Structured Review

Roche tcda
Toxin titers in cultures of parent and codY null mutant strains. R elative levels of <t>TcdA</t> and <t>TcdB</t> in culture supernatants of UK1, UK1 codY (LB-CD16) and UK1 codY/codY + (ND-CD10) collected after 24 hrs of bacterial growth were determined by ELISA. Two samples were assayed for each toxin for each strain; toxin levels were averaged and normalized to the values in the wild-type strain (UK1) set at 1.0. Error bars were created for all pairs of samples, but for some pairs the difference was so small that the bars are not visible.
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1) Product Images from "Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027"

Article Title: Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027

Journal: PLoS ONE

doi: 10.1371/journal.pone.0206896

Toxin titers in cultures of parent and codY null mutant strains. R elative levels of TcdA and TcdB in culture supernatants of UK1, UK1 codY (LB-CD16) and UK1 codY/codY + (ND-CD10) collected after 24 hrs of bacterial growth were determined by ELISA. Two samples were assayed for each toxin for each strain; toxin levels were averaged and normalized to the values in the wild-type strain (UK1) set at 1.0. Error bars were created for all pairs of samples, but for some pairs the difference was so small that the bars are not visible.
Figure Legend Snippet: Toxin titers in cultures of parent and codY null mutant strains. R elative levels of TcdA and TcdB in culture supernatants of UK1, UK1 codY (LB-CD16) and UK1 codY/codY + (ND-CD10) collected after 24 hrs of bacterial growth were determined by ELISA. Two samples were assayed for each toxin for each strain; toxin levels were averaged and normalized to the values in the wild-type strain (UK1) set at 1.0. Error bars were created for all pairs of samples, but for some pairs the difference was so small that the bars are not visible.

Techniques Used: Mutagenesis, Enzyme-linked Immunosorbent Assay

Effect of a codY null mutation on tcdA and tcdB transcription in strain UK1. Cultures of strains UK1, LB-CD16 ( codY :: intron :: erm ) and ND-CD10 ( codY :: intron :: erm codY + ) were grown in CDMM medium and samples were removed at 8 hrs (late exponential growth phase) and 24 hrs (stationary phase). RNA was extracted and assayed for tcdA and tcdB expression by qRT-PCR (see Materials and Methods ). Results for the toxin genes were normalized to those obtained for the rpoA gene.
Figure Legend Snippet: Effect of a codY null mutation on tcdA and tcdB transcription in strain UK1. Cultures of strains UK1, LB-CD16 ( codY :: intron :: erm ) and ND-CD10 ( codY :: intron :: erm codY + ) were grown in CDMM medium and samples were removed at 8 hrs (late exponential growth phase) and 24 hrs (stationary phase). RNA was extracted and assayed for tcdA and tcdB expression by qRT-PCR (see Materials and Methods ). Results for the toxin genes were normalized to those obtained for the rpoA gene.

Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR

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

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

Journal: Molecular Cancer

doi: 10.1186/1476-4598-4-14

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

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

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

Techniques Used: Expressing, Quantitative RT-PCR

3) Product Images from "Enhancement of mitochondrial biogenesis with polyphenols: combined effects of resveratrol and equol in human endothelial cells"

Article Title: Enhancement of mitochondrial biogenesis with polyphenols: combined effects of resveratrol and equol in human endothelial cells

Journal: Immunity & Ageing : I & A

doi: 10.1186/1742-4933-10-28

The combined treatment of resveratrol and equol strongly increased the number of mitochondria in HUVEC cells. (A) Mitotracker fluorescent intensities were analysed to assess the mitochondrial biogenesis. (B) Relative mitochondrial DNA (mtDNA) content was estimated by qRT-PCR. Representative data of at least 3 experiments each performed in triplicate. (*= P
Figure Legend Snippet: The combined treatment of resveratrol and equol strongly increased the number of mitochondria in HUVEC cells. (A) Mitotracker fluorescent intensities were analysed to assess the mitochondrial biogenesis. (B) Relative mitochondrial DNA (mtDNA) content was estimated by qRT-PCR. Representative data of at least 3 experiments each performed in triplicate. (*= P

Techniques Used: Quantitative RT-PCR

Effect of resveratrol and equol on mRNA expression of PGC1-α (A), NRF-1 (B), TFAM (C) in HUVEC. qRT-PCR measurement to assess the mRNA expression of the mitochondrial biogenesis factors. Representative data of at least 3 experiments each performed in triplicate. (*= P
Figure Legend Snippet: Effect of resveratrol and equol on mRNA expression of PGC1-α (A), NRF-1 (B), TFAM (C) in HUVEC. qRT-PCR measurement to assess the mRNA expression of the mitochondrial biogenesis factors. Representative data of at least 3 experiments each performed in triplicate. (*= P

Techniques Used: Expressing, Quantitative RT-PCR

4) Product Images from "Enhancement of mitochondrial biogenesis with polyphenols: combined effects of resveratrol and equol in human endothelial cells"

Article Title: Enhancement of mitochondrial biogenesis with polyphenols: combined effects of resveratrol and equol in human endothelial cells

Journal: Immunity & Ageing : I & A

doi: 10.1186/1742-4933-10-28

Effect of resveratrol and equol on mRNA expression of PGC1-α (A), NRF-1 (B), TFAM (C) in HUVEC. qRT-PCR measurement to assess the mRNA expression of the mitochondrial biogenesis factors. Representative data of at least 3 experiments each performed in triplicate. (*= P
Figure Legend Snippet: Effect of resveratrol and equol on mRNA expression of PGC1-α (A), NRF-1 (B), TFAM (C) in HUVEC. qRT-PCR measurement to assess the mRNA expression of the mitochondrial biogenesis factors. Representative data of at least 3 experiments each performed in triplicate. (*= P

Techniques Used: Expressing, Quantitative RT-PCR

5) Product Images from "Influence of untranslated regions on retroviral mRNA transfer and expression"

Article Title: Influence of untranslated regions on retroviral mRNA transfer and expression

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-13-35

mRNA cellular persistence: quantitative RT-PCR of Non-viral (A and B) and Truncated-retroviral (C and D) mRNA. 293FT cells were transfected with constructs. One day after transfection, transcription was blocked by actinomycin D, and luciferase-mRNA levels were measured at 24, 48 and 72 h. Luciferase mRNA was normalized to that of actin. ( A and C ) Comparison of luciferase mRNA level in transfected cells, for each condition the amount was normalized to that of b-actin in cells transfected with pcDNA-Luc-psi or pCMV-5′LTR-psi-Luc and pcDNA-Luc-psi-UTR or pCMV-5′LTR-psi-Luc-UTR. The bars indicate relative amounts compared to control condition (in A pcDNA-Luc-psi and in C pCMV-5′LTR-psi-Luc). Number of experiments, N = 2. ( B and D ) Evolution of the specific amount of the different RNAs. RNA extracted from cells immediately after the addition of actinomycin D (time-point 0) was used to define the initial level of mRNA and arbitrarily set to 100%, at 24, 48 and 72 hours the amount of the different RNAs are compared to this initial point. Number of experiments, N = 2.
Figure Legend Snippet: mRNA cellular persistence: quantitative RT-PCR of Non-viral (A and B) and Truncated-retroviral (C and D) mRNA. 293FT cells were transfected with constructs. One day after transfection, transcription was blocked by actinomycin D, and luciferase-mRNA levels were measured at 24, 48 and 72 h. Luciferase mRNA was normalized to that of actin. ( A and C ) Comparison of luciferase mRNA level in transfected cells, for each condition the amount was normalized to that of b-actin in cells transfected with pcDNA-Luc-psi or pCMV-5′LTR-psi-Luc and pcDNA-Luc-psi-UTR or pCMV-5′LTR-psi-Luc-UTR. The bars indicate relative amounts compared to control condition (in A pcDNA-Luc-psi and in C pCMV-5′LTR-psi-Luc). Number of experiments, N = 2. ( B and D ) Evolution of the specific amount of the different RNAs. RNA extracted from cells immediately after the addition of actinomycin D (time-point 0) was used to define the initial level of mRNA and arbitrarily set to 100%, at 24, 48 and 72 hours the amount of the different RNAs are compared to this initial point. Number of experiments, N = 2.

Techniques Used: Quantitative RT-PCR, Transfection, Construct, Luciferase

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

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

Journal: Molecular Cytogenetics

doi: 10.1186/1755-8166-7-42

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

Techniques Used: Expressing, Fluorescence, MANN-WHITNEY

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

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

7) 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

8) Product Images from "Modulation of NKG2D Expression in Human CD8+ T Cells Corresponding with Tuberculosis Drug Cure"

Article Title: Modulation of NKG2D Expression in Human CD8+ T Cells Corresponding with Tuberculosis Drug Cure

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070063

NKG2D ex vivo blood gene expression prior to and during chemotherapy. RNA was extracted from whole blood Tempus tubes and gene expression investigated by qRT-PCR. (A) NKG2D expression in ex vivo venous blood from untreated active TB cases at diagnosis (n = 26), in latently-infected household contacts (Contacts (+): n = 13) and in uninfected contacts (Contacts (−): n = 11), all recruited in Lahore, Pakistan. The Wilcoxon ranksum test was used to compare the clinical groups. (B) NKG2D expression after the end of the intensive phase in 16 patients who were eventually successfully cured (closed circles) and in 2 patients who subsequently died (open circles). (C) NKG2D expression during the entire course of TB chemotherapy in a different 6 patients, for whom data were available at the end of treatment. The Wilcoxon signrank test was used to analyse the paired data in (B) and (C). The lines in the centre of each group represent medians. NKG2D mRNA expression was determined by qRT-PCR, with results shown normalised against the housekeeping gene Cyclophilin A.
Figure Legend Snippet: NKG2D ex vivo blood gene expression prior to and during chemotherapy. RNA was extracted from whole blood Tempus tubes and gene expression investigated by qRT-PCR. (A) NKG2D expression in ex vivo venous blood from untreated active TB cases at diagnosis (n = 26), in latently-infected household contacts (Contacts (+): n = 13) and in uninfected contacts (Contacts (−): n = 11), all recruited in Lahore, Pakistan. The Wilcoxon ranksum test was used to compare the clinical groups. (B) NKG2D expression after the end of the intensive phase in 16 patients who were eventually successfully cured (closed circles) and in 2 patients who subsequently died (open circles). (C) NKG2D expression during the entire course of TB chemotherapy in a different 6 patients, for whom data were available at the end of treatment. The Wilcoxon signrank test was used to analyse the paired data in (B) and (C). The lines in the centre of each group represent medians. NKG2D mRNA expression was determined by qRT-PCR, with results shown normalised against the housekeeping gene Cyclophilin A.

Techniques Used: Ex Vivo, Expressing, Quantitative RT-PCR, Infection

Regulation of NKG2D and DAP10 mRNA following mycobacterial stimulation in vitro . CD4 + and CD8 + T cells were isolated from ten healthy BCG-vaccinated donors after 7 days of PBMC stimulation with live M. tuberculosis H37Rv, on ten separate occasions. NKG2D (A) and DAP10 (B) mRNA expression levels in each T cell subset were determined by qRT-PCR. Data are normalised against the HPRT housekeeping gene, with the mean of duplicate technical replicates shown. (C) Diluted whole blood cultures from five healthy donors were incubated on five separate occasions with live M. bovis BCG in the absence or presence of IL-2 for the time indicated. NKG2D mRNA expression was determined for each sample in duplicate by qRT-PCR and is shown normalised against HPRT and normalised against the unstimulated control at each time point, with mean and standard error of the mean for the five donors shown. *P
Figure Legend Snippet: Regulation of NKG2D and DAP10 mRNA following mycobacterial stimulation in vitro . CD4 + and CD8 + T cells were isolated from ten healthy BCG-vaccinated donors after 7 days of PBMC stimulation with live M. tuberculosis H37Rv, on ten separate occasions. NKG2D (A) and DAP10 (B) mRNA expression levels in each T cell subset were determined by qRT-PCR. Data are normalised against the HPRT housekeeping gene, with the mean of duplicate technical replicates shown. (C) Diluted whole blood cultures from five healthy donors were incubated on five separate occasions with live M. bovis BCG in the absence or presence of IL-2 for the time indicated. NKG2D mRNA expression was determined for each sample in duplicate by qRT-PCR and is shown normalised against HPRT and normalised against the unstimulated control at each time point, with mean and standard error of the mean for the five donors shown. *P

Techniques Used: In Vitro, Isolation, Expressing, Quantitative RT-PCR, Incubation

NKG2D gene expression at diagnosis and during chemotherapy in in vitro stimulated RNA samples. PBMC were isolated and stimulated in vitro with M. bovis BCG (MOI 1∶1) or anti-CD3 mAb for 16 hours and NKG2D mRNA expression analysed by qRT-PCR. (A) NKG2D mRNA expression at TB diagnosis in 26 untreated TB patients (P), 13 latently infected contacts (C+) and 11 uninfected contacts (C-). (B) Modulation of NKG2D mRNA expression during the intensive phase of treatment in 16 successfully cured TB patients. (C) NKG2D mRNA expression following stimulation in 2 patients who subsequently died. (D) NKG2D mRNA expression in a separate group of 6 successfully cured patients throughout the full treatment course. The line in the centre of the box and whisker plots represents the median whereas the top and bottom lines represent the 75 th and 25 th quartile respectively, and whiskers represent minimum and maximum data points. The Wilcoxon ranksum test (A.) and the Wilcoxon signrank test (B) and (D) were used for statistical analyses of unpaired and paired data respectively.
Figure Legend Snippet: NKG2D gene expression at diagnosis and during chemotherapy in in vitro stimulated RNA samples. PBMC were isolated and stimulated in vitro with M. bovis BCG (MOI 1∶1) or anti-CD3 mAb for 16 hours and NKG2D mRNA expression analysed by qRT-PCR. (A) NKG2D mRNA expression at TB diagnosis in 26 untreated TB patients (P), 13 latently infected contacts (C+) and 11 uninfected contacts (C-). (B) Modulation of NKG2D mRNA expression during the intensive phase of treatment in 16 successfully cured TB patients. (C) NKG2D mRNA expression following stimulation in 2 patients who subsequently died. (D) NKG2D mRNA expression in a separate group of 6 successfully cured patients throughout the full treatment course. The line in the centre of the box and whisker plots represents the median whereas the top and bottom lines represent the 75 th and 25 th quartile respectively, and whiskers represent minimum and maximum data points. The Wilcoxon ranksum test (A.) and the Wilcoxon signrank test (B) and (D) were used for statistical analyses of unpaired and paired data respectively.

