitors of HCV production (Ezetimibe (30 µM), LMB (18 nM), BMS-986094 (10 nM) and DGAT 1 inhibitor (10 nM)) and control (Pen peptide 100 µM, DMSO 2%, no treatment, and no infection) were examined. Groups of NLS and NES peptides appeared to yield similar inhibitory effects and are grouped in panels B-D (B) a group of NLS peptides (core NLS 1-4, NS2 NLS and SV40 NLS) inhibiting intracellular and extracellular viral RNA to approximately the same level, (C) the NLS peptides (NS3 NLS 1-2, NS5A NLS and HIV-1 Rev NLS) which further inhibited the extracellular virus and (D) the NES peptides (core NES 1-2, NS2 NES and HIV-1 Rev NES). Error bars represent ± standard deviation, n = 3.

Techniques Used: Infection, Functional Assay, Standard Deviation

22) Product Images from "NrsZ: a novel, processed, nitrogen-dependent, small non-coding RNA that regulates Pseudomonas aeruginosa PAO1 virulence"

Article Title: NrsZ: a novel, processed, nitrogen-dependent, small non-coding RNA that regulates Pseudomonas aeruginosa PAO1 virulence

Journal: Environmental Microbiology

doi: 10.1111/1462-2920.12272

NrsZ is processed in three small forms. The RNA deep sequencing profile of NrsZ was determined from RNA extracted from PAO1 grown in minimum medium supplemented with nitrate and fractioned in two parts, (A, left panel) the medium fraction ranging from 150 nt to 450 nt, and (B, left panel) the small fraction ranging from 30 nt to 200 nt. (A and B, right panels): insert length distributions of the transcripts corresponding to NrsZ in both fractions. Base numbering starts from the +1 transcription start site represented by a bent arrow, and horizontal arrows underline the major transcripts positions and sizes. (C) Predicted secondary structure of the NrsZ primary transcript. Each black dot represents one 3′ extremity of NrsZ obtained by 3′ RACE experiment. 5′-matured terminal nucleotides determined by RNA deep sequencing are indicated in bold. Conserved motifs in the stem-loop sequences are shown in red. SL: stem-loop structure. The M fold program was used to predict RNA secondary structures ( Zuker, 2003 ).
Figure Legend Snippet: NrsZ is processed in three small forms. The RNA deep sequencing profile of NrsZ was determined from RNA extracted from PAO1 grown in minimum medium supplemented with nitrate and fractioned in two parts, (A, left panel) the medium fraction ranging from 150 nt to 450 nt, and (B, left panel) the small fraction ranging from 30 nt to 200 nt. (A and B, right panels): insert length distributions of the transcripts corresponding to NrsZ in both fractions. Base numbering starts from the +1 transcription start site represented by a bent arrow, and horizontal arrows underline the major transcripts positions and sizes. (C) Predicted secondary structure of the NrsZ primary transcript. Each black dot represents one 3′ extremity of NrsZ obtained by 3′ RACE experiment. 5′-matured terminal nucleotides determined by RNA deep sequencing are indicated in bold. Conserved motifs in the stem-loop sequences are shown in red. SL: stem-loop structure. The M fold program was used to predict RNA secondary structures ( Zuker, 2003 ).

Techniques Used: Sequencing

The sRNA NrsZ of P. aeruginosa PAO1 is induced during nitrogen limitation by the NtrB/C-RpoN cascade. A. β-Galactosidase activities of the chromosomal reporter fusion sRNA-lacZ ( nrsZ-lacZ ) under various nitrogen-limited conditions. The PAO1 WT strain carrying nrsZ-lacZ (PAO6750) was grown in NYB, MMP supplemented with glucose as carbon source and ammonia, nitrate or casamino acids (0.1%) as nitrogen source. Activity of the nrsZ-lacZ chromosomal fusion was measured in exponential phase and when stationary phase was reached. Each value represents the average of triplicate cultures ± standard deviation. B. Northern blot detection of the sRNA: RNA was isolated from PAO1 (WT) grown to stationary phase in NYB, MMP supplemented with succinate as carbon source and ammonium or nitrate as nitrogen source. 7.5 μg of cross-linked total RNA was hybridized with the ssRNA probe NrsRNA . As loading control, the membrane was re-probed with the 5SDNA , which detects 5S rRNA. C. Northern blot detection of the sRNA: Total RNA was extracted from strains PAO1 WT, Δ rpoN (PAO6358), Δ ntrC (PAO6764), and from the strain mutated in the RpoN box of the sRNA promoter (PAO6846) grown to stationary phase in MMP supplemented with glucose and casamino acids (0.1%). 5 μg of cross-linked total RNA was hybridized with the ssRNA probe NrsRNA . As loading control, the membranes were re-probed with the 5SDNA detecting the 5S rRNA. D.β-Galactosidase activities of the chromosomal reporter fusion nrsZ-lacZ in different strains. The WT (PAO6750), Δ rpoN (PAO6847) and Δ ntrC (PAO6842) strains carrying the pME6001 empty vector, the strain Δ rpoN complemented with rpoN + (pME6001:: rpoN , pME10389) and the strain ΔntrC complemented with ntrC + (pME6001:: ntrC , pME10390) were grown in MMP supplemented with glucose and casamino acids (0.1%). nrsZ-lacZ activity was measured when stationary phase was reached. Each value represents the average of triplicate cultures ± standard deviation.
Figure Legend Snippet: The sRNA NrsZ of P. aeruginosa PAO1 is induced during nitrogen limitation by the NtrB/C-RpoN cascade. A. β-Galactosidase activities of the chromosomal reporter fusion sRNA-lacZ ( nrsZ-lacZ ) under various nitrogen-limited conditions. The PAO1 WT strain carrying nrsZ-lacZ (PAO6750) was grown in NYB, MMP supplemented with glucose as carbon source and ammonia, nitrate or casamino acids (0.1%) as nitrogen source. Activity of the nrsZ-lacZ chromosomal fusion was measured in exponential phase and when stationary phase was reached. Each value represents the average of triplicate cultures ± standard deviation. B. Northern blot detection of the sRNA: RNA was isolated from PAO1 (WT) grown to stationary phase in NYB, MMP supplemented with succinate as carbon source and ammonium or nitrate as nitrogen source. 7.5 μg of cross-linked total RNA was hybridized with the ssRNA probe NrsRNA . As loading control, the membrane was re-probed with the 5SDNA , which detects 5S rRNA. C. Northern blot detection of the sRNA: Total RNA was extracted from strains PAO1 WT, Δ rpoN (PAO6358), Δ ntrC (PAO6764), and from the strain mutated in the RpoN box of the sRNA promoter (PAO6846) grown to stationary phase in MMP supplemented with glucose and casamino acids (0.1%). 5 μg of cross-linked total RNA was hybridized with the ssRNA probe NrsRNA . As loading control, the membranes were re-probed with the 5SDNA detecting the 5S rRNA. D.β-Galactosidase activities of the chromosomal reporter fusion nrsZ-lacZ in different strains. The WT (PAO6750), Δ rpoN (PAO6847) and Δ ntrC (PAO6842) strains carrying the pME6001 empty vector, the strain Δ rpoN complemented with rpoN + (pME6001:: rpoN , pME10389) and the strain ΔntrC complemented with ntrC + (pME6001:: ntrC , pME10390) were grown in MMP supplemented with glucose and casamino acids (0.1%). nrsZ-lacZ activity was measured when stationary phase was reached. Each value represents the average of triplicate cultures ± standard deviation.