Techniques Used: Expressing, In Vitro, Isolation, Quantitative RT-PCR, Infection, Whisker Assay

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

Article Title: NUCKS overexpression in breast cancer

Journal: Cancer Cell International

doi: 10.1186/1475-2867-9-19

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

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

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

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

10) 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

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

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

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.10773

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

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

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

Techniques Used: Injection, Incubation, Over Expression

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

Techniques Used: Injection, Quantitative RT-PCR

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

Techniques Used: Injection, Antibody Labeling

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

Techniques Used: Injection, Antibody Labeling

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

Techniques Used: Expressing, Incubation, Quantitative RT-PCR

12) Product Images from "Transcriptome analysis of an apple (Malus × domestica) yellow fruit somatic mutation identifies a gene network module highly associated with anthocyanin and epigenetic regulation"

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

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erv433

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

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

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

Techniques Used: Expressing, Quantitative RT-PCR

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

Techniques Used: Quantitative RT-PCR

13) Product Images from "Ixodes scapularis and Ixodes ricinus tick cell lines respond to infection with tick-borne encephalitis virus: transcriptomic and proteomic analysis"

Article Title: Ixodes scapularis and Ixodes ricinus tick cell lines respond to infection with tick-borne encephalitis virus: transcriptomic and proteomic analysis

Journal: Parasites & Vectors

doi: 10.1186/s13071-015-1210-x

Gene knockdown and the effect on LGTV replication and production in IRE/CTVM19 cells. IRE/CTVM19 cells were treated with dsRNA to silence differentially-expressed transcripts and were subsequently infected with LGTV at an MOI of 0.5. a Transcripts coding for Argonaute (Ago 30) and Dicer (Dcr 90) were amplified by RT-PCR using dsT7-Ago 30 or dsT7-Dcr90 primers and visualised by agarose gel electrophoresis. A representative 1 % agarose gel from one of the three experiments is shown; upper lanes show Ago 30 and Dcr 90 PCR products, lower lanes show beta actin PCR products. b Gel-electrophoresis images were used to semi-quantify mRNA knockdown of Ago 30 and Dcr 90 with Image Lab software (BioRad) normalised to beta actin control. c Knockdown of mRNA was quantified using qRT-PCR. Gene expression was normalised to beta actin and is shown relative to eGFP-dsRNA controls. d Viral RNA levels were determined by qRT-PCR using LGTV NS5 primers at 24 h p.i.. The data was normalised to beta actin and is presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold changes relative to eGFP dsRNA controls. e Infectious virus present in the supernatant was titrated by plaque assay at 24 h p.i. and the titres are presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to titres in the eGFP-dsRNA control. The mean with standard error of three independent experiments is shown, including only those replicates in which the knockdown was validated. Statistical significance was calculated using two-way ANOVA Fisher’s LSD test (* p
Figure Legend Snippet: Gene knockdown and the effect on LGTV replication and production in IRE/CTVM19 cells. IRE/CTVM19 cells were treated with dsRNA to silence differentially-expressed transcripts and were subsequently infected with LGTV at an MOI of 0.5. a Transcripts coding for Argonaute (Ago 30) and Dicer (Dcr 90) were amplified by RT-PCR using dsT7-Ago 30 or dsT7-Dcr90 primers and visualised by agarose gel electrophoresis. A representative 1 % agarose gel from one of the three experiments is shown; upper lanes show Ago 30 and Dcr 90 PCR products, lower lanes show beta actin PCR products. b Gel-electrophoresis images were used to semi-quantify mRNA knockdown of Ago 30 and Dcr 90 with Image Lab software (BioRad) normalised to beta actin control. c Knockdown of mRNA was quantified using qRT-PCR. Gene expression was normalised to beta actin and is shown relative to eGFP-dsRNA controls. d Viral RNA levels were determined by qRT-PCR using LGTV NS5 primers at 24 h p.i.. The data was normalised to beta actin and is presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold changes relative to eGFP dsRNA controls. e Infectious virus present in the supernatant was titrated by plaque assay at 24 h p.i. and the titres are presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to titres in the eGFP-dsRNA control. The mean with standard error of three independent experiments is shown, including only those replicates in which the knockdown was validated. Statistical significance was calculated using two-way ANOVA Fisher’s LSD test (* p

Techniques Used: Infection, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Software, Quantitative RT-PCR, Expressing, Plaque Assay

Gene knockdown and the effect on LGTV replication and production in IDE8 cells. IDE8 cells were treated with dsRNA to silence selected transcripts and subsequently infected with LGTV at MOI 0.01. a Transcripts coding for Argonaute (Ago 30) and Dicer (Dcr 90) were amplified by RT-PCR using dsT7-Ago 30 or dsT7-Dcr 90 primers and visualised by agarose gel electrophoresis. A representative 1 % agarose gel from one of the three experiments is shown; upper lanes show Ago 30 and Dcr 90 PCR products, lower lanes show beta actin PCR products. b Gel-electrophoresis images were used to semi-quantify mRNA knockdown of Ago 30 and Dcr 90 with Image Lab software (BioRad) normalised to beta actin control. c Knockdown of mRNA of the genes listed in the x-axis was quantified using qRT-PCR with qRT-PCR primers (Additional file 1 ). Gene expression was normalised to beta actin and is shown relative to eGFP-dsRNA controls. d Viral RNA levels were determined by qRT-PCR using LGTV NS5 primers at 48 h p.i.. The data was normalised to beta actin and is presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to eGFP dsRNA controls. e Infectious virus present in the supernatant was titrated by plaque assay at 48 h p.i. and the titres are presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to titres in the eGFP-dsRNA control. The mean with standard error of three independent experiments is shown, including only those replicates in which the knockdown was validated. Statistical significance was calculated using two-way ANOVA Fisher’s LSD test (* p
Figure Legend Snippet: Gene knockdown and the effect on LGTV replication and production in IDE8 cells. IDE8 cells were treated with dsRNA to silence selected transcripts and subsequently infected with LGTV at MOI 0.01. a Transcripts coding for Argonaute (Ago 30) and Dicer (Dcr 90) were amplified by RT-PCR using dsT7-Ago 30 or dsT7-Dcr 90 primers and visualised by agarose gel electrophoresis. A representative 1 % agarose gel from one of the three experiments is shown; upper lanes show Ago 30 and Dcr 90 PCR products, lower lanes show beta actin PCR products. b Gel-electrophoresis images were used to semi-quantify mRNA knockdown of Ago 30 and Dcr 90 with Image Lab software (BioRad) normalised to beta actin control. c Knockdown of mRNA of the genes listed in the x-axis was quantified using qRT-PCR with qRT-PCR primers (Additional file 1 ). Gene expression was normalised to beta actin and is shown relative to eGFP-dsRNA controls. d Viral RNA levels were determined by qRT-PCR using LGTV NS5 primers at 48 h p.i.. The data was normalised to beta actin and is presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to eGFP dsRNA controls. e Infectious virus present in the supernatant was titrated by plaque assay at 48 h p.i. and the titres are presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to titres in the eGFP-dsRNA control. The mean with standard error of three independent experiments is shown, including only those replicates in which the knockdown was validated. Statistical significance was calculated using two-way ANOVA Fisher’s LSD test (* p

Techniques Used: Infection, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Software, Quantitative RT-PCR, Expressing, Plaque Assay

14) Product Images from "STAT1 inhibits human hepatocellular carcinoma cell growth through induction of p53 and Fbxw7"

Article Title: STAT1 inhibits human hepatocellular carcinoma cell growth through induction of p53 and Fbxw7

Journal: Cancer Cell International

doi: 10.1186/s12935-015-0253-6

Effects of STAT1 knockdown on SMMC7721 and HepG2 cells growth. a , b , c qRT-PCR and western blot assays were used to determine siRNAs efficiency; d cell proliferation was quantified by MTT assay. STAT1 siRNA-transfected SMMC7721 and HepG2 cells grew significantly faster than control siRNA-transfected cells (P
Figure Legend Snippet: Effects of STAT1 knockdown on SMMC7721 and HepG2 cells growth. a , b , c qRT-PCR and western blot assays were used to determine siRNAs efficiency; d cell proliferation was quantified by MTT assay. STAT1 siRNA-transfected SMMC7721 and HepG2 cells grew significantly faster than control siRNA-transfected cells (P

Techniques Used: Quantitative RT-PCR, Western Blot, MTT Assay, Transfection

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

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

Journal: Oncotarget

doi:

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

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

16) 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

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0140786

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

Techniques Used: Expressing, Quantitative RT-PCR

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

Techniques Used: Expressing, Quantitative RT-PCR

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

Techniques Used: Incubation, Software

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

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

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

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

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

Techniques Used: Staining, Flow Cytometry, Cytometry

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

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

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

Techniques Used: Staining, Flow Cytometry, Cytometry

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

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005568

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

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

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

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

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17040499

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

Techniques Used:

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

Techniques Used:

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

Techniques Used: Construct, Expressing

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

Techniques Used: Expressing

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

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

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-016-0340-1

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Journal: Cell Transplantation

doi: 10.1177/0963689717741139

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

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

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

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

Journal: Oncotarget

doi: 10.18632/oncotarget.22708

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

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

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

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

23) Product Images from "Insights into the complex regulation of rpoS in Borrelia burgdorferi"

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2007.05813.x

Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.
Figure Legend Snippet: Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

Techniques Used: Quantitative RT-PCR, Standard Deviation, Plasmid Preparation

Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

Techniques Used: Quantitative RT-PCR, Standard Deviation

Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.
Figure Legend Snippet: Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

Techniques Used: Quantitative RT-PCR, Standard Deviation

Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.
Figure Legend Snippet: Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

Techniques Used: Quantitative RT-PCR, Standard Deviation

Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

Techniques Used: Quantitative RT-PCR, Standard Deviation

24) Product Images from "Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane"

Article Title: Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-14-92

Co-bombardment and qRT-PCR assays reveal the synthetic luc * and Ren luc * transgenes produce increased levels of steady state mRNA relative to the native luciferase transgenes. A . qRT-PCR on equimolar serial dilutions of pUbi- luc (orange lines) and pUbi- luc * (blue lines) plasmid template using the corresponding gene-specific primer pair (Additional file 4 Figure S4A B). B . Amplification from cDNA synthesised from suspension cells co-bombarded with equimolar amounts of pUbi- luc and pUbi- luc * plasmid. C . luc * transcript levels were on average 8.9-fold higher than luc transcript levels in the co-bombarded suspension cells cDNA. Error bars represent the standard error of the mean across three independent co-bombardments. D . qRT-PCR on equimolar serial dilutions of pUbi-Ren luc (green lines) and pUbi-Ren luc* green lines) plasmid using the corresponding gene specific primer pair (Additional file 4 : Figure S4A B). E . Amplification from cDNA synthesised from suspension cells co-bombarded with equimolar amounts of pUbi-Ren luc and pUbi-Ren luc * plasmid. F . Synthetic Ren luc * transcript levels were on average 6.5-fold higher than the native Ren luc transcript levels in the co-bombarded suspension cells cDNA. Error bars represent the standard error of the mean across three independent co-bombardments.
Figure Legend Snippet: Co-bombardment and qRT-PCR assays reveal the synthetic luc * and Ren luc * transgenes produce increased levels of steady state mRNA relative to the native luciferase transgenes. A . qRT-PCR on equimolar serial dilutions of pUbi- luc (orange lines) and pUbi- luc * (blue lines) plasmid template using the corresponding gene-specific primer pair (Additional file 4 Figure S4A B). B . Amplification from cDNA synthesised from suspension cells co-bombarded with equimolar amounts of pUbi- luc and pUbi- luc * plasmid. C . luc * transcript levels were on average 8.9-fold higher than luc transcript levels in the co-bombarded suspension cells cDNA. Error bars represent the standard error of the mean across three independent co-bombardments. D . qRT-PCR on equimolar serial dilutions of pUbi-Ren luc (green lines) and pUbi-Ren luc* green lines) plasmid using the corresponding gene specific primer pair (Additional file 4 : Figure S4A B). E . Amplification from cDNA synthesised from suspension cells co-bombarded with equimolar amounts of pUbi-Ren luc and pUbi-Ren luc * plasmid. F . Synthetic Ren luc * transcript levels were on average 6.5-fold higher than the native Ren luc transcript levels in the co-bombarded suspension cells cDNA. Error bars represent the standard error of the mean across three independent co-bombardments.

Techniques Used: Quantitative RT-PCR, Luciferase, Plasmid Preparation, Amplification

25) Product Images from "Larval diet affects mosquito development and permissiveness to Plasmodium infection"

Article Title: Larval diet affects mosquito development and permissiveness to Plasmodium infection

Journal: Scientific Reports

doi: 10.1038/srep38230

Impact of the larval diet on larval microbiota. ( a ) bacterial load in larvae fed with the three diets, quantified by 16S rRNA using primers binding to generic sequences conserved in all bacteria (All) or to family-specific sequences ( Entero . – Enterobacteriaceae, Flavo. – Flavobacteriaceae ) and normalised to the housekeeping mosquito gene AgS7 . In ( b ) the same family-specific 16S rRNA quantifications are normalised to the generic 16S rRNA quantification, and grouped significance bar shows that difference between pelleted and flaked diets. Data show averages (±s.e.m.) from 3 independent replicates and statistical analyses are ANOVA following lmer model fitting, *p
Figure Legend Snippet: Impact of the larval diet on larval microbiota. ( a ) bacterial load in larvae fed with the three diets, quantified by 16S rRNA using primers binding to generic sequences conserved in all bacteria (All) or to family-specific sequences ( Entero . – Enterobacteriaceae, Flavo. – Flavobacteriaceae ) and normalised to the housekeeping mosquito gene AgS7 . In ( b ) the same family-specific 16S rRNA quantifications are normalised to the generic 16S rRNA quantification, and grouped significance bar shows that difference between pelleted and flaked diets. Data show averages (±s.e.m.) from 3 independent replicates and statistical analyses are ANOVA following lmer model fitting, *p

Techniques Used: Binding Assay

Impact of larval diet on the adult mosquito microbiota. ( a,b ) Bacterial load quantified by qRT-PCR on 16S rRNA in the midguts ( a ) and ovaries ( b ) of females emerged from larvae fed on the three diets prior to or 24 h after a blood meal (sugar-fed - SF and blood-fed - BF, respectively). ( c–f ) Family-specific bacterial load in adult midguts normalised to AgS7 ( c,d ) and to the total bacterial load ( e,f ), quantified by qRT-PCR on family specific 16S rRNA amplicons. SF and BF mosquitoes had significantly different results in ( a) (***), ( c) (*), ( d) (***), ( f) (**). In ( b ) grouped significance bar shows that difference between pelleted and flaked diets. Data show averages (±s.e.m.) from 3 independent replicates and statistical analyses are ANOVA following lmer model fitting, *p
Figure Legend Snippet: Impact of larval diet on the adult mosquito microbiota. ( a,b ) Bacterial load quantified by qRT-PCR on 16S rRNA in the midguts ( a ) and ovaries ( b ) of females emerged from larvae fed on the three diets prior to or 24 h after a blood meal (sugar-fed - SF and blood-fed - BF, respectively). ( c–f ) Family-specific bacterial load in adult midguts normalised to AgS7 ( c,d ) and to the total bacterial load ( e,f ), quantified by qRT-PCR on family specific 16S rRNA amplicons. SF and BF mosquitoes had significantly different results in ( a) (***), ( c) (*), ( d) (***), ( f) (**). In ( b ) grouped significance bar shows that difference between pelleted and flaked diets. Data show averages (±s.e.m.) from 3 independent replicates and statistical analyses are ANOVA following lmer model fitting, *p

Techniques Used: Quantitative RT-PCR

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

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

Journal: Biology Open

doi: 10.1242/bio.027532

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

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

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

Techniques Used: Expressing, Transfection, Microscopy, Staining

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

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

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

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

27) Product Images from "Novel Wolbachia-transinfected Aedes aegypti mosquitoes possess diverse fitness and vector competence phenotypes"

Article Title: Novel Wolbachia-transinfected Aedes aegypti mosquitoes possess diverse fitness and vector competence phenotypes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006751

w MelCS and w Ri Wolbachia strains inhibit DENV-3 replication and dissemination following an infectious blood meal. Seven-day old female mosquitoes were fed a blood meal of fresh DENV-3 (2.0 x 10 6 TCID 50 /ml) mixed 1:1 with sheep blood (n = 500 per Wolbachia -infected line, n = 200 per respective Tet control line). Mosquitoes were sorted immediately to identify those that took a blood meal, and incubated for 14 days. Mosquito heads were then separated from the thorax and total RNA was extracted from each head and remaining mosquito body. DENV genome copies were determined by qRT-PCR for each body as a measure of infection, and for each head as a measure of viral dissemination. Data are the mean genome copies per mosquito body (top) or head (bottom) ± SEM, and are representative of 3 independent experiments. Statistical analyses were performed using a Mann-Whitney test where ** p
Figure Legend Snippet: w MelCS and w Ri Wolbachia strains inhibit DENV-3 replication and dissemination following an infectious blood meal. Seven-day old female mosquitoes were fed a blood meal of fresh DENV-3 (2.0 x 10 6 TCID 50 /ml) mixed 1:1 with sheep blood (n = 500 per Wolbachia -infected line, n = 200 per respective Tet control line). Mosquitoes were sorted immediately to identify those that took a blood meal, and incubated for 14 days. Mosquito heads were then separated from the thorax and total RNA was extracted from each head and remaining mosquito body. DENV genome copies were determined by qRT-PCR for each body as a measure of infection, and for each head as a measure of viral dissemination. Data are the mean genome copies per mosquito body (top) or head (bottom) ± SEM, and are representative of 3 independent experiments. Statistical analyses were performed using a Mann-Whitney test where ** p

Techniques Used: Infection, Incubation, Quantitative RT-PCR, MANN-WHITNEY

w MelCS provides superior blocking of DENV-3 genome replication in a mosquito injection challenge model. (A) DENV-3 was injected into the thorax of 6 or 7-day old female mosquitoes at 2.5 x10 6 TCID 50 /ml (undiluted) or 10, 100 or 1000-fold dilutions thereof. RNA was extracted from whole mosquito bodies 7-days post infection and virus replication was quantified by qRT-PCR. Data are the mean number of genome copies per mosquito ± SEM. Number of DENV-3 positive mosquitoes/total n are indicated above each bar. ** p
Figure Legend Snippet: w MelCS provides superior blocking of DENV-3 genome replication in a mosquito injection challenge model. (A) DENV-3 was injected into the thorax of 6 or 7-day old female mosquitoes at 2.5 x10 6 TCID 50 /ml (undiluted) or 10, 100 or 1000-fold dilutions thereof. RNA was extracted from whole mosquito bodies 7-days post infection and virus replication was quantified by qRT-PCR. Data are the mean number of genome copies per mosquito ± SEM. Number of DENV-3 positive mosquitoes/total n are indicated above each bar. ** p

Techniques Used: Blocking Assay, Injection, Infection, Quantitative RT-PCR

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

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

Journal: Nature Communications

doi: 10.1038/ncomms15480

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

Techniques Used: Lysis, Transmission Electron Microscopy

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

Techniques Used: Mouse Assay, Ex Vivo

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

Techniques Used: Mouse Assay

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

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

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2017.00236

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

Techniques Used: Infection, Expressing, Cell Culture, Cytometry

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

Techniques Used: Expressing, Infection, Plex Assay

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-017-00220-w

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

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

31) Product Images from "miR-200a-5p regulates myocardial necroptosis induced by Se deficiency via targeting RNF11"

Article Title: miR-200a-5p regulates myocardial necroptosis induced by Se deficiency via targeting RNF11

Journal: Redox Biology

doi: 10.1016/j.redox.2017.11.025

miR-200a-5p-mediated modulation of MAPKs activation. (A-C) The mRNA levels of ERK, JNK and p38 were detected in vivo and vitro. Cardiomyocytes were treated with z-VAD-fmk (added TNF-α) with/without BHA or Nec-1 after transfected with mimic and inhibitor. Bars represent the mean ± SD. Bars that do not share the same letters are significantly different (p
Figure Legend Snippet: miR-200a-5p-mediated modulation of MAPKs activation. (A-C) The mRNA levels of ERK, JNK and p38 were detected in vivo and vitro. Cardiomyocytes were treated with z-VAD-fmk (added TNF-α) with/without BHA or Nec-1 after transfected with mimic and inhibitor. Bars represent the mean ± SD. Bars that do not share the same letters are significantly different (p

Techniques Used: Activation Assay, In Vivo, Transfection

Different miRNA and mRNA expression profiles and qRT-PCR detection in vivo and in vitro and luciferase activity assay. (A) Heat map with intuitive reflection of the features of expression profiles changes in the control group and Se-deficient group. (B) Myocardial tissue miRNA levels of miR-200a-5p in the control group and Se-deficient group. Bars represent the mean ± SD of 30 individuals. Bars represent the mean ± SD of triplicate cell cultures. *p
Figure Legend Snippet: Different miRNA and mRNA expression profiles and qRT-PCR detection in vivo and in vitro and luciferase activity assay. (A) Heat map with intuitive reflection of the features of expression profiles changes in the control group and Se-deficient group. (B) Myocardial tissue miRNA levels of miR-200a-5p in the control group and Se-deficient group. Bars represent the mean ± SD of 30 individuals. Bars represent the mean ± SD of triplicate cell cultures. *p

Techniques Used: Expressing, Quantitative RT-PCR, In Vivo, In Vitro, Luciferase, Activity Assay

miR-200a-mediated modulation of RIP3-mediated necroptosis. (A) The ultrastructural changes of cardiomyocytes transfected with mimic, inhibitor and control cardiomyocytes. (B) The mRNA expression of TNF-α, MLKL, RIP1, RIP3, Caspase8 and FADD in the control myocardial tissue and Se-deficient myocardial tissue. Bars represent the mean ± SD of 30 individuals. *p
Figure Legend Snippet: miR-200a-mediated modulation of RIP3-mediated necroptosis. (A) The ultrastructural changes of cardiomyocytes transfected with mimic, inhibitor and control cardiomyocytes. (B) The mRNA expression of TNF-α, MLKL, RIP1, RIP3, Caspase8 and FADD in the control myocardial tissue and Se-deficient myocardial tissue. Bars represent the mean ± SD of 30 individuals. *p

Techniques Used: Transfection, Expressing

32) Product Images from "Genetic Control of Lithium Sensitivity and Regulation of Inositol Biosynthetic Genes"

Article Title: Genetic Control of Lithium Sensitivity and Regulation of Inositol Biosynthetic Genes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0011151

Loss of dpoA reduces Li + sensitivity. A , PO activity peaks at 8 hours of Dictyostelium development, and then declines to levels only slightly higher than in growing cells (0 hours of development). Error Bars: mean ± standard error of 3 independent experiments. Peak activity corresponds to streaming and mound developmental stages. B , Cell tracks of wild type and mutant cells in 7 mM LiCl (or control of 7 mM NaCl) during chemotaxis along a 1 nM/µm cAMP gradient (direction of arrow). D = Directionality, S = cell speed (µm/min). dpoA null mutants have a reduced Li + sensitivity, showing higher Directionality and speed than wild type in Li + .
Figure Legend Snippet: Loss of dpoA reduces Li + sensitivity. A , PO activity peaks at 8 hours of Dictyostelium development, and then declines to levels only slightly higher than in growing cells (0 hours of development). Error Bars: mean ± standard error of 3 independent experiments. Peak activity corresponds to streaming and mound developmental stages. B , Cell tracks of wild type and mutant cells in 7 mM LiCl (or control of 7 mM NaCl) during chemotaxis along a 1 nM/µm cAMP gradient (direction of arrow). D = Directionality, S = cell speed (µm/min). dpoA null mutants have a reduced Li + sensitivity, showing higher Directionality and speed than wild type in Li + .