Techniques Used: Activity Assay, Standard Deviation, Northern Blot, Isolation, Plasmid Preparation

23) Product Images from "Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization"

Article Title: Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization

Journal: Planta

doi: 10.1007/s00425-013-1990-1

Cyclin expression levels during maize endosperm development. a Cyclin and actin RNA levels during endosperm development. RT-PCR was performed on total RNA isolated from endosperm at different developmental stages (indicated by DAP) with cyclin- or ACT1 -specific primers. Reaction products were separated by electrophoresis and exposed to a Phosphorscreen. b Cyclin RNA levels relative to actin RNA in developing endosperms. Signal from amplicons from two independent experiments was quantified, and expression was normalized relative to 7 DAP, which is considered one expression unit. Error bars show standard error of the means. c Immunoblot analysis of cyclins during maize endosperm development. Total soluble protein extracted from endosperm at different developmental stages (indicated by DAP) was separated by SDS-PAGE and immunoblotted with affinity-purified antibodies as indicated on right. An immunoblot with actin antibody is shown as the loading control
Figure Legend Snippet: Cyclin expression levels during maize endosperm development. a Cyclin and actin RNA levels during endosperm development. RT-PCR was performed on total RNA isolated from endosperm at different developmental stages (indicated by DAP) with cyclin- or ACT1 -specific primers. Reaction products were separated by electrophoresis and exposed to a Phosphorscreen. b Cyclin RNA levels relative to actin RNA in developing endosperms. Signal from amplicons from two independent experiments was quantified, and expression was normalized relative to 7 DAP, which is considered one expression unit. Error bars show standard error of the means. c Immunoblot analysis of cyclins during maize endosperm development. Total soluble protein extracted from endosperm at different developmental stages (indicated by DAP) was separated by SDS-PAGE and immunoblotted with affinity-purified antibodies as indicated on right. An immunoblot with actin antibody is shown as the loading control

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Electrophoresis, SDS Page, Affinity Purification

CDK expression levels during maize endosperm development. a CDK and actin RNA levels during endosperm development. RT-PCR was performed on total RNA isolated from endosperm at different developmental stages (indicated by DAP) with CDK- or ACT1 -specific primers. Reaction products were separated by electrophoresis and exposed to a Phosphorscreen. b CDK RNA levels relative to actin RNA in developing endosperms. Signal from amplicons from two independent experiments was quantified and expression was normalized relative to 7 DAP, which is considered one expression unit. Error bars show standard error of the means. c Immunoblot analysis of CDKs present during maize endosperm development. Total soluble protein extracted from endosperm at different developmental stages (indicated by DAP) was separated by SDS-PAGE and immunoblotted with affinity-purified antibodies as indicated on the right. An immunoblot with actin antibody is shown as the loading control
Figure Legend Snippet: CDK expression levels during maize endosperm development. a CDK and actin RNA levels during endosperm development. RT-PCR was performed on total RNA isolated from endosperm at different developmental stages (indicated by DAP) with CDK- or ACT1 -specific primers. Reaction products were separated by electrophoresis and exposed to a Phosphorscreen. b CDK RNA levels relative to actin RNA in developing endosperms. Signal from amplicons from two independent experiments was quantified and expression was normalized relative to 7 DAP, which is considered one expression unit. Error bars show standard error of the means. c Immunoblot analysis of CDKs present during maize endosperm development. Total soluble protein extracted from endosperm at different developmental stages (indicated by DAP) was separated by SDS-PAGE and immunoblotted with affinity-purified antibodies as indicated on the right. An immunoblot with actin antibody is shown as the loading control

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Electrophoresis, SDS Page, Affinity Purification

24) Product Images from "BioDry: An Inexpensive, Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature"

Article Title: BioDry: An Inexpensive, Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144686

Assessment of protein and m RNA integrity for seawater field samples preserved with BioDry. Gene expression for rbcL was assayed using clade-specific primers for diatoms (A) and haptophytes (B). Protein was assayed by western blot using an RbcL antibody (C). Red arrows represent assay positives for Emiliania huxleyi CCMP Glycine max (soy) leaf extract respectively. Frozen controls (T 0 ), preserved samples (T 10 T 30 days), as well as the representative DNA/contamination controls are indicated.
Figure Legend Snippet: Assessment of protein and m RNA integrity for seawater field samples preserved with BioDry. Gene expression for rbcL was assayed using clade-specific primers for diatoms (A) and haptophytes (B). Protein was assayed by western blot using an RbcL antibody (C). Red arrows represent assay positives for Emiliania huxleyi CCMP Glycine max (soy) leaf extract respectively. Frozen controls (T 0 ), preserved samples (T 10 T 30 days), as well as the representative DNA/contamination controls are indicated.

Techniques Used: Expressing, Western Blot

25) Product Images from "Transforming Growth Factor-β1 Induced Epithelial Mesenchymal Transition is blocked by a chemical antagonist of translation factor eIF4E"

Article Title: Transforming Growth Factor-β1 Induced Epithelial Mesenchymal Transition is blocked by a chemical antagonist of translation factor eIF4E

Journal: Scientific Reports

doi: 10.1038/srep18233

Pharmacological antagonism of eIF4E profoundly suppresses TGF-β1-mediated ribosome recruitment to the Snail1 transcript and nuclear accumulation of Snail1. Following pretreatment with 4Ei-1 (200 μM) for 4 h, or no pretreatment, RLE-6TN cells were treated with TGF-β1 (2.5 ng/ml) for 2 h, and processed for total and polysome bound RNA. ( a ) Shown are values for Snail1 mRNA and β-actin mRNA by qRT-PCR found in the total RNA samples normalized to the untreated sample. Shown are mean values for two independent experiments. ( b ) Relative Snail1 mRNA values across the 10 gradient fractions of polysome bound mRNA as analyzed by qRT-PCR. ( c ) To determine Snail1 intercellular localization, RLE-6TN cells were changed to medium with 0.6% serum. After 4 h, cells were treated with 4Ei-1 (100 μM) or vehicle (control) for an additional 4 h followed by TGF-β1 (2.5 ng/ml) or vehicle for 6h. Shown are representative images demonstrating nuclear Snail1 in TGF-β1-treated cells and cytoplasmic Snail1 in control, 4Ei-1, and TGF-β1 ± 4Ei-1-treated cells. d. Quantification of cells with nuclear Snail1: TGF-β1, p
Figure Legend Snippet: Pharmacological antagonism of eIF4E profoundly suppresses TGF-β1-mediated ribosome recruitment to the Snail1 transcript and nuclear accumulation of Snail1. Following pretreatment with 4Ei-1 (200 μM) for 4 h, or no pretreatment, RLE-6TN cells were treated with TGF-β1 (2.5 ng/ml) for 2 h, and processed for total and polysome bound RNA. ( a ) Shown are values for Snail1 mRNA and β-actin mRNA by qRT-PCR found in the total RNA samples normalized to the untreated sample. Shown are mean values for two independent experiments. ( b ) Relative Snail1 mRNA values across the 10 gradient fractions of polysome bound mRNA as analyzed by qRT-PCR. ( c ) To determine Snail1 intercellular localization, RLE-6TN cells were changed to medium with 0.6% serum. After 4 h, cells were treated with 4Ei-1 (100 μM) or vehicle (control) for an additional 4 h followed by TGF-β1 (2.5 ng/ml) or vehicle for 6h. Shown are representative images demonstrating nuclear Snail1 in TGF-β1-treated cells and cytoplasmic Snail1 in control, 4Ei-1, and TGF-β1 ± 4Ei-1-treated cells. d. Quantification of cells with nuclear Snail1: TGF-β1, p