Techniques Used: Activity Assay, Mutagenesis, Chemotaxis Assay

33) Product Images from "Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize"

Article Title: Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061757

Validation of RNA-Seq data using qRT-PCR. Correlation of expression levels of 12 selected DEGs derived from log 2 [fold change] in G_GG, G_GB, B_BB, and B_BG pairwise comparisons were determined by linear fitting the RNA-Seq and qRT-PCR data. Error bars represent the SD ( n = 3).
Figure Legend Snippet: Validation of RNA-Seq data using qRT-PCR. Correlation of expression levels of 12 selected DEGs derived from log 2 [fold change] in G_GG, G_GB, B_BB, and B_BG pairwise comparisons were determined by linear fitting the RNA-Seq and qRT-PCR data. Error bars represent the SD ( n = 3).

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

34) Product Images from "Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage"

Article Title: Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage

Journal: Molecular Cell

doi: 10.1016/j.molcel.2018.07.034

cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. .
Figure Legend Snippet: cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. .

Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Confocal Microscopy, Translocation Assay, Liquid Chromatography with Mass Spectroscopy

Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. .
Figure Legend Snippet: Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. .

Techniques Used: Activation Assay, Activity Assay, Transfection, Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. A–S3F.
Figure Legend Snippet: STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. A–S3F.

Techniques Used: Expressing, Clonogenic Cell Survival Assay, Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy, Translocation Assay, Reverse Transcription Polymerase Chain Reaction

TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. .
Figure Legend Snippet: TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. .

Techniques Used: Immunoprecipitation, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Molecular Weight, Modification, SDS Page

The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. G–S3L.
Figure Legend Snippet: The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. G–S3L.

Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Translocation Assay, Luciferase

Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). .
Figure Legend Snippet: Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). .

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Cytotoxicity Assay, Expressing

Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). .
Figure Legend Snippet: Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). .

Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Quantitative RT-PCR

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

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

Journal: Biology Open

doi: 10.1242/bio.010405

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

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

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

Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR

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

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

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

Techniques Used: Mutagenesis, Incubation, Quantitative RT-PCR

36) Product Images from "The Arabidopsis ABA-Activated Kinase OST1 Phosphorylates the bZIP Transcription Factor ABF3 and Creates a 14-3-3 Binding Site Involved in Its Turnover"

Article Title: The Arabidopsis ABA-Activated Kinase OST1 Phosphorylates the bZIP Transcription Factor ABF3 and Creates a 14-3-3 Binding Site Involved in Its Turnover

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013935

OST1 interacts with ABF3 in the nucleus of guard cell. (A) qRT-PCR analysis of ABF genes expression in epidermal peels in response to 50 µM ABA for 3 h. Data were normalized with AtACTIN2 expression and the expression of ABF3 gene without ABA was set to 1 as reference. Error bars indicate standard deviation for two technical replicates. A representative experiment out of three independent biological repetitions is shown (B) qRT-PCR analysis of At2g36640 gene expression in epidermal peels of wild type (Col), ost1 and abf3 plant genotypes in response to 50 µM ABA for 3 h. At2g36640 expression was normalized to the expression of AtACTIN2 in all samples and the value corresponding to non-treated wild type plants (Col) set to 1. A representative experiment out of three independent biological repetitions is shown. (C) Confocal images of guard cells expressing YFP-ABF3 (left) and YFP-OST1 (right). On the bottom panel, the confocal images were merged with the corresponding bright field images. Scale bar corresponds to 8 µm. (D) ABF3 and OST1 interaction is analyzed by Bimolecular Fluorescent Complementation in N. benthamiana tobacco leaves expressing the given constructs. The YFP fluorescent signal is located in the nucleus of epidermal cells. In these experiments, the nuclear localized AKINβ protein is used as negative control. Scale bar corresponds to 20 µm.
Figure Legend Snippet: OST1 interacts with ABF3 in the nucleus of guard cell. (A) qRT-PCR analysis of ABF genes expression in epidermal peels in response to 50 µM ABA for 3 h. Data were normalized with AtACTIN2 expression and the expression of ABF3 gene without ABA was set to 1 as reference. Error bars indicate standard deviation for two technical replicates. A representative experiment out of three independent biological repetitions is shown (B) qRT-PCR analysis of At2g36640 gene expression in epidermal peels of wild type (Col), ost1 and abf3 plant genotypes in response to 50 µM ABA for 3 h. At2g36640 expression was normalized to the expression of AtACTIN2 in all samples and the value corresponding to non-treated wild type plants (Col) set to 1. A representative experiment out of three independent biological repetitions is shown. (C) Confocal images of guard cells expressing YFP-ABF3 (left) and YFP-OST1 (right). On the bottom panel, the confocal images were merged with the corresponding bright field images. Scale bar corresponds to 8 µm. (D) ABF3 and OST1 interaction is analyzed by Bimolecular Fluorescent Complementation in N. benthamiana tobacco leaves expressing the given constructs. The YFP fluorescent signal is located in the nucleus of epidermal cells. In these experiments, the nuclear localized AKINβ protein is used as negative control. Scale bar corresponds to 20 µm.

Techniques Used: Quantitative RT-PCR, Expressing, Standard Deviation, Construct, Negative Control

37) Product Images from "Molecular and structural assessment of alveolar bone during tooth eruption and function in the miniature pig, Sus scrofa"

Article Title: Molecular and structural assessment of alveolar bone during tooth eruption and function in the miniature pig, Sus scrofa

Journal: Anatomia, histologia, embryologia

doi: 10.1111/j.1439-0264.2011.01067.x

qRT PCR
Figure Legend Snippet: qRT PCR

Techniques Used: Quantitative RT-PCR

38) Product Images from "Cold tolerance and silencing of three cold-tolerance genes of overwintering Chinese white pine larvae"

Article Title: Cold tolerance and silencing of three cold-tolerance genes of overwintering Chinese white pine larvae

Journal: Scientific Reports

doi: 10.1038/srep34698

qRT-PCR analysis of DarmSDH, DarmTPS and DarmGLK transcript patterns from D. armandi larvae; after injected for 24 h, 48 h and 72 h. The standard errors of the means of three biological replicates are represented by error bars. Transcript patterns of DarmSDH and DarmTPS were analyzed on December 2014, and DarmGLK on November 2014.
Figure Legend Snippet: qRT-PCR analysis of DarmSDH, DarmTPS and DarmGLK transcript patterns from D. armandi larvae; after injected for 24 h, 48 h and 72 h. The standard errors of the means of three biological replicates are represented by error bars. Transcript patterns of DarmSDH and DarmTPS were analyzed on December 2014, and DarmGLK on November 2014.

Techniques Used: Quantitative RT-PCR, Injection

39) Product Images from "Transmigration across activated endothelium induces transcriptional changes, inhibits apoptosis, and decreases antimicrobial protein expression in human monocytes"

Article Title: Transmigration across activated endothelium induces transcriptional changes, inhibits apoptosis, and decreases antimicrobial protein expression in human monocytes

Journal: Journal of Leukocyte Biology

doi: 10.1189/jlb.0209062

qRT-PCR confirmation
Figure Legend Snippet: qRT-PCR confirmation

Techniques Used: Quantitative RT-PCR

40) Product Images from "Up-Regulated Expression and Aberrant DNA Methylation of LEP and SH3PXD2A in Pre-Eclampsia"

Article Title: Up-Regulated Expression and Aberrant DNA Methylation of LEP and SH3PXD2A in Pre-Eclampsia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0059753

Validation the mRNA expression of LEP and SH3PXD2A in preeclamptic (n = 7) versus normal (n = 6) placentas. (A) Expression of LEP mRNA measured by qRT-PCR. The difference between preeclamptic placentas and normal controls is highly significant ( p = 0.003). ** p
Figure Legend Snippet: Validation the mRNA expression of LEP and SH3PXD2A in preeclamptic (n = 7) versus normal (n = 6) placentas. (A) Expression of LEP mRNA measured by qRT-PCR. The difference between preeclamptic placentas and normal controls is highly significant ( p = 0.003). ** p

Techniques Used: Expressing, Quantitative RT-PCR

41) Product Images from "Transcriptome analysis of rice root heterosis by RNA-Seq"

Article Title: Transcriptome analysis of rice root heterosis by RNA-Seq

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-19

Comparison of the log 2 (FC) of 18 selected transcripts using RNA-Seq and qRT-PCR.
Figure Legend Snippet: Comparison of the log 2 (FC) of 18 selected transcripts using RNA-Seq and qRT-PCR.

Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

42) Product Images from "Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus 1Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus 1 [OPEN]"

Article Title: Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus 1Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus 1 [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.16.00116

HMRm requires a transit peptide to regulate HMR-dependent genes in the nucleus. A, qRT-PCR analyses of the relative expression levels of representative HMR-repressed and PIF-induced Class A genes in 4-d-old Col-0, Col-0 with lincomycin (linc) treatment,
Figure Legend Snippet: HMRm requires a transit peptide to regulate HMR-dependent genes in the nucleus. A, qRT-PCR analyses of the relative expression levels of representative HMR-repressed and PIF-induced Class A genes in 4-d-old Col-0, Col-0 with lincomycin (linc) treatment,

Techniques Used: Quantitative RT-PCR, Expressing

43) Product Images from "Autophagy mediates epithelial cancer chemoresistance by reducing p62/SQSTM1 accumulation"

Article Title: Autophagy mediates epithelial cancer chemoresistance by reducing p62/SQSTM1 accumulation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201621

Increased p62 and Nrf2 expression in TDR HEp-2 cells. ( a ) mRNA abundance of the indicated genes representative of the canonical ( i . e ., ER stress-induced) UPR, Nrf2-dependent anti-oxidant response, autophagic factors, proteasome subunits, and the mitochondrial UPR (mtUPR), were assessed by Sybr Green qRT-PCR in parental and TDR HEp-2 cells and normalized to histone H3 levels (fold change TDR/HEp-2 ± SEM, Wilcoxon signed-rank test, * p
Figure Legend Snippet: Increased p62 and Nrf2 expression in TDR HEp-2 cells. ( a ) mRNA abundance of the indicated genes representative of the canonical ( i . e ., ER stress-induced) UPR, Nrf2-dependent anti-oxidant response, autophagic factors, proteasome subunits, and the mitochondrial UPR (mtUPR), were assessed by Sybr Green qRT-PCR in parental and TDR HEp-2 cells and normalized to histone H3 levels (fold change TDR/HEp-2 ± SEM, Wilcoxon signed-rank test, * p

Techniques Used: Expressing, SYBR Green Assay, Quantitative RT-PCR

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

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

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2013/527561

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

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

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023334

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

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

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0121524

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

Techniques Used: Polymerase Chain Reaction

47) Product Images from "Transcription Elongation Factor GreA Plays a Key Role in Cellular Invasion and Virulence of Francisella tularensis subsp. novicida"

Article Title: Transcription Elongation Factor GreA Plays a Key Role in Cellular Invasion and Virulence of Francisella tularensis subsp. novicida

Journal: Scientific Reports

doi: 10.1038/s41598-018-25271-5

Verification of RNA-seq data. ( A ) Detection of gene transcription in the wild-type U112 strain and the Δ greA mutant. Transcription levels of genes by RNA-seq were shown with solid bars. Relative level of each target gene (open bars) by qRT-PCR was normalized to that of the 16S rRNA gene. Data are presented as mean fold changes relative to the wild-type U112 strain ± SD of the results from triplicate samples. The experiment was repeated twice. ( B ) Detection of protein expression in the wild-type U112 strain and the Δ greA mutant. Mid-log bacteria were resuspended in PBS to OD 600 = 1.0. The suspensions were concentrated 10-fold, separated with SDS-PAGE, and detected with western blotting, using antiserum specific for each target protein. The left panel is a section of a coomassie stained gel as a loading control. The coomassie stained gel image was acquired with the digital camera (Canon, Janpan). The experiment was repeated twice. Size of each protein is indicated on the left in kDa.
Figure Legend Snippet: Verification of RNA-seq data. ( A ) Detection of gene transcription in the wild-type U112 strain and the Δ greA mutant. Transcription levels of genes by RNA-seq were shown with solid bars. Relative level of each target gene (open bars) by qRT-PCR was normalized to that of the 16S rRNA gene. Data are presented as mean fold changes relative to the wild-type U112 strain ± SD of the results from triplicate samples. The experiment was repeated twice. ( B ) Detection of protein expression in the wild-type U112 strain and the Δ greA mutant. Mid-log bacteria were resuspended in PBS to OD 600 = 1.0. The suspensions were concentrated 10-fold, separated with SDS-PAGE, and detected with western blotting, using antiserum specific for each target protein. The left panel is a section of a coomassie stained gel as a loading control. The coomassie stained gel image was acquired with the digital camera (Canon, Janpan). The experiment was repeated twice. Size of each protein is indicated on the left in kDa.

Techniques Used: RNA Sequencing Assay, Mutagenesis, Quantitative RT-PCR, Expressing, SDS Page, Western Blot, Staining

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

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

Journal: American journal of physiology. Heart and circulatory physiology

doi: 10.1152/ajpheart.00595.2010

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

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

49) Product Images from "UBE2C Overexpression Aggravates Patient Outcome by Promoting Estrogen-Dependent/Independent Cell Proliferation in Early Hormone Receptor-Positive and HER2-Negative Breast Cancer"

Article Title: UBE2C Overexpression Aggravates Patient Outcome by Promoting Estrogen-Dependent/Independent Cell Proliferation in Early Hormone Receptor-Positive and HER2-Negative Breast Cancer

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2019.01574

UBE2C is expressed in HR+/HER2– breast cancer tissues and cell lines. (A) UBE2C mRNA expression in FFPE breast cancer tissue was evaluated in a dataset of 819 samples from our cohort according to LN status, pN0 (left panels), pN1 (middle panel), and pN2/N3 (right panel). (B) UBE2C mRNA expression in FFPE breast cancer tissue was evaluated in a dataset of 410 samples from patients with HR+/HER2– breast cancer according to LN status, pN0, pN1, and pN2/N3. The bar indicates the median value. (C) UBE2C protein expression was quantified by immunoblotting in breast cancer cell lines. * P
Figure Legend Snippet: UBE2C is expressed in HR+/HER2– breast cancer tissues and cell lines. (A) UBE2C mRNA expression in FFPE breast cancer tissue was evaluated in a dataset of 819 samples from our cohort according to LN status, pN0 (left panels), pN1 (middle panel), and pN2/N3 (right panel). (B) UBE2C mRNA expression in FFPE breast cancer tissue was evaluated in a dataset of 410 samples from patients with HR+/HER2– breast cancer according to LN status, pN0, pN1, and pN2/N3. The bar indicates the median value. (C) UBE2C protein expression was quantified by immunoblotting in breast cancer cell lines. * P

Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded

UBE2C depletion in combination with tamoxifen increases apoptosis of HR+/HER2– breast cancer cells. (A) Chou–Talalay drug interaction analysis in MCF-7 cells treated with UBE2C siRNA and tamoxifen. (B) The apoptotic potential of treated cells was determined by flow cytometry and Annexin V assay. The cells were stained with propidium iodide and Annexin V antibody, and DNA content and intensity were analyzed by flow cytometry. Numbers indicate the percentage of apoptotic cells. (C) The viability of treated MCF-7 cells was evaluated using a 3D Matrigel assay (40×). (D) Lysates from treated MCF-7 cells were subjected to western blotting to monitor the protein levels of cleaved PARP and cleaved CASPASE 3. Fold changes were determined by comparing the protein levels with those of β-ACTIN using ImageJ. Tam, tamoxifen.
Figure Legend Snippet: UBE2C depletion in combination with tamoxifen increases apoptosis of HR+/HER2– breast cancer cells. (A) Chou–Talalay drug interaction analysis in MCF-7 cells treated with UBE2C siRNA and tamoxifen. (B) The apoptotic potential of treated cells was determined by flow cytometry and Annexin V assay. The cells were stained with propidium iodide and Annexin V antibody, and DNA content and intensity were analyzed by flow cytometry. Numbers indicate the percentage of apoptotic cells. (C) The viability of treated MCF-7 cells was evaluated using a 3D Matrigel assay (40×). (D) Lysates from treated MCF-7 cells were subjected to western blotting to monitor the protein levels of cleaved PARP and cleaved CASPASE 3. Fold changes were determined by comparing the protein levels with those of β-ACTIN using ImageJ. Tam, tamoxifen.

Techniques Used: Flow Cytometry, Annexin V Assay, Staining, Matrigel Assay, Western Blot

UBE2C mRNA expression is strongly associated with poor survival in patients with HR+/HER2– breast cancer. The impact of UBE2C mRNA expression on DFS (left panels), DMFS (middle panels), and OS (right panels) of (A) all, (B) pN0, (C) pN1, or (D) pN2/N3 patients with HR+/HER2 – breast cancer was analyzed using the Kaplan–Meier method.
Figure Legend Snippet: UBE2C mRNA expression is strongly associated with poor survival in patients with HR+/HER2– breast cancer. The impact of UBE2C mRNA expression on DFS (left panels), DMFS (middle panels), and OS (right panels) of (A) all, (B) pN0, (C) pN1, or (D) pN2/N3 patients with HR+/HER2 – breast cancer was analyzed using the Kaplan–Meier method.

Techniques Used: Expressing

UBE2C contributes to the tumorigenicity of HR+/HER2– breast cancer cells. (A) The efficacy of siRNA-mediated UBE2C knockdown was evaluated by immunoblotting in T47D cells (left panel). NT, non-targeting; N, siNT; U, si UBE2C . The effects of UBE2C knockdown on cell proliferation were evaluated using an IncuCyte analyzer (middle panel) and clonogenic assay (right panel). (B) Ectopic UBE2C overexpression was confirmed by immunoblotting in MCF-7 cells (upper left panel). The effects of UBE2C overexpression on cell proliferation were evaluated using an IncuCyte analyzer (lower left panel) and clonogenic assay in 2D (upper right panel) and 3D Matrigel (100×, lower right panel). (C) The effects of UBE2C knockdown on cell migration were evaluated with the wound healing assay using the IncuCyte analyzer in T47D cells. (D) The effects of ectopic UBE2C overexpression on cell migration were evaluated with the Boyden chamber assay in MCF-7 cells. n = 3; Bars, SE; * P
Figure Legend Snippet: UBE2C contributes to the tumorigenicity of HR+/HER2– breast cancer cells. (A) The efficacy of siRNA-mediated UBE2C knockdown was evaluated by immunoblotting in T47D cells (left panel). NT, non-targeting; N, siNT; U, si UBE2C . The effects of UBE2C knockdown on cell proliferation were evaluated using an IncuCyte analyzer (middle panel) and clonogenic assay (right panel). (B) Ectopic UBE2C overexpression was confirmed by immunoblotting in MCF-7 cells (upper left panel). The effects of UBE2C overexpression on cell proliferation were evaluated using an IncuCyte analyzer (lower left panel) and clonogenic assay in 2D (upper right panel) and 3D Matrigel (100×, lower right panel). (C) The effects of UBE2C knockdown on cell migration were evaluated with the wound healing assay using the IncuCyte analyzer in T47D cells. (D) The effects of ectopic UBE2C overexpression on cell migration were evaluated with the Boyden chamber assay in MCF-7 cells. n = 3; Bars, SE; * P

Techniques Used: Clonogenic Assay, Over Expression, Migration, Wound Healing Assay, Boyden Chamber Assay

UBE2C is a target of ERα and is required for estrogen-induced cell proliferation. (A) The induction of UBE2C mRNA (upper panel) and protein (lower panel) expression by estrogen in MCF-7 cells was quantified by qRT-PCR and immunoblotting, respectively. (B) The ERα-bound UBE2C promoter region was assayed by conventional ChIP-PCR. The GREB1 and GAPDH promoters served as ER α-bound positive and negative controls, respectively. (C) MCF-7 cells were transfected with UBE2C siRNAs for 48 h, and estrogen was added to the medium at 48 h intervals. Cell proliferation was evaluated using an IncuCyte analyzer (upper panel). MCF-7 cells were transfected with UBE2C siRNAs for 48 h, and estrogen was added to the medium for 24 h. Then, cells were stained with propidium iodide, and DNA content and intensity were analyzed by flow cytometry. (D) Hormone-independent cell proliferation of UBE2C-overexpressing MCF-7 cells was monitored by clonogenic assay under DMEM (upper and lower panels) or charcoal stripped DMEM (middle and lower panels) culture conditions. n = 3; Bars: SE. * P
Figure Legend Snippet: UBE2C is a target of ERα and is required for estrogen-induced cell proliferation. (A) The induction of UBE2C mRNA (upper panel) and protein (lower panel) expression by estrogen in MCF-7 cells was quantified by qRT-PCR and immunoblotting, respectively. (B) The ERα-bound UBE2C promoter region was assayed by conventional ChIP-PCR. The GREB1 and GAPDH promoters served as ER α-bound positive and negative controls, respectively. (C) MCF-7 cells were transfected with UBE2C siRNAs for 48 h, and estrogen was added to the medium at 48 h intervals. Cell proliferation was evaluated using an IncuCyte analyzer (upper panel). MCF-7 cells were transfected with UBE2C siRNAs for 48 h, and estrogen was added to the medium for 24 h. Then, cells were stained with propidium iodide, and DNA content and intensity were analyzed by flow cytometry. (D) Hormone-independent cell proliferation of UBE2C-overexpressing MCF-7 cells was monitored by clonogenic assay under DMEM (upper and lower panels) or charcoal stripped DMEM (middle and lower panels) culture conditions. n = 3; Bars: SE. * P

Techniques Used: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Transfection, Staining, Flow Cytometry, Clonogenic Assay

50) Product Images from "Transcriptome analysis reveals the roles of stem nodes in cadmium transport to rice grain"

Article Title: Transcriptome analysis reveals the roles of stem nodes in cadmium transport to rice grain

Journal: BMC Genomics

doi: 10.1186/s12864-020-6474-7

Consistency results of qRT-PCR and RNA-seq. a Expression of mRNAs in different group. b Expression of miRNAs in different group. c Consistency results of qRT-PCR and RNA-seq (including mRNAs and miRNAs). The figure was based on the fold change of qRT-PCR and log2 fold change of RNA-seq. R 2 represented the correlation coefficient between qRT-PCR and RNA-seq
Figure Legend Snippet: Consistency results of qRT-PCR and RNA-seq. a Expression of mRNAs in different group. b Expression of miRNAs in different group. c Consistency results of qRT-PCR and RNA-seq (including mRNAs and miRNAs). The figure was based on the fold change of qRT-PCR and log2 fold change of RNA-seq. R 2 represented the correlation coefficient between qRT-PCR and RNA-seq

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

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

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

Journal: Carcinogenesis

doi: 10.1093/carcin/bgs285

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

Techniques Used: Quantitative RT-PCR, Expressing

52) Product Images from "Chromatin Modulation at the FLO11 Promoter of Saccharomyces cerevisiae by HDAC and Swi/Snf Complexes"

Article Title: Chromatin Modulation at the FLO11 Promoter of Saccharomyces cerevisiae by HDAC and Swi/Snf Complexes

Journal: Genetics

doi: 10.1534/genetics.112.140301

Sfl1-mediated repression is important for FLO11 regulation but is absent in the 133d strain. (A) Northern blot analysis of FLO11 mRNA, as described in , for the indicated wild-type and mutant strains. (B) qRT-PCR analysis of FLO11 and SFL1 expression
Figure Legend Snippet: Sfl1-mediated repression is important for FLO11 regulation but is absent in the 133d strain. (A) Northern blot analysis of FLO11 mRNA, as described in , for the indicated wild-type and mutant strains. (B) qRT-PCR analysis of FLO11 and SFL1 expression

Techniques Used: Northern Blot, Mutagenesis, Quantitative RT-PCR, Expressing

Rpd3 counteracts the action of Sfl1 on the laboratory FLO11 allele by regulating ICR1 ncRNA. (A) Northern blot analysis of FLO11 mRNA, as described in , for the indicated wild-type and mutant strains. (B) qRT-PCR analysis of ICR1 ncRNA expression
Figure Legend Snippet: Rpd3 counteracts the action of Sfl1 on the laboratory FLO11 allele by regulating ICR1 ncRNA. (A) Northern blot analysis of FLO11 mRNA, as described in , for the indicated wild-type and mutant strains. (B) qRT-PCR analysis of ICR1 ncRNA expression

Techniques Used: Northern Blot, Mutagenesis, Quantitative RT-PCR, Expressing

53) Product Images from "Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients ▿"

Article Title: Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients ▿

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.00785-11

Bland-Altman representation of CMV DNA loads (copies/ml) measured using the Abbott CMV PCR kit in the m2000RT (ABBOTT), the LightCycler CMV Quant kit in the Light Cycler 2.0 instrument (ROCHE), and the Q-CMV complete kit in the LightCycler 2.0 instrument
Figure Legend Snippet: Bland-Altman representation of CMV DNA loads (copies/ml) measured using the Abbott CMV PCR kit in the m2000RT (ABBOTT), the LightCycler CMV Quant kit in the Light Cycler 2.0 instrument (ROCHE), and the Q-CMV complete kit in the LightCycler 2.0 instrument