Techniques Used: Quantitative RT-PCR

26) Product Images from "The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation"

Article Title: The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw474

Deregulation of feedback inhibitors of inflammation through TTP and TTP-AA. ( A–C ) Genomic tracks for the feedback inhibitors Dusp1 (A), Ier3 (B) and Tnfaip3 (C) containing information for iCLIP, RNASeq and RiboSeq data for GFP-, GFP–TTP- and GFP–TTP-AA-expressing cells are shown in the format as introduced in Figure 3 . ATTTA and TATTTAT repeats are indicated below the iCLIP tracks. Dimensions are indicated by the numbers in brackets for each track. The graph to the right of each track shows the RNA-IPs for GFP–TTP and GFP–TTP-AA in relation to the GFP/EV Control IP. Asterisks (*) indicate P -values ≤ 0.05 for GFP–TTP-AA IP compared to GFP–TTP IP. ( D ) Relative mRNA levels of the indicated transcripts were determined by qPCR upon different stimulations. The control conditions of GFP/EV-expressing cells were set to 1. Fold changes were calculated in relation to this condition. ( E ) Log 2 -fold changes in translation versus changes in mean expression (log 10 ) for a set of feedback inhibitors of the inflammatory response ( 14 ) and selected other mRNAs in GFP–TTP-AA- versus GFP–TTP-expressing cells. The position of individual transcripts is marked. TTP-bound iCLIP target mRNAs are shown as red dots, whereas non-targets are black. ( F ) Levels of the Dusp1-, Ier3- and Tnfaip3-protein were determined by western blot in the TTP BMDM cell lines upon different stimulations. GAPDH served as a loading control. Quantifications of western blots are given in Supplementary Figure S8.
Figure Legend Snippet: Deregulation of feedback inhibitors of inflammation through TTP and TTP-AA. ( A–C ) Genomic tracks for the feedback inhibitors Dusp1 (A), Ier3 (B) and Tnfaip3 (C) containing information for iCLIP, RNASeq and RiboSeq data for GFP-, GFP–TTP- and GFP–TTP-AA-expressing cells are shown in the format as introduced in Figure 3 . ATTTA and TATTTAT repeats are indicated below the iCLIP tracks. Dimensions are indicated by the numbers in brackets for each track. The graph to the right of each track shows the RNA-IPs for GFP–TTP and GFP–TTP-AA in relation to the GFP/EV Control IP. Asterisks (*) indicate P -values ≤ 0.05 for GFP–TTP-AA IP compared to GFP–TTP IP. ( D ) Relative mRNA levels of the indicated transcripts were determined by qPCR upon different stimulations. The control conditions of GFP/EV-expressing cells were set to 1. Fold changes were calculated in relation to this condition. ( E ) Log 2 -fold changes in translation versus changes in mean expression (log 10 ) for a set of feedback inhibitors of the inflammatory response ( 14 ) and selected other mRNAs in GFP–TTP-AA- versus GFP–TTP-expressing cells. The position of individual transcripts is marked. TTP-bound iCLIP target mRNAs are shown as red dots, whereas non-targets are black. ( F ) Levels of the Dusp1-, Ier3- and Tnfaip3-protein were determined by western blot in the TTP BMDM cell lines upon different stimulations. GAPDH served as a loading control. Quantifications of western blots are given in Supplementary Figure S8.

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

Examples of established and novel TTP targets identified by iCLIP and their validation by RNA-IP and expression analysis. ( A–C ) Genomic tracks for TNF (A), Cxcl10 (B) and Gdf15 (C) are shown, containing information for iCLIP, RNASeq and RiboSeq for GFP-, GFP–TTP- and GFP–TTP-AA-expressing cells. ATTTA and TATTTAT repeats are indicated as red dots below the iCLIP tracks. Dimensions are indicated by the numbers in brackets for each track. At the right of the tracks, RNA-IPs for GFP–TTP and GFP–TTP-AA are shown in relation to the GFP/EV control IP. The asterisk (*) indicates P -values ≤ 0.05 for GFP–TTP-AA IP compared to GFP–TTP IP. ( D ) Cxcl10 mRNA level in the three different cell lines were determined by qPCR. ( E ) Cxcl10 protein levels were determined by ELISA for the indicated stimulatory conditions. 1 × 10 4 cells were seeded in triplicates in a 96-well plate. After attachment of the cells, stimulation was started and Cxcl10 protein in supernatants was measured. The asterisk (*) indicates P -values ≤ 0.03; n.d.: not detectable.
Figure Legend Snippet: Examples of established and novel TTP targets identified by iCLIP and their validation by RNA-IP and expression analysis. ( A–C ) Genomic tracks for TNF (A), Cxcl10 (B) and Gdf15 (C) are shown, containing information for iCLIP, RNASeq and RiboSeq for GFP-, GFP–TTP- and GFP–TTP-AA-expressing cells. ATTTA and TATTTAT repeats are indicated as red dots below the iCLIP tracks. Dimensions are indicated by the numbers in brackets for each track. At the right of the tracks, RNA-IPs for GFP–TTP and GFP–TTP-AA are shown in relation to the GFP/EV control IP. The asterisk (*) indicates P -values ≤ 0.05 for GFP–TTP-AA IP compared to GFP–TTP IP. ( D ) Cxcl10 mRNA level in the three different cell lines were determined by qPCR. ( E ) Cxcl10 protein levels were determined by ELISA for the indicated stimulatory conditions. 1 × 10 4 cells were seeded in triplicates in a 96-well plate. After attachment of the cells, stimulation was started and Cxcl10 protein in supernatants was measured. The asterisk (*) indicates P -values ≤ 0.03; n.d.: not detectable.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Correlation of iCLIP, mRNA abundance (RNASeq) and translation (RiboSeq) to identify global consequences of TTP- and TTP-AA-binding for the myeloid transcriptome. ( A ) Scheme for the correlations made between RNA levels or abundance (RNASeq), ribosomal occupation of mRNAs (RibosSeq) and the iCLIP data. RNASeq and RiboSeq measurements were performed in triplicate for each GFP-protein. See Supplementary Table S1 for number of replicates and conditions. ( B ) Cluster analysis for the RNASeq samples. Three replicates of cells each expressing GFP/EV, GFP–TTP or GFP–TTP-AA following 6 h Dox/1h LPS, are numbered 1, 2 and 3. ( C and D ) Overall changes in mRNA abundance (RNASeq (c)) and mRNA translation (RiboSeq (d)) between the indicated genotypes comparing the following TTP targets: iCLIP targets (white boxes) and non-bound mRNAs (gray boxes) are shown as a kernel density distribution in boxplots. A Wilcoxon rank sum test with continuity correction was used to determine the P -values shown for each comparison. Statistically significant differences in abundance and translation between targets and non-targets are indicated in red. For each genotype n = 3 sequencing libraries were analyzed and the mean of each group is shown. The red lines indicate for y = 0. Highly statistically significant differences are indicated in red. ( E and F ) For the comparisons between TTP/GFP and TTP-AA/GFP changed targets obtained from (C) and (D) were assigned to the mRNA region (5′UTR, ORF and 3′UTR) leading to detailed (E) RNASeq and (F) RiboSeq blots. A Wilcoxon rank sum test with continuity correction was used to determine the P -values shown for every comparison. The red lines indicate for y = 0. Highly statistically significant differences in abundance (E) and translation (F) between targets (white boxes) and non-targets (gray boxes) are indicated in red and could only be detected for targets where TTP binds in the 3′UTR. ( G ) The overlap of DE genes from RNASeq and RiboSeq experiments is visualized by Venn diagrams for the different comparisons. The number of genes for the RNASeq (changes in mRNA abundance), RiboSeq (changes in translation) and RNASeq/RiboSeq (changes in abundance and translation) are given together with the number of iCLIP targets in each group (in brackets).
Figure Legend Snippet: Correlation of iCLIP, mRNA abundance (RNASeq) and translation (RiboSeq) to identify global consequences of TTP- and TTP-AA-binding for the myeloid transcriptome. ( A ) Scheme for the correlations made between RNA levels or abundance (RNASeq), ribosomal occupation of mRNAs (RibosSeq) and the iCLIP data. RNASeq and RiboSeq measurements were performed in triplicate for each GFP-protein. See Supplementary Table S1 for number of replicates and conditions. ( B ) Cluster analysis for the RNASeq samples. Three replicates of cells each expressing GFP/EV, GFP–TTP or GFP–TTP-AA following 6 h Dox/1h LPS, are numbered 1, 2 and 3. ( C and D ) Overall changes in mRNA abundance (RNASeq (c)) and mRNA translation (RiboSeq (d)) between the indicated genotypes comparing the following TTP targets: iCLIP targets (white boxes) and non-bound mRNAs (gray boxes) are shown as a kernel density distribution in boxplots. A Wilcoxon rank sum test with continuity correction was used to determine the P -values shown for each comparison. Statistically significant differences in abundance and translation between targets and non-targets are indicated in red. For each genotype n = 3 sequencing libraries were analyzed and the mean of each group is shown. The red lines indicate for y = 0. Highly statistically significant differences are indicated in red. ( E and F ) For the comparisons between TTP/GFP and TTP-AA/GFP changed targets obtained from (C) and (D) were assigned to the mRNA region (5′UTR, ORF and 3′UTR) leading to detailed (E) RNASeq and (F) RiboSeq blots. A Wilcoxon rank sum test with continuity correction was used to determine the P -values shown for every comparison. The red lines indicate for y = 0. Highly statistically significant differences in abundance (E) and translation (F) between targets (white boxes) and non-targets (gray boxes) are indicated in red and could only be detected for targets where TTP binds in the 3′UTR. ( G ) The overlap of DE genes from RNASeq and RiboSeq experiments is visualized by Venn diagrams for the different comparisons. The number of genes for the RNASeq (changes in mRNA abundance), RiboSeq (changes in translation) and RNASeq/RiboSeq (changes in abundance and translation) are given together with the number of iCLIP targets in each group (in brackets).