Techniques Used: Polymerase Chain Reaction

Correlation and linear regression analysis of cytomegalovirus (CMV) DNA load values (copies/ml) obtained for all positive specimens by the Abbott CMV PCR kit following DNA extraction using the Abbott mSample preparation system DNA kit on the m24 SP instrument
Figure Legend Snippet: Correlation and linear regression analysis of cytomegalovirus (CMV) DNA load values (copies/ml) obtained for all positive specimens by the Abbott CMV PCR kit following DNA extraction using the Abbott mSample preparation system DNA kit on the m24 SP instrument

Techniques Used: Polymerase Chain Reaction, DNA Extraction

Bland-Altman representation of CMV DNA loads (copies/ml) measured using the Abbott CMV PCR kit in the m2000RT (ABBOTT) after extraction by the Abbott mSample preparation system DNA kit on the m24 SP instrument and those measured by the LightCycler CMV
Figure Legend Snippet: Bland-Altman representation of CMV DNA loads (copies/ml) measured using the Abbott CMV PCR kit in the m2000RT (ABBOTT) after extraction by the Abbott mSample preparation system DNA kit on the m24 SP instrument and those measured by the LightCycler CMV

Techniques Used: Polymerase Chain Reaction

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

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

Journal: Journal of Bacteriology

doi: 10.1128/JB.00481-10

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

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

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

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

Journal: Biochemistry

doi: 10.1021/bi501388y

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

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

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

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

Journal: Current Genomics

doi: 10.2174/1389202918666170228134205

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

Techniques Used: Quantitative RT-PCR, Expressing

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

Techniques Used: Quantitative RT-PCR, Expressing

57) Product Images from "Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia"

Article Title: Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01661

ZNF554 down-regulation in the villous trophoblast (VT) in preeclampsia. (A) Tissue qRT-PCR array revealed the highest ZNF554 expression in the placenta among 48 human tissues. Color code depicts gene expression levels relative to that of the placenta (100%). (B) In situ hybridization of a third trimester control placenta (GW29) and (C) immunohistochemistry of a first trimester placenta (GW12) shows mainly syncytiotrophoblastic ZNF554 expression (hematoxylin counterstaining, 1,400 and 400× magnifications, respectively). Black or white arrowheads depict syncytiotrophoblast or cytotrophoblast, while black arrow depicts fetal endothelium, respectively. (D) qRT-PCR revealed that ZNF554 expression is up-regulated during VT differentiation in parallel with CSH1 . (E,F) ZNF554 immunopositivity was faint in the syncytiotrophoblast in preeclampsia [ (F) , GW35] compared to gestational-age matched controls [ (E) , GW36] (hematoxylin counterstaining, 400× magnifications). Arrow and arrowhead depict syncytiotrophoblast and villous endothelium, respectively. (G) ZNF554 mRNA expression was 74% lower in ZNF554 -silenced BeWo cells compared to controls used for the microarrays ( p = 5.24 × 10 −6 ). (H) Nuclear and cytoplasmic ZNF554 immunofluorescence decreased in BeWo cells treated with ZNF554 siRNA compared to control cells (3,500× magnifications). (I) Molecular functions and one Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (glycolysis/gluconeogenesis) affected in ZNF554 -silenced BeWo cells. Colors denote the proportions of up- or down-regulated genes (red: > 0.5 up-regulated; blue: > 0.5 down-regulated; black: 0.5–0.5 up- and down-regulated). Letter sizes represent the minus log 10 of p -values of the given functions or pathway. (J) qRT-PCR validated FSTL3 up-regulation (2.7-fold, p
Figure Legend Snippet: ZNF554 down-regulation in the villous trophoblast (VT) in preeclampsia. (A) Tissue qRT-PCR array revealed the highest ZNF554 expression in the placenta among 48 human tissues. Color code depicts gene expression levels relative to that of the placenta (100%). (B) In situ hybridization of a third trimester control placenta (GW29) and (C) immunohistochemistry of a first trimester placenta (GW12) shows mainly syncytiotrophoblastic ZNF554 expression (hematoxylin counterstaining, 1,400 and 400× magnifications, respectively). Black or white arrowheads depict syncytiotrophoblast or cytotrophoblast, while black arrow depicts fetal endothelium, respectively. (D) qRT-PCR revealed that ZNF554 expression is up-regulated during VT differentiation in parallel with CSH1 . (E,F) ZNF554 immunopositivity was faint in the syncytiotrophoblast in preeclampsia [ (F) , GW35] compared to gestational-age matched controls [ (E) , GW36] (hematoxylin counterstaining, 400× magnifications). Arrow and arrowhead depict syncytiotrophoblast and villous endothelium, respectively. (G) ZNF554 mRNA expression was 74% lower in ZNF554 -silenced BeWo cells compared to controls used for the microarrays ( p = 5.24 × 10 −6 ). (H) Nuclear and cytoplasmic ZNF554 immunofluorescence decreased in BeWo cells treated with ZNF554 siRNA compared to control cells (3,500× magnifications). (I) Molecular functions and one Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (glycolysis/gluconeogenesis) affected in ZNF554 -silenced BeWo cells. Colors denote the proportions of up- or down-regulated genes (red: > 0.5 up-regulated; blue: > 0.5 down-regulated; black: 0.5–0.5 up- and down-regulated). Letter sizes represent the minus log 10 of p -values of the given functions or pathway. (J) qRT-PCR validated FSTL3 up-regulation (2.7-fold, p

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

Gene modules associated with blood pressure (BP) and birthweight (BW). (A) Hierarchical clustering of qRT-PCR data obtained with 100 samples and 47 genes. Pearson correlation was used for similarity analysis and average method for linkage calculation. Samples were colored according to patient groups and maturity status. M1 (green) and M2 (red) module genes and 34 of 60 samples from women with preeclampsia clustered together. (B) Association of gene expression with BP and BW. The significance p -values for these coefficients were plotted for all genes, colored according to module classification (black: not changed on the microarray). Filled circles represent predominantly placenta-expressed genes and dashed lines the significance threshold at p = 0.05. Seven of 9 genes related to BW belong to module M1, while 10 of 15 genes related to BP are from module M2. Gene expression (C) and protein immunostaining (D) in Supplementary Material. (E) Representative images of the same placenta from a preterm control (left, 29 weeks) and a patient with preterm preeclampsia associated with SGA (right, 31 weeks) are shown for the immunostainings (hematoxylin counterstaining, 40× magnification).
Figure Legend Snippet: Gene modules associated with blood pressure (BP) and birthweight (BW). (A) Hierarchical clustering of qRT-PCR data obtained with 100 samples and 47 genes. Pearson correlation was used for similarity analysis and average method for linkage calculation. Samples were colored according to patient groups and maturity status. M1 (green) and M2 (red) module genes and 34 of 60 samples from women with preeclampsia clustered together. (B) Association of gene expression with BP and BW. The significance p -values for these coefficients were plotted for all genes, colored according to module classification (black: not changed on the microarray). Filled circles represent predominantly placenta-expressed genes and dashed lines the significance threshold at p = 0.05. Seven of 9 genes related to BW belong to module M1, while 10 of 15 genes related to BP are from module M2. Gene expression (C) and protein immunostaining (D) in Supplementary Material. (E) Representative images of the same placenta from a preterm control (left, 29 weeks) and a patient with preterm preeclampsia associated with SGA (right, 31 weeks) are shown for the immunostainings (hematoxylin counterstaining, 40× magnification).

Techniques Used: Quantitative RT-PCR, Expressing, Microarray, Immunostaining

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

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

Journal: Experimental & Molecular Medicine

doi: 10.1038/s12276-018-0033-1

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

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

59) Product Images from "Regulation of Nucleotide Excision Repair by Nuclear Lamin B1"

Article Title: Regulation of Nucleotide Excision Repair by Nuclear Lamin B1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069169

Transient silencing of LB1 induces growth arrest in U-2 OS cells. A. The protein levels of LB1, LB2, and LA and C were assayed by immunoblotting at day 3 after electroporation with the vector encoding shRNA (shLB1) or a scrambled sequence (Sc). B. Relative expression levels of LMNB1 , LMNB2 , and LMNA mRNA in cells were determined by qRT-PCR at day 3 after silencing using GAPDH as a reference gene. The error bars represent standard deviation of the mean (n = 5). C. Growth rate of shLB1 and Sc cells were compared for 5 days following silencing. Growth rate was evaluated as previously described [17] (n = 6, p = 5.24 ×10 −7 ); error bars represent standard deviations.
Figure Legend Snippet: Transient silencing of LB1 induces growth arrest in U-2 OS cells. A. The protein levels of LB1, LB2, and LA and C were assayed by immunoblotting at day 3 after electroporation with the vector encoding shRNA (shLB1) or a scrambled sequence (Sc). B. Relative expression levels of LMNB1 , LMNB2 , and LMNA mRNA in cells were determined by qRT-PCR at day 3 after silencing using GAPDH as a reference gene. The error bars represent standard deviation of the mean (n = 5). C. Growth rate of shLB1 and Sc cells were compared for 5 days following silencing. Growth rate was evaluated as previously described [17] (n = 6, p = 5.24 ×10 −7 ); error bars represent standard deviations.

Techniques Used: Electroporation, Plasmid Preparation, shRNA, Sequencing, Expressing, Quantitative RT-PCR, Standard Deviation

60) Product Images from "Methylation of promoter of RBL1 enhances the radioresistance of three dimensional cultured carcinoma cells"

Article Title: Methylation of promoter of RBL1 enhances the radioresistance of three dimensional cultured carcinoma cells

Journal: Oncotarget

doi: 10.18632/oncotarget.12647

Overexpression of RBL1 sensitizes the 3D cultured A549 and MCF7 cells to X-rays A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 3D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 3D A549 and MCF7 cells. C D . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 3D A549 and MCF7 cells. F . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 3D A549 and MCF7 cells. G H . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1-siRNA or NC, were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
Figure Legend Snippet: Overexpression of RBL1 sensitizes the 3D cultured A549 and MCF7 cells to X-rays A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 3D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 3D A549 and MCF7 cells. C D . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 3D A549 and MCF7 cells. F . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 3D A549 and MCF7 cells. G H . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1-siRNA or NC, were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

Techniques Used: Over Expression, Cell Culture, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Colony Assay, Negative Control

Expression of cell cycle regulation genes in irradiated 2D and 3D A549 cells A . Microarray analysis of 84 cell cycle regulation genes expression profile in 2D and 3D A549 cells 12h after irradiation with 4Gy X-rays. B . Venn diagrams of different expression of 84 cell cycle genes. C . Relative expression levels of RBL1, CCND1 and CCNF were measured by qRT-PCR at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells. GAPDH were used as internal control. D . The expressions of RBL1, CCND1 and CCNF at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells by western blot assay. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
Figure Legend Snippet: Expression of cell cycle regulation genes in irradiated 2D and 3D A549 cells A . Microarray analysis of 84 cell cycle regulation genes expression profile in 2D and 3D A549 cells 12h after irradiation with 4Gy X-rays. B . Venn diagrams of different expression of 84 cell cycle genes. C . Relative expression levels of RBL1, CCND1 and CCNF were measured by qRT-PCR at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells. GAPDH were used as internal control. D . The expressions of RBL1, CCND1 and CCNF at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells by western blot assay. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

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

Knockdown of RBL1 enhances the radioresistance of 2D cultured A549 and MCF7 cells and decreases the G2/M arrest induced by X-ray A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 2D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 2D A549 and MCF7 cells. C D . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1-siRNA or NC, was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E F . Cell cycle distribution of 2D A549 cells transfected with RBL1-siRNA or NC and then exposed 4 Gy X-rays. G . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 2D A549 and MCF7 cells. H . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 2D A549 and MCF7 cells. I J . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
Figure Legend Snippet: Knockdown of RBL1 enhances the radioresistance of 2D cultured A549 and MCF7 cells and decreases the G2/M arrest induced by X-ray A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 2D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 2D A549 and MCF7 cells. C D . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1-siRNA or NC, was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E F . Cell cycle distribution of 2D A549 cells transfected with RBL1-siRNA or NC and then exposed 4 Gy X-rays. G . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 2D A549 and MCF7 cells. H . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 2D A549 and MCF7 cells. I J . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Western Blot, Colony Assay, Over Expression, Plasmid Preparation

61) Product Images from "Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread"

Article Title: Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

Journal: Journal of Virology

doi: 10.1128/JVI.03241-13

Inhibiting cell-to-cell spread affects antiviral synergy potential between HCV entry/egress inhibitors and interferon. Chronically HCV-infected Huh7 cells were seeded in 96-well plates. (A) Cultures were mock treated (black) or treated with 100 units/ml IFN-α (brown), 200 μM naringenin (NG; orange), or 30 μM ezetimibe (EZE; blue) either alone (solid lines) or in combination (dotted lines) as indicated. (B) Cultures were mock treated (black) or treated with 100 units/ml IFN-α (brown), 50 μM ferristatin (TF; orange), an inhibitor of transferrin receptor-1, or 30 μM EZE (blue) either alone (solid lines) or in combination (dotted lines) as indicated. At 24, 48, 72, or 96 h posttreatment (hpt), intracellular RNA was extracted. HCV and cellular GAPDH RNA levels were quantified by qRT-PCR. HCV copies were normalized to cellular GAPDH and are expressed as average number of HCV copies/μg RNA in triplicate samples ± SD. Significant differences between the interferon-treated samples and the IFN-EZE treatment are indicated (**, P
Figure Legend Snippet: Inhibiting cell-to-cell spread affects antiviral synergy potential between HCV entry/egress inhibitors and interferon. Chronically HCV-infected Huh7 cells were seeded in 96-well plates. (A) Cultures were mock treated (black) or treated with 100 units/ml IFN-α (brown), 200 μM naringenin (NG; orange), or 30 μM ezetimibe (EZE; blue) either alone (solid lines) or in combination (dotted lines) as indicated. (B) Cultures were mock treated (black) or treated with 100 units/ml IFN-α (brown), 50 μM ferristatin (TF; orange), an inhibitor of transferrin receptor-1, or 30 μM EZE (blue) either alone (solid lines) or in combination (dotted lines) as indicated. At 24, 48, 72, or 96 h posttreatment (hpt), intracellular RNA was extracted. HCV and cellular GAPDH RNA levels were quantified by qRT-PCR. HCV copies were normalized to cellular GAPDH and are expressed as average number of HCV copies/μg RNA in triplicate samples ± SD. Significant differences between the interferon-treated samples and the IFN-EZE treatment are indicated (**, P

Techniques Used: Infection, Quantitative RT-PCR

ApoB, ApoE, and MTP are not required for HCV cell-to-cell spread. Nongrowing Huh7 cells were transfected with siRNAs targeting ApoB, ApoE, MTP, claudin-1, or GFP (negative control). After 24 h, cultures were inoculated with 100 FFU JFH-1 HCVcc. At 16 hpi, cells were washed and medium containing neutralizing anti-E2 was added to limit cell-free virus spread. Cells were fixed at 5 days postinfection and stained for HCV E2 to detect infected cells. (A) Levels of target gene mRNA were quantified by qRT-PCR at days 1 and 6 posttransfection, and the averages of duplicate samples ± SD are graphed as percentages of levels in siGFP-silenced cells. (B) Number of foci observed in cultures transfected with indicated siRNA. Averages of duplicate wells ± SD are graphed as percentages of number of foci observed in cells transfected with control siGFP. (C) Morphology of foci observed in cultures transfected with the indicated siRNA. Insets show magnification of a single focus. Wells were photographed using a Nikon Eclipse TE2000U with a 4× objective lens or a 10× objective lens. (D) Average number of cells per focus ± SD. The number of cells in the foci under each condition were not significantly different from the control (as determined by ANOVA). (E) Effect of indicated siRNA-mediated knockdown on HCV secretion at 48 hpi (72 h posttransfection) and 72 hpi (96 h posttransfection). Infectious virus was quantified by titration of medium from the silenced cells on naive Huh7 cells. Average FFUs per ml ± SD are graphed as a percentage of infectious virus released from control siGFP cells. Results are representative of three experiments performed in both growing and nongrowing Huh7 cells.
Figure Legend Snippet: ApoB, ApoE, and MTP are not required for HCV cell-to-cell spread. Nongrowing Huh7 cells were transfected with siRNAs targeting ApoB, ApoE, MTP, claudin-1, or GFP (negative control). After 24 h, cultures were inoculated with 100 FFU JFH-1 HCVcc. At 16 hpi, cells were washed and medium containing neutralizing anti-E2 was added to limit cell-free virus spread. Cells were fixed at 5 days postinfection and stained for HCV E2 to detect infected cells. (A) Levels of target gene mRNA were quantified by qRT-PCR at days 1 and 6 posttransfection, and the averages of duplicate samples ± SD are graphed as percentages of levels in siGFP-silenced cells. (B) Number of foci observed in cultures transfected with indicated siRNA. Averages of duplicate wells ± SD are graphed as percentages of number of foci observed in cells transfected with control siGFP. (C) Morphology of foci observed in cultures transfected with the indicated siRNA. Insets show magnification of a single focus. Wells were photographed using a Nikon Eclipse TE2000U with a 4× objective lens or a 10× objective lens. (D) Average number of cells per focus ± SD. The number of cells in the foci under each condition were not significantly different from the control (as determined by ANOVA). (E) Effect of indicated siRNA-mediated knockdown on HCV secretion at 48 hpi (72 h posttransfection) and 72 hpi (96 h posttransfection). Infectious virus was quantified by titration of medium from the silenced cells on naive Huh7 cells. Average FFUs per ml ± SD are graphed as a percentage of infectious virus released from control siGFP cells. Results are representative of three experiments performed in both growing and nongrowing Huh7 cells.

Techniques Used: Transfection, Negative Control, Staining, Infection, Quantitative RT-PCR, Titration

62) Product Images from "Use of Anti-Aedes aegypti Salivary Extract Antibody Concentration to Correlate Risk of Vector Exposure and Dengue Transmission Risk in Colombia"

Article Title: Use of Anti-Aedes aegypti Salivary Extract Antibody Concentration to Correlate Risk of Vector Exposure and Dengue Transmission Risk in Colombia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081211

Concentration of anti- Ae. aegypti SGE antibodies according to the DENV serotype in participants from Los Patios diagnosed by RT-PCR. Figure also represents the distribution of the different SGE-antibody concentrations for each DENV serotype by age group. Statistical significance (*) p
Figure Legend Snippet: Concentration of anti- Ae. aegypti SGE antibodies according to the DENV serotype in participants from Los Patios diagnosed by RT-PCR. Figure also represents the distribution of the different SGE-antibody concentrations for each DENV serotype by age group. Statistical significance (*) p

Techniques Used: Concentration Assay, Reverse Transcription Polymerase Chain Reaction

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

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

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1555-6

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

Techniques Used:

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

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

Journal: BMC Systems Biology

doi: 10.1186/1752-0509-4-159

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

Techniques Used: Quantitative RT-PCR, Expressing, Microarray

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

Techniques Used: Quantitative RT-PCR, Inhibition

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

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku485

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

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

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

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

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

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

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

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

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

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

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

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

66) Product Images from "Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts"

Article Title: Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Journal: Scientific Reports

doi: 10.1038/srep39149

Determination of the hBMP2 mRNA decay kinetics via qRT-PCR, quantification of hBMP2 protein and mRNA productivity. C2C12 cells were transfected with hBMP2-CYBA 2 × 3 and hBMP2-CYBA 5 + 3 transcripts and were compared to the control without UTRs. Absolute mRNA quantification ( a ) as well as the corresponding protein amounts ( b ) were determined by qRT-PCR and ELISA, respectively. Mean fold induction of the mRNA productivity of the different CYBA UTR combinations at 12 and 24 hours post-transfection ( c ). Data represent means ± SEM of three replicates. Statistical significance was assessed by 2-way ANOVA test with p values: ***p
Figure Legend Snippet: Determination of the hBMP2 mRNA decay kinetics via qRT-PCR, quantification of hBMP2 protein and mRNA productivity. C2C12 cells were transfected with hBMP2-CYBA 2 × 3 and hBMP2-CYBA 5 + 3 transcripts and were compared to the control without UTRs. Absolute mRNA quantification ( a ) as well as the corresponding protein amounts ( b ) were determined by qRT-PCR and ELISA, respectively. Mean fold induction of the mRNA productivity of the different CYBA UTR combinations at 12 and 24 hours post-transfection ( c ). Data represent means ± SEM of three replicates. Statistical significance was assessed by 2-way ANOVA test with p values: ***p

Techniques Used: Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay

Determination of mRNA quantity, the corresponding protein amounts and the resulting mRNA productivity ( Met Luc). mRNA decay kinetics in NIH3T3 ( a ) and A549 cells ( b ). mRNA amounts over time were quantified by qRT-PCR. The corresponding Met Luc protein translation data at 4, 24, 48, 72, 96 and 120 hours post-transfection in NIH3T3 ( c ) and in A549 cells ( d ). Mean fold-induction of transcript productivity of the different CYBA UTR combinations in NIH3T3 ( e ) and A549 ( f ) cells, respectively. Data represent means ± standard error of the mean (SEM) of three replicates. Statistical significance was assessed by 2-way ANOVA test with p values: *p
Figure Legend Snippet: Determination of mRNA quantity, the corresponding protein amounts and the resulting mRNA productivity ( Met Luc). mRNA decay kinetics in NIH3T3 ( a ) and A549 cells ( b ). mRNA amounts over time were quantified by qRT-PCR. The corresponding Met Luc protein translation data at 4, 24, 48, 72, 96 and 120 hours post-transfection in NIH3T3 ( c ) and in A549 cells ( d ). Mean fold-induction of transcript productivity of the different CYBA UTR combinations in NIH3T3 ( e ) and A549 ( f ) cells, respectively. Data represent means ± standard error of the mean (SEM) of three replicates. Statistical significance was assessed by 2-way ANOVA test with p values: *p

Techniques Used: Quantitative RT-PCR, Transfection

Osteogenic differentiation of hAMSC cells after transfection with hBMP2 transcripts as confirmed by alizarin red staining, ALP activity and qRT-PCR of RunX2 and OPN. ( a ) Mineralization of hAMSCs after recombinant hBMP2-CYBA mRNA transfection was visualized after 0, 7, 14 and 21 days using alizarin red staining. The scale bars represent 200 μm. ( b ) ALP activity was quantified at 3, 7 and 14 days post-transfection ( c ) Total RNA was extracted and qRT-PCR was performed at 3 and 7 days for RunX2 and 14 and 21 days for OPN after transfection. Expression is reported as fold induction compared to untransfected controls. All values were normalized to the housekeeping gene ß-tubulin. Data represent means ± SEM of three replicates. Statistical significance was assessed by 2-way ANOVA test with p values: *p
Figure Legend Snippet: Osteogenic differentiation of hAMSC cells after transfection with hBMP2 transcripts as confirmed by alizarin red staining, ALP activity and qRT-PCR of RunX2 and OPN. ( a ) Mineralization of hAMSCs after recombinant hBMP2-CYBA mRNA transfection was visualized after 0, 7, 14 and 21 days using alizarin red staining. The scale bars represent 200 μm. ( b ) ALP activity was quantified at 3, 7 and 14 days post-transfection ( c ) Total RNA was extracted and qRT-PCR was performed at 3 and 7 days for RunX2 and 14 and 21 days for OPN after transfection. Expression is reported as fold induction compared to untransfected controls. All values were normalized to the housekeeping gene ß-tubulin. Data represent means ± SEM of three replicates. Statistical significance was assessed by 2-way ANOVA test with p values: *p

Techniques Used: Transfection, Staining, ALP Assay, Activity Assay, Quantitative RT-PCR, Recombinant, Expressing

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

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

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0203-1

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

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

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

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

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

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

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2014.00233

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

Techniques Used:

69) Product Images from "Physical and Functional Interplay between MazF1Bif and Its Noncognate Antitoxins from Bifidobacterium longum"

Article Title: Physical and Functional Interplay between MazF1Bif and Its Noncognate Antitoxins from Bifidobacterium longum

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03232-16

MazF 1 Bif is an mRNA interferase that is inhibited by its cognate antitoxin, MazE 1 Bif . Relative transcript levels of tufA Bif were determined in E. coli expressing tufA Bif with pACYCDuet-1, pAD-F 1 , or pAD-F 1 E 1 . The strains were grown with 0.2% arabinose
Figure Legend Snippet: MazF 1 Bif is an mRNA interferase that is inhibited by its cognate antitoxin, MazE 1 Bif . Relative transcript levels of tufA Bif were determined in E. coli expressing tufA Bif with pACYCDuet-1, pAD-F 1 , or pAD-F 1 E 1 . The strains were grown with 0.2% arabinose

Techniques Used: Expressing

Noncognate antitoxin proteins counteract the mRNA interferase activity of MazF 1 Bif . Relative transcript levels of tufA Bif were determined in E. coli expressing tufA Bif with pACYCDuet-1, pAD-F 1 , and pAD-F 1 E 2 (A) or pAD-F 1 B (B). The strains were grown with
Figure Legend Snippet: Noncognate antitoxin proteins counteract the mRNA interferase activity of MazF 1 Bif . Relative transcript levels of tufA Bif were determined in E. coli expressing tufA Bif with pACYCDuet-1, pAD-F 1 , and pAD-F 1 E 2 (A) or pAD-F 1 B (B). The strains were grown with

Techniques Used: Activity Assay, Expressing

Interactions between MazF 1 Bif and its cognate antitoxin, MazE 1 Bif , or noncognate antitoxins affect cell growth. The growth characteristics of E. coli carrying pACYCDuet-1, pAD-F 1 , and pAD-F 1 E 1 (A), pAD-F 1 E 2 (B), or pAD-F 1 B (C) were analyzed by measuring
Figure Legend Snippet: Interactions between MazF 1 Bif and its cognate antitoxin, MazE 1 Bif , or noncognate antitoxins affect cell growth. The growth characteristics of E. coli carrying pACYCDuet-1, pAD-F 1 , and pAD-F 1 E 1 (A), pAD-F 1 E 2 (B), or pAD-F 1 B (C) were analyzed by measuring

Techniques Used:

70) Product Images from "Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase"

Article Title: Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw200

Rational design of RT-KTQ-LSIM libraries. Amino acids in immediate proximity to the 2′-oxygen of the nucleotide paired to the incoming dNTP were selected for saturation mutagenesis (namely G668, V669, G672, R746, K747 and N750). Adapted from PDB 4BWM ( 24 ) using PyMOL (Schrödinger, LLC, New York, NY, USA).
Figure Legend Snippet: Rational design of RT-KTQ-LSIM libraries. Amino acids in immediate proximity to the 2′-oxygen of the nucleotide paired to the incoming dNTP were selected for saturation mutagenesis (namely G668, V669, G672, R746, K747 and N750). Adapted from PDB 4BWM ( 24 ) using PyMOL (Schrödinger, LLC, New York, NY, USA).