Techniques Used: Binding Assay, Expressing, Sequencing

27) Product Images from "CD26 Expression on T-Anaplastic Large Cell Lymphoma (ALCL) Line Karpas 299 is associated with increased expression of Versican and MT1-MMP and enhanced adhesion"

Article Title: CD26 Expression on T-Anaplastic Large Cell Lymphoma (ALCL) Line Karpas 299 is associated with increased expression of Versican and MT1-MMP and enhanced adhesion

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-517

Confirmation of Versican expression in Karpas 299 cells and in CD26-depleted and Versican-depleted Karpas cells. A . Western blots confirmed versican expression in Karpas cell lines and clones resulting from knockdown of versican in parental Karpas 299 cells using shRNA. Whole cell lysates (30 μg) of Karpas, Dep1, Dep2, and two clones derived from knock down of versican in parental Karpas cells, 1A12 and 6RD3 were run on 7.5% gels. The top of the gel and 250 kD marker are indicated. Blots were probed with anti-versican antibody at 1:100 dilution, followed by anti-mouse HRP at 1:10,000 dilution. B . RT-PCR using V0 and V1 specific primers show product was present when RNA from the parental Karpas 299 cells was used but barely detectable when RNA from Dep1 or Dep2 was used as the template. Results from Western blots and RT-PCR were obtained from two independent experiments.
Figure Legend Snippet: Confirmation of Versican expression in Karpas 299 cells and in CD26-depleted and Versican-depleted Karpas cells. A . Western blots confirmed versican expression in Karpas cell lines and clones resulting from knockdown of versican in parental Karpas 299 cells using shRNA. Whole cell lysates (30 μg) of Karpas, Dep1, Dep2, and two clones derived from knock down of versican in parental Karpas cells, 1A12 and 6RD3 were run on 7.5% gels. The top of the gel and 250 kD marker are indicated. Blots were probed with anti-versican antibody at 1:100 dilution, followed by anti-mouse HRP at 1:10,000 dilution. B . RT-PCR using V0 and V1 specific primers show product was present when RNA from the parental Karpas 299 cells was used but barely detectable when RNA from Dep1 or Dep2 was used as the template. Results from Western blots and RT-PCR were obtained from two independent experiments.

Techniques Used: Expressing, Western Blot, Clone Assay, shRNA, Derivative Assay, Marker, Reverse Transcription Polymerase Chain Reaction

28) Product Images from "Computational Detection and Functional Analysis of Human Tissue-Specific A-to-I RNA Editing"

Article Title: Computational Detection and Functional Analysis of Human Tissue-Specific A-to-I RNA Editing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018129

Experimental validation of the predicted brain-specific and ovary-specific RNA editing sites. (A) The up/downstream region of the brain-specific RNA editing site was amplified successfully from cDNAs and gDNA of two adjacent non-cancerous brain tissues, as well as the HepG2, K562, MDA-MB-231, 293T, Raji, SW480 and SF126 cell lines. (B) Sequencing results of paired genomic DNA (control) and cDNA from the same human brain specimens and seven human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in both adjacent non-cancerous brain tissues and the human glioma cell line SF126. (C) The up/downstream region of the ovary-specific RNA editing site was amplified successfully from cDNAs and gDNA of the OVCAR3, SKOV3, SF126, HepG2, Raji, 293T, SW480, U2OS, K562 and MDA-MB-231 cell lines. (D) Sequencing results of paired genomic DNA (control) and cDNA from the same ten human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in the two human ovarian cancer cell lines SKOV3 and OVCAR3.
Figure Legend Snippet: Experimental validation of the predicted brain-specific and ovary-specific RNA editing sites. (A) The up/downstream region of the brain-specific RNA editing site was amplified successfully from cDNAs and gDNA of two adjacent non-cancerous brain tissues, as well as the HepG2, K562, MDA-MB-231, 293T, Raji, SW480 and SF126 cell lines. (B) Sequencing results of paired genomic DNA (control) and cDNA from the same human brain specimens and seven human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in both adjacent non-cancerous brain tissues and the human glioma cell line SF126. (C) The up/downstream region of the ovary-specific RNA editing site was amplified successfully from cDNAs and gDNA of the OVCAR3, SKOV3, SF126, HepG2, Raji, 293T, SW480, U2OS, K562 and MDA-MB-231 cell lines. (D) Sequencing results of paired genomic DNA (control) and cDNA from the same ten human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in the two human ovarian cancer cell lines SKOV3 and OVCAR3.

Techniques Used: Amplification, Multiple Displacement Amplification, Sequencing

29) Product Images from "The Base Excision Repair Pathway Is Required for Efficient Lentivirus Integration"

Article Title: The Base Excision Repair Pathway Is Required for Efficient Lentivirus Integration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017862

Infection of BER cell lines with HIV with and without accessory genes or the cPPT. Wild type (WT), Myh −/−, and Ogg1 −/− cell lines, wild type ( Neil1 +/+) and Neil1 −/− cell lines, or wild type ( Polß +/+) and Polß −/− cell lines were infected with HIV retroviral vectors. Wild type murine embryonic fibroblasts were from littermates. Cells were analyzed at 72 hpi by flow cytometry for GFP expression. (A) Cells were infected with an HIV vector with accessory genes or without accessory genes (Δ vif Δ vpr Δ vpu Δ nef ). (B) Total RNA was isolated from wild type HIV and HIV(Δ vif Δ vpr Δ vpu Δ nef ) vector producer cells, treated with DNaseI to digest producer plasmids, and re-isolated. RNA fractions were amplified with the same conditions by RT-PCR or PCR, to confirm the absence of contaminating producer plasmids. RT-PCR and PCR targets were Lane 1 water negative control, Lane 2 HIV(Δ vif Δ vpr Δ vpu Δ nef ) RNA, Lane 3 wild type HIV RNA, and Lane 4 wild type HIV producer plasmid DNA, positive control for PCR. Primers amplified the accessory genes vpu , vpr , vif , and nef as well as the gag gene. (C) Cells were infected with an HIV vector with or without the cPPT. Infections were performed at two MOI in duplicate at least three times. Error bars indicate the standard deviation after normalization.
Figure Legend Snippet: Infection of BER cell lines with HIV with and without accessory genes or the cPPT. Wild type (WT), Myh −/−, and Ogg1 −/− cell lines, wild type ( Neil1 +/+) and Neil1 −/− cell lines, or wild type ( Polß +/+) and Polß −/− cell lines were infected with HIV retroviral vectors. Wild type murine embryonic fibroblasts were from littermates. Cells were analyzed at 72 hpi by flow cytometry for GFP expression. (A) Cells were infected with an HIV vector with accessory genes or without accessory genes (Δ vif Δ vpr Δ vpu Δ nef ). (B) Total RNA was isolated from wild type HIV and HIV(Δ vif Δ vpr Δ vpu Δ nef ) vector producer cells, treated with DNaseI to digest producer plasmids, and re-isolated. RNA fractions were amplified with the same conditions by RT-PCR or PCR, to confirm the absence of contaminating producer plasmids. RT-PCR and PCR targets were Lane 1 water negative control, Lane 2 HIV(Δ vif Δ vpr Δ vpu Δ nef ) RNA, Lane 3 wild type HIV RNA, and Lane 4 wild type HIV producer plasmid DNA, positive control for PCR. Primers amplified the accessory genes vpu , vpr , vif , and nef as well as the gag gene. (C) Cells were infected with an HIV vector with or without the cPPT. Infections were performed at two MOI in duplicate at least three times. Error bars indicate the standard deviation after normalization.

Techniques Used: Infection, Flow Cytometry, Cytometry, Expressing, Plasmid Preparation, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Positive Control, Standard Deviation

30) Product Images from "Expression and Activity of a Novel Cathelicidin from Domestic Cats"

Article Title: Expression and Activity of a Novel Cathelicidin from Domestic Cats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018756

Expression and immunolocalization of feline cathelicidin. (A) Quantitative RT-PCR (qPCR) analysis of absolute copy number of mRNA transcripts encoding feCath in RNA isolated from duodenum (n = 8), proximal jejunum (n = 7), distal jejunum (n = 8), ileum (n = 8), colon (n = 8), skin (n = 2) and bone marrow (n = 7). Non-template control (NTC) in qPCR yielded
Figure Legend Snippet: Expression and immunolocalization of feline cathelicidin. (A) Quantitative RT-PCR (qPCR) analysis of absolute copy number of mRNA transcripts encoding feCath in RNA isolated from duodenum (n = 8), proximal jejunum (n = 7), distal jejunum (n = 8), ileum (n = 8), colon (n = 8), skin (n = 2) and bone marrow (n = 7). Non-template control (NTC) in qPCR yielded

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

3′ RACE Analysis and Identification of feCath. (A) Schematic of 3′ RACE strategy targeting the signal sequence region and the propeptide (cathelin) domain with sense primers and using antisense adaptor primer, AP1. Feline bone marrow RNA was reverse transcribed with oligo-dT conjugated to adaptor primer 1/2 (AP1/2). Sense primers (sequences found in Table 1 ) were used to amplify cathelicidin related sequences in the pool of bone marrow cDNA. (B) Agarose gel electrophoresis analysis of 3′ RACE PCR products from (A). HaeIII digested Phi-X174 phage DNA used as the marker.
Figure Legend Snippet: 3′ RACE Analysis and Identification of feCath. (A) Schematic of 3′ RACE strategy targeting the signal sequence region and the propeptide (cathelin) domain with sense primers and using antisense adaptor primer, AP1. Feline bone marrow RNA was reverse transcribed with oligo-dT conjugated to adaptor primer 1/2 (AP1/2). Sense primers (sequences found in Table 1 ) were used to amplify cathelicidin related sequences in the pool of bone marrow cDNA. (B) Agarose gel electrophoresis analysis of 3′ RACE PCR products from (A). HaeIII digested Phi-X174 phage DNA used as the marker.

Techniques Used: Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

31) Product Images from "Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse"

Article Title: Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019839

Phisiological parameters. A, B : Analysis of blood glucose and Insulin levels after intra-peritoneal glucose tolerance test (upper panels). Glycemia was measured by glucometer, while Insulin concentration was assessed by ELISA assay, as described in the Materials and Methods section. Oral administration of T3 significantly reduces severity and progression of STZ-induced diabetes in Balb/c mice and assured normal Insulin responsiveness. C : Insulin tolerance was performed (lower panel) after intra peritoneal glucose injection. Insulin was injected intraperitoneally after glucose to the different experimental groups of animals. Glycemia was measured by glucometer. Results represent the mean ± SE of three separate experiments. Grey: control black:STZ white:STZ+T3. D RT-PCR: Total RNA was extracted from liver from mice of the different experimental groups and RTPCR was performed as described in the Materials and Methods section. A single product was obtained for each gene, as showed by agarose electrophoresis. All PCR products were of the expected size and sequence. The presence of T3 did not induce any change in the DIO1 expression, as normalized to 18s.
Figure Legend Snippet: Phisiological parameters. A, B : Analysis of blood glucose and Insulin levels after intra-peritoneal glucose tolerance test (upper panels). Glycemia was measured by glucometer, while Insulin concentration was assessed by ELISA assay, as described in the Materials and Methods section. Oral administration of T3 significantly reduces severity and progression of STZ-induced diabetes in Balb/c mice and assured normal Insulin responsiveness. C : Insulin tolerance was performed (lower panel) after intra peritoneal glucose injection. Insulin was injected intraperitoneally after glucose to the different experimental groups of animals. Glycemia was measured by glucometer. Results represent the mean ± SE of three separate experiments. Grey: control black:STZ white:STZ+T3. D RT-PCR: Total RNA was extracted from liver from mice of the different experimental groups and RTPCR was performed as described in the Materials and Methods section. A single product was obtained for each gene, as showed by agarose electrophoresis. All PCR products were of the expected size and sequence. The presence of T3 did not induce any change in the DIO1 expression, as normalized to 18s.

Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Polymerase Chain Reaction, Sequencing, Expressing

32) Product Images from "Analyses and interpretation of whole-genome gene expression from formalin-fixed paraffin-embedded tissue: an illustration with breast cancer tissues"

Article Title: Analyses and interpretation of whole-genome gene expression from formalin-fixed paraffin-embedded tissue: an illustration with breast cancer tissues

Journal: BMC Genomics

doi: 10.1186/1471-2164-11-622

Principle components analysis (PCA) and sources of variation . Principle components analysis (PCA) of 71 samples displaying spatial separation (expression level clustering) of RNA extracted from FF samples (shown in red on left) and FFPE samples (shown in blue on right); The three principle components (PC#1, #2 #3 and respective contributable variations are presented on x-, y- and z-axis along). A Statistical significance of the different
Figure Legend Snippet: Principle components analysis (PCA) and sources of variation . Principle components analysis (PCA) of 71 samples displaying spatial separation (expression level clustering) of RNA extracted from FF samples (shown in red on left) and FFPE samples (shown in blue on right); The three principle components (PC#1, #2 #3 and respective contributable variations are presented on x-, y- and z-axis along). A Statistical significance of the different "sources of variation" in the total gene expression estimated by 3-way ANOVA models. F ratio for each factor (source) represents the F-statistics for that factor/F-statistics for error (noise). The mean of the F Ratio for all the genes for a particular factor are shown on Y-axis and the different factors (sources of variation) are shown in x-axis.

Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded

Scatter plot of the log 2 -transformed signal intensities . Scatter plot of the log 2 -transformed signal intensities of representative samples are shown in 1A - 1E. The central straight line represents the regression line and the two lines on the either sides mark the boundaries for 2-fold changes. Pearson correlation coefficient squared (r 2 ) for each scatter plot is shown on the top. Figure-1A shows the signal intensities of two technical replicates of RNA samples. Figure-1B 1C show correlations between corresponding FF (x-axis) FFPE samples (y-axis) in normal breast tissue and tumor tissue respectively. Figure 1D 1E show correlations between corresponding tumor (y-axis) and normal (x-axis) tissue from same patient using FF and FFPE samples respectively.
Figure Legend Snippet: Scatter plot of the log 2 -transformed signal intensities . Scatter plot of the log 2 -transformed signal intensities of representative samples are shown in 1A - 1E. The central straight line represents the regression line and the two lines on the either sides mark the boundaries for 2-fold changes. Pearson correlation coefficient squared (r 2 ) for each scatter plot is shown on the top. Figure-1A shows the signal intensities of two technical replicates of RNA samples. Figure-1B 1C show correlations between corresponding FF (x-axis) FFPE samples (y-axis) in normal breast tissue and tumor tissue respectively. Figure 1D 1E show correlations between corresponding tumor (y-axis) and normal (x-axis) tissue from same patient using FF and FFPE samples respectively.

Techniques Used: Transformation Assay, Formalin-fixed Paraffin-Embedded

33) Product Images from "Spermidine Promotes Human Hair Growth and Is a Novel Modulator of Human Epithelial Stem Cell Functions"

Article Title: Spermidine Promotes Human Hair Growth and Is a Novel Modulator of Human Epithelial Stem Cell Functions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022564

Spermidine up-regulates expression of the epithelial stem cell-associated keratins K15 and K19. A . Spermidine treatment for 6d increased K15-like immunoreactivity in the 0.5 µM and 1 µM doses, as assessed by quantitative immunoreactivity. n = 10–15 HFs/group; cumulative results of two different experiments. B. Spermidine significantly upregulated K15 mRNA expression in cultured ORS keratinocytes after 48 h of treatment. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. C. DFMO (400 mg/l) significantly decreased K15-like immunoreactivity after 6d. n = 6–8 HFs/group . D. Spermidine treatment for 6d increased K19-like immunoreactivity in the 0.1 µM and 0.5 µM doses, as assessed by quantitative immunoreactivity. n = 30–36 HFs/group; cumulative results of two different experiments. E. By Q-PCR, spermidine treatment for 48 h significantly increased the K19 transcript steady-state levels in human anagen HF mRNA extracts. F. Treatment of ORS keratinocytes for 48 h showed a tendency of K19 mRNA upregulation, but these results were not statistically significant. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. Scale bars, 50 µm. Columns represent means±SEM; * P
Figure Legend Snippet: Spermidine up-regulates expression of the epithelial stem cell-associated keratins K15 and K19. A . Spermidine treatment for 6d increased K15-like immunoreactivity in the 0.5 µM and 1 µM doses, as assessed by quantitative immunoreactivity. n = 10–15 HFs/group; cumulative results of two different experiments. B. Spermidine significantly upregulated K15 mRNA expression in cultured ORS keratinocytes after 48 h of treatment. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. C. DFMO (400 mg/l) significantly decreased K15-like immunoreactivity after 6d. n = 6–8 HFs/group . D. Spermidine treatment for 6d increased K19-like immunoreactivity in the 0.1 µM and 0.5 µM doses, as assessed by quantitative immunoreactivity. n = 30–36 HFs/group; cumulative results of two different experiments. E. By Q-PCR, spermidine treatment for 48 h significantly increased the K19 transcript steady-state levels in human anagen HF mRNA extracts. F. Treatment of ORS keratinocytes for 48 h showed a tendency of K19 mRNA upregulation, but these results were not statistically significant. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. Scale bars, 50 µm. Columns represent means±SEM; * P

Techniques Used: Expressing, Cell Culture, Polymerase Chain Reaction

34) Product Images from "Transcriptional properties of human NANOG1 and NANOG2 in acute leukemic cells"

Article Title: Transcriptional properties of human NANOG1 and NANOG2 in acute leukemic cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq307

Gene expression profiling experiment. ( A ) The cell line 293 was transfected with episomal pEPI-EGFP vector containing expression cassettes for NANOG1A, NANOG2D2c or NANOG2E. After 10 weeks of culture, GPF-positive cells were sorted and used for RNA isolation. These RNA samples were used for Affymetrix chip experiments, resulting in few deregulated genes shown in the right. White boxed areas: up-regulated genes. Light grey boxed areas: down-regulated genes. Common genes identified in all three experiments using the different NANOG1/2 protein variants are highlighted. ( B ) Mode of action for the investigated NANOG protein variants. NANOG protein is able to induce transcription of EGR1 (directly or indirectly). EGR1 protein is directly binding to the promoter regions of FOS and p21 . P21 protein is necessary for stem cell maintenance, while the FOS transcription factor is necessary for cell proliferation.
Figure Legend Snippet: Gene expression profiling experiment. ( A ) The cell line 293 was transfected with episomal pEPI-EGFP vector containing expression cassettes for NANOG1A, NANOG2D2c or NANOG2E. After 10 weeks of culture, GPF-positive cells were sorted and used for RNA isolation. These RNA samples were used for Affymetrix chip experiments, resulting in few deregulated genes shown in the right. White boxed areas: up-regulated genes. Light grey boxed areas: down-regulated genes. Common genes identified in all three experiments using the different NANOG1/2 protein variants are highlighted. ( B ) Mode of action for the investigated NANOG protein variants. NANOG protein is able to induce transcription of EGR1 (directly or indirectly). EGR1 protein is directly binding to the promoter regions of FOS and p21 . P21 protein is necessary for stem cell maintenance, while the FOS transcription factor is necessary for cell proliferation.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Isolation, Chromatin Immunoprecipitation, Binding Assay

35) Product Images from "Deciphering Variability of PKD1 and PKD2 in an Italian Cohort of 643 Patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD)"

Article Title: Deciphering Variability of PKD1 and PKD2 in an Italian Cohort of 643 Patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Journal: Scientific Reports

doi: 10.1038/srep30850

( A ) Pedigree. In this family, the proband (arrowed) inherited a synonymous PKD1 variant (c.2097G > T, p. Ser699=) from his mother. ( B ) RT-PCR from whole blood RNA. In the control sample (Wt), only the expected 585 bp fragment was present. In the patient sample (P), an additional 1038 bp abnormal fragment was amplified. ( C ) Sequencing of the regular and abnormal fragments from the patient sample. In the upper panel, the sequence of the 585 bp normal c.2097G allele cDNA showing the adjacent exons 10 and 11, correctly spliced; in the lower panel, the sequence of the abnormal 1038 bp cDNA showing that in the mutated allele c.2097T, intron 10 has been completely retained in the transcript. ( D ) A scheme of the spliced regions in the normal and mutant alleles. Filled symbol: ADPKD; empty symbol: healthy subject.
Figure Legend Snippet: ( A ) Pedigree. In this family, the proband (arrowed) inherited a synonymous PKD1 variant (c.2097G > T, p. Ser699=) from his mother. ( B ) RT-PCR from whole blood RNA. In the control sample (Wt), only the expected 585 bp fragment was present. In the patient sample (P), an additional 1038 bp abnormal fragment was amplified. ( C ) Sequencing of the regular and abnormal fragments from the patient sample. In the upper panel, the sequence of the 585 bp normal c.2097G allele cDNA showing the adjacent exons 10 and 11, correctly spliced; in the lower panel, the sequence of the abnormal 1038 bp cDNA showing that in the mutated allele c.2097T, intron 10 has been completely retained in the transcript. ( D ) A scheme of the spliced regions in the normal and mutant alleles. Filled symbol: ADPKD; empty symbol: healthy subject.

Techniques Used: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Mutagenesis

36) Product Images from "Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA"

Article Title: Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA

Journal: Scientific Reports

doi: 10.1038/s41598-017-12186-w

Ribonucleolytic site of Zm ASCH. ( a ) Simulated Zm ASCH structure in complex with an ssRNA AGGACAU. The Zm ASCH apo -structure and two representative simulated complex structures were superimposed. Three protein structures were displayed with coils of different colors. The bound ssRNA is depicted as blue lines; 5′- and 3′-ends are labeled. Some key residues that interact with ssRNA, which were mutated, are depicted as stick models. ( b , c ) Interaction of Zm ASCH with an ssRNA at two sites. The bound ssRNA (cyan-colored carbon) and the protein residues (green-colored carbon) were drawn as thick stick models. Water molecules are displayed as red spheres and polar interactions among atoms are indicated with black-dotted lines. ( d ) 32 P-labelled RNA or DNA probes (0.3 μM) were incubated with wild-type and mutant proteins of Zm ASCH (3 μM) for 30 min at 310 K in the presence of 10 mM MgCl 2 . The bracket on the left panel indicates the degradation products.
Figure Legend Snippet: Ribonucleolytic site of Zm ASCH. ( a ) Simulated Zm ASCH structure in complex with an ssRNA AGGACAU. The Zm ASCH apo -structure and two representative simulated complex structures were superimposed. Three protein structures were displayed with coils of different colors. The bound ssRNA is depicted as blue lines; 5′- and 3′-ends are labeled. Some key residues that interact with ssRNA, which were mutated, are depicted as stick models. ( b , c ) Interaction of Zm ASCH with an ssRNA at two sites. The bound ssRNA (cyan-colored carbon) and the protein residues (green-colored carbon) were drawn as thick stick models. Water molecules are displayed as red spheres and polar interactions among atoms are indicated with black-dotted lines. ( d ) 32 P-labelled RNA or DNA probes (0.3 μM) were incubated with wild-type and mutant proteins of Zm ASCH (3 μM) for 30 min at 310 K in the presence of 10 mM MgCl 2 . The bracket on the left panel indicates the degradation products.

Techniques Used: Labeling, Incubation, Mutagenesis

37) Product Images from "Mycobacterium tuberculosis PPE32 promotes cytokines production and host cell apoptosis through caspase cascade accompanying with enhanced ER stress response"

Article Title: Mycobacterium tuberculosis PPE32 promotes cytokines production and host cell apoptosis through caspase cascade accompanying with enhanced ER stress response

Journal: Oncotarget

doi: 10.18632/oncotarget.12030

ER stress is involved in PPE32-inducedt macrophage apoptosis ( A ) Macrophages were stimulated with 5 μg/ml PPE32 for 6 and 12 h. Total RNA was isolated and 1.0 μg of total RNA was used to generate cDNAs by reverse transcription PCR and amplified using XBP-1 specific primers that were used to amplify products of unspliced and spliced mRNA. After RT-PCR, the amplified products were separated by electrophoresis on a 3% agarose gel and visualized by ethidium bromide staining. ( B ) The expression of CHOP mRNA in macrophages after incubation with PPE32. ( C ) The expression of GRP78 mRNA in macrophages after treatment with PPE32. The statistical significance of observed differences between antigen stimulated and unstimulated groups were verified by student t -test.
Figure Legend Snippet: ER stress is involved in PPE32-inducedt macrophage apoptosis ( A ) Macrophages were stimulated with 5 μg/ml PPE32 for 6 and 12 h. Total RNA was isolated and 1.0 μg of total RNA was used to generate cDNAs by reverse transcription PCR and amplified using XBP-1 specific primers that were used to amplify products of unspliced and spliced mRNA. After RT-PCR, the amplified products were separated by electrophoresis on a 3% agarose gel and visualized by ethidium bromide staining. ( B ) The expression of CHOP mRNA in macrophages after incubation with PPE32. ( C ) The expression of GRP78 mRNA in macrophages after treatment with PPE32. The statistical significance of observed differences between antigen stimulated and unstimulated groups were verified by student t -test.

Techniques Used: Isolation, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Staining, Expressing, Incubation

38) Product Images from "Inhibition of HIV-1 infection in humanized mice and metabolic stability of protein phosphatase-1-targeting small molecule 1E7-03"

Article Title: Inhibition of HIV-1 infection in humanized mice and metabolic stability of protein phosphatase-1-targeting small molecule 1E7-03

Journal: Oncotarget

doi: 10.18632/oncotarget.19999

Antiviral efficacy of 1E7-03 in HIV-1 89. 6-infected NSG mice Mice were treated with 3 mg/kg of 1E7-03 or 1.5 mg/kg F07#13 by a single dose intraperitoneal (i.p.) injection After 48 hrs, mice were sacrificed; plasma samples were collected and tested for levels of HIV-1 TAR RNA (A) and HIV-1 TAR-gag RNA (B) . For quantitative analysis of HIV-1 RNA, total RNA was isolated from blood specimens, treated with RNase-free DNase I and reverse transcribed. Real-time PCR reactions were carried out in triplicates. Each data point represents the blood from a single animal. For all figures, * p
Figure Legend Snippet: Antiviral efficacy of 1E7-03 in HIV-1 89. 6-infected NSG mice Mice were treated with 3 mg/kg of 1E7-03 or 1.5 mg/kg F07#13 by a single dose intraperitoneal (i.p.) injection After 48 hrs, mice were sacrificed; plasma samples were collected and tested for levels of HIV-1 TAR RNA (A) and HIV-1 TAR-gag RNA (B) . For quantitative analysis of HIV-1 RNA, total RNA was isolated from blood specimens, treated with RNase-free DNase I and reverse transcribed. Real-time PCR reactions were carried out in triplicates. Each data point represents the blood from a single animal. For all figures, * p

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

39) Product Images from "Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells. Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells"

Article Title: Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells. Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells

Journal: Journal of Cellular Physiology

doi: 10.1002/jcp.26222

qPCR validation of RNA sequencing results. Gene expression (log 2 fold change) of selected target genes is altered upon 16 hr fluid shear stress, as measured by quantitative PCR. Parallel plate flow‐chamber induced fluid shear stress at 2.0 dyn/cm 2 in PTECs; n = 13 per condition; Hprt served as housekeeping gene to correct for cDNA input; data were normalized to static controls (log 2 fold change = 0). *Indicates significantly altered expression by flow versus no flow ( p
Figure Legend Snippet: qPCR validation of RNA sequencing results. Gene expression (log 2 fold change) of selected target genes is altered upon 16 hr fluid shear stress, as measured by quantitative PCR. Parallel plate flow‐chamber induced fluid shear stress at 2.0 dyn/cm 2 in PTECs; n = 13 per condition; Hprt served as housekeeping gene to correct for cDNA input; data were normalized to static controls (log 2 fold change = 0). *Indicates significantly altered expression by flow versus no flow ( p

Techniques Used: Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Expressing, Flow Cytometry

40) Product Images from "Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling"

Article Title: Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02622

Abundance distributions of COGs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant COGs that constituted > 0.5% in either library are shown.
Figure Legend Snippet: Abundance distributions of COGs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant COGs that constituted > 0.5% in either library are shown.

Techniques Used:

Glycoside hydrolases (GHs) detected in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant GHs that constituted > 0.05% in either library are shown.
Figure Legend Snippet: Glycoside hydrolases (GHs) detected in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant GHs that constituted > 0.05% in either library are shown.

Techniques Used:

Genus-level taxonomic compositions of metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Top 30 abundant genera that constituted > 0.5% in either library are shown, with their phyla in parentheses.
Figure Legend Snippet: Genus-level taxonomic compositions of metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Top 30 abundant genera that constituted > 0.5% in either library are shown, with their phyla in parentheses.

Techniques Used:

Abundance distributions of KOs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant KOs that constituted > 0.5% in either library are shown.
Figure Legend Snippet: Abundance distributions of KOs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant KOs that constituted > 0.5% in either library are shown.

Techniques Used:

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Transfection:

Article Title: Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact
Article Snippet: .. Extraction of DNA and RNA from Cells DNA and RNA were extracted from transfected and non-transfected hepatoma cells in a Magnapure robot (Roche Applied Science, Germany) using the Total NA protocol. .. Harvested cells were washed in 1 mL PBS and after centrifugation at 5000 rpm for 3 min the pellet was re-suspended in 800 µL RLT lysis buffer (Qiagen Sciences, MD, USA) before extraction.

Synthesized:

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

Isolation:

Article Title: MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-? treatment
Article Snippet: .. RNA from 200 µl culture medium of CoV-infected cells was isolated with a MagnaPure LC Total Nucleic Acid Isolation kit (Roche) and eluted in 100 µl. .. RT-PCR conditions for quantifying MERS-CoV and SARS-CoV RNA and amplification parameters have been described previously ( ; ).

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

Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

Quantitative RT-PCR:

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

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

Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

Purification:

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

Real-time Polymerase Chain Reaction:

Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

Reverse Transcription Polymerase Chain Reaction:

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

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

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    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si <t>RNA</t> ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative <t>PCR</t> using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P
    Rna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche qrt pcr analysis total rna
    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 <t>qRT-PCR.</t> 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
    Qrt Pcr Analysis Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Journal: Cancer Science

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1

    doi: 10.1111/cas.13754

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

    Article Snippet: 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA).

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

    T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Article Snippet: Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche).

    Techniques: Activation Assay, Transformation Assay, Purification, Selection, RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Activity Assay, In Vitro, Expressing, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Staining, FACS

    Genes downregulated upon GADL1 overexpression. ( a ) RNA expression array analyses were used to determine the levels of GADL1 , FN1 , ITGA2 , ITGAV and CCL2 mRNAs in the GADL1 -overexpressing cells (GADL1) relative to the parental cell line, SH-SY5Y (5Y). ( b ) Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for GADL1 , FN1 , ITGA2 , ITGAV and CCL2 . Data were normalized to that for ACTB in each sample, and the fold-change value for each gene is shown for GADL1 -overexpressing cells relative to SH-SY5Y cells. RNA samples for expression microarray analysis and RT-qPCR validation were prepared independently. ( c–g ) GADL1 -overexpressing cells were transfected with RISC-free negative control siRNA or siRNA targeting GADL1 (siGADL1) at 0.1 μM using DharmaFECT1 (FECT1) transfection reagent. Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for ( c ) GADL1 , ( d ) FN1 , ( e ) ITGA2 , ( f ) ITGAV and ( g ) CCL2 . The fold-change value for each gene was normalized to ACTB expression. ( b–g ) Data were combined from two independent experiments.

    Journal: Scientific Reports

    Article Title: Effects of GADL1 overexpression on cell migration and the associated morphological changes

    doi: 10.1038/s41598-019-41689-x

    Figure Lengend Snippet: Genes downregulated upon GADL1 overexpression. ( a ) RNA expression array analyses were used to determine the levels of GADL1 , FN1 , ITGA2 , ITGAV and CCL2 mRNAs in the GADL1 -overexpressing cells (GADL1) relative to the parental cell line, SH-SY5Y (5Y). ( b ) Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for GADL1 , FN1 , ITGA2 , ITGAV and CCL2 . Data were normalized to that for ACTB in each sample, and the fold-change value for each gene is shown for GADL1 -overexpressing cells relative to SH-SY5Y cells. RNA samples for expression microarray analysis and RT-qPCR validation were prepared independently. ( c–g ) GADL1 -overexpressing cells were transfected with RISC-free negative control siRNA or siRNA targeting GADL1 (siGADL1) at 0.1 μM using DharmaFECT1 (FECT1) transfection reagent. Total RNA from cells was reverse transcribed into cDNA and subjected to RT-qPCR analysis for ( c ) GADL1 , ( d ) FN1 , ( e ) ITGA2 , ( f ) ITGAV and ( g ) CCL2 . The fold-change value for each gene was normalized to ACTB expression. ( b–g ) Data were combined from two independent experiments.

    Article Snippet: The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Roche, Switzerland).

    Techniques: Over Expression, RNA Expression, Quantitative RT-PCR, Expressing, Microarray, Transfection, Negative Control

    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

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms19061786

    Figure Lengend 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

    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).

    Techniques: 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

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms19061786

    Figure Lengend 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

    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).

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