Techniques Used: Mutagenesis

DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.
Figure Legend Snippet: DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. ( A ) Structures of relevant nucleotides. ( B ) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. ( C ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.

Techniques Used: DNA Synthesis, Methylation, Quantitative RT-PCR, Amplification

RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).
Figure Legend Snippet: RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. ( A ) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. ( B ) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. ( C ) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. ( D ) The ΔC T -method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).

Techniques Used: Methylation, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Concentration Assay

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

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

Journal: Acta Neuropathologica Communications

doi: 10.1186/s40478-016-0289-4

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

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

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

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

72) Product Images from "Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression"

Article Title: Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2008.00619.x

Analysis of the MMP expression profile of mMSC. ( A ) mMSC were analysed for their transcriptional expression of MMP‐2, MMP‐9 and MT1‐MMP relative to the expression of the housekeeping gene ALAS1 (set as 100%). ( B ) MMP‐2, MMP‐9 and MT1‐MMP transcript levels in Wnt3a‐stimulated (150 ng/ml) TCF/LEF‐reporter‐mMSC were determined after 4 days using qRT‐PCR and compared with those of unstimulated cells (set as 100%). ( C ) The gelatinolytic activity of MMP‐2 and MMP‐9 was examined in supernatants of TCF/LEF‐reporter‐mMSC stimulated with different Wnt3a concentrations over a time period of 3 days. HT1080‐conditioned medium containing pro‐MMP‐9, pro‐MMP‐2 and active forms of MMP‐2 and MMP‐9 was used as a marker. Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P
Figure Legend Snippet: Analysis of the MMP expression profile of mMSC. ( A ) mMSC were analysed for their transcriptional expression of MMP‐2, MMP‐9 and MT1‐MMP relative to the expression of the housekeeping gene ALAS1 (set as 100%). ( B ) MMP‐2, MMP‐9 and MT1‐MMP transcript levels in Wnt3a‐stimulated (150 ng/ml) TCF/LEF‐reporter‐mMSC were determined after 4 days using qRT‐PCR and compared with those of unstimulated cells (set as 100%). ( C ) The gelatinolytic activity of MMP‐2 and MMP‐9 was examined in supernatants of TCF/LEF‐reporter‐mMSC stimulated with different Wnt3a concentrations over a time period of 3 days. HT1080‐conditioned medium containing pro‐MMP‐9, pro‐MMP‐2 and active forms of MMP‐2 and MMP‐9 was used as a marker. Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P

Techniques Used: Expressing, Quantitative RT-PCR, Activity Assay, Marker

73) Product Images from "Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs"

Article Title: Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095240

Validation for microarray. qRT-PCR validation of the microarray data of the five miRNAs with consistent expression changes in A/exo and D/exo. Compared to S/exo, the levels of miR-100 , miR-17 , miR-222 , miR-342-3p and miR-451 were significantly up-regulated in A/exo and D/exo using qRT-PCR. Data are normalized to U6 . Fold changes from microarray and qRT-PCR are expressed as the mean ± SD, n = 3.
Figure Legend Snippet: Validation for microarray. qRT-PCR validation of the microarray data of the five miRNAs with consistent expression changes in A/exo and D/exo. Compared to S/exo, the levels of miR-100 , miR-17 , miR-222 , miR-342-3p and miR-451 were significantly up-regulated in A/exo and D/exo using qRT-PCR. Data are normalized to U6 . Fold changes from microarray and qRT-PCR are expressed as the mean ± SD, n = 3.

Techniques Used: Microarray, Quantitative RT-PCR, Expressing

74) Product Images from "Rubella Virus Strain-Associated Differences in the Induction of Oxidative Stress Are Independent of Their Interferon Activation"

Article Title: Rubella Virus Strain-Associated Differences in the Induction of Oxidative Stress Are Independent of Their Interferon Activation

Journal: Viruses

doi: 10.3390/v10100540

In contrast to the induction of oxidative stress, the RV-associated interferon (IFN) response on A549 cells was comparable among RV strains. ( A ) Western blot analysis of mock- and RV-infected A549 cells was performed at indicated time points with an antibody against RV E1 protein. The image shown is representative for three Western blot runs with independent samples; ( B ) Virus progeny generation was assessed by plaque (Wb-12) and focus forming assay (03-03703); ( C ) At 3 dpi the expression level of the antioxidant enzyme SOD2 and the oxidative stress-sensitive protein p21 was determined at mRNA expression level by qRT-PCR. H 2 O 2 applied over night at a 1:5000 dilution was used as positive control; ( D ) The LEGENDplex IFN panel was used to obtain the IFN profile for Wb-12- and 03-03703-infected A549 cells at 3 dpi. The lack of error bars is due to the upper detection limit of the assay, which was present despite analysis of a 1:4 dilution of the supernatants collected from mock- and RV-infected A549 cells; ( E ) The type I and III IFNs mRNA expression level was determined by qRT-PCR for samples extracted from Wb-12- and 03-03703-infected A549 cells at 1 and 3 dpi; ( F ) Activation of IFN-stimulated genes (ISGs) was validated by qRT-PCR for Wb-12- and 03-03703-infected A549 cells (obtained at 3 dpi); ( E , F ) Expression level was normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT1) and expression is given relative to the corresponding mock- or untreated control (CTL); ( D – F ) As a positive control, RNA extracted at 6 h post-transfection (hpst) from polyinosinic-polycytidylic acid (poly I:C)-transfected A549 cells was used. As a negative control mock-infected and lipofectamine2000-transfected samples were employed for RV infection and poly I:C transfection, respectively. *, p
Figure Legend Snippet: In contrast to the induction of oxidative stress, the RV-associated interferon (IFN) response on A549 cells was comparable among RV strains. ( A ) Western blot analysis of mock- and RV-infected A549 cells was performed at indicated time points with an antibody against RV E1 protein. The image shown is representative for three Western blot runs with independent samples; ( B ) Virus progeny generation was assessed by plaque (Wb-12) and focus forming assay (03-03703); ( C ) At 3 dpi the expression level of the antioxidant enzyme SOD2 and the oxidative stress-sensitive protein p21 was determined at mRNA expression level by qRT-PCR. H 2 O 2 applied over night at a 1:5000 dilution was used as positive control; ( D ) The LEGENDplex IFN panel was used to obtain the IFN profile for Wb-12- and 03-03703-infected A549 cells at 3 dpi. The lack of error bars is due to the upper detection limit of the assay, which was present despite analysis of a 1:4 dilution of the supernatants collected from mock- and RV-infected A549 cells; ( E ) The type I and III IFNs mRNA expression level was determined by qRT-PCR for samples extracted from Wb-12- and 03-03703-infected A549 cells at 1 and 3 dpi; ( F ) Activation of IFN-stimulated genes (ISGs) was validated by qRT-PCR for Wb-12- and 03-03703-infected A549 cells (obtained at 3 dpi); ( E , F ) Expression level was normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT1) and expression is given relative to the corresponding mock- or untreated control (CTL); ( D – F ) As a positive control, RNA extracted at 6 h post-transfection (hpst) from polyinosinic-polycytidylic acid (poly I:C)-transfected A549 cells was used. As a negative control mock-infected and lipofectamine2000-transfected samples were employed for RV infection and poly I:C transfection, respectively. *, p

Techniques Used: Western Blot, Infection, Focus Forming Assay, Expressing, Quantitative RT-PCR, Positive Control, Activation Assay, CTL Assay, Transfection, Negative Control

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0177899

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

Techniques Used: Staining, Expressing, Quantitative RT-PCR

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

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

Related Articles

Centrifugation:

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Amplification:

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Mass Spectrometry:

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

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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).

Cytometry:

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

Quantitative RT-PCR:

Article Title: Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer
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Article Title: Effects of GADL1 overexpression on cell migration and the associated morphological changes
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Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
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Real-time Polymerase Chain Reaction:

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Article Title: Effects of GADL1 overexpression on cell migration and the associated morphological changes
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Article Title: LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p
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Article Snippet: .. Primers for qRT-PCR were designed using the online PrimerQuest tool from Integrated DNA Technologies ( http://www.idtdna.com/Scitools/Applications/Primerquest ), and amplification efficiencies for each primer set were determined prior to use. cDNA samples were used as templates for quantitative PCR of rpoA (defined as that of R20291_0096) (primers oLB273/oLB274), tcdA (primers oLB131/oLB132) and tcdB (primers oND32/oND33) using Roche SYBR Green I PCR mix and a Roche LightCycler 480 II thermocycler. ..

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. .. This was carried out using an ABI real-time PCR system 7300 (Applied Biosystems) with Power SYBR Green PCR Master Mix.

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). ..

Incubation:

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

Expressing:

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

Article Title: 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. .. Real-time RT-PCR was used to quantify HR and meiotic specific mRNA expression of E. invadens and E. histolytica during encystation and serum starvation respectively.

Flow Cytometry:

Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
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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.

Cell Cycle Assay:

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

Northern Blot:

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). .. For Northern blot analysis, total RNA (10 μg) was used for each lanes and blotted onto positively charged nylon membranes (Roche Diagnostics).

Reverse Transcription Polymerase Chain Reaction:

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

Article Title: 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: MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-? treatment
Article Snippet: Paragraph title: Real-time reverse transcription-PCR (RT-PCR). ... 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.

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). ..

Isolation:

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

Article Title: Depletion of the cdk Inhibitor p16INK4a Differentially Affects Proliferation of Established Cervical Carcinoma Cells
Article Snippet: RNA was isolated using the RNeasy Minikit (Qiagen) and quantified as described above. .. For cDNA synthesis, 1 μg of total RNA was reverse transcribed with the Transcriptor first-strand cDNA synthesis kit (Roche Applied Science) and diluted 1:4.

Article Title: Effects of GADL1 overexpression on cell migration and the associated morphological changes
Article Snippet: Extraction of mRNA from cells and real-time quantitative PCR (RT-qPCR) Total RNA was extracted SH-SY5Y or GADL1 -overexpressing cells pooling from sextuplicate wells using the NucleoSpin RNA/protein isolation kit (MACHEREY-NAGEL, Germany). .. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Roche, Switzerland).

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

Article Title: 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: Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases
Article Snippet: Nucleic acid analysis Genomic DNA was isolated using Isoplant II (Nippon Gene). .. RNA (1 μg) was used in each RT-PCR following the manufacturer's protocol (Titan One Tube RT-PCR System, Roche Diagnostics).

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). ..

Size-exclusion Chromatography:

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). .. PCR amplification was performed for a first round of 10 cycles as follows: denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 68°C for 45 sec. Then the second round of cycles was performed as follows: denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 68°C for 45 sec + 5/cycle sec.

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.

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). .. PCR amplification was performed for a first round of 10 cycles as follows: denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 68°C for 45 sec. Then the second round of cycles was performed as follows: denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 68°C for 45 sec + 5/cycle sec.

Article Title: Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027
Article Snippet: .. Primers for qRT-PCR were designed using the online PrimerQuest tool from Integrated DNA Technologies ( http://www.idtdna.com/Scitools/Applications/Primerquest ), and amplification efficiencies for each primer set were determined prior to use. cDNA samples were used as templates for quantitative PCR of rpoA (defined as that of R20291_0096) (primers oLB273/oLB274), tcdA (primers oLB131/oLB132) and tcdB (primers oND32/oND33) using Roche SYBR Green I PCR mix and a Roche LightCycler 480 II thermocycler. ..

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. .. This was carried out using an ABI real-time PCR system 7300 (Applied Biosystems) with Power SYBR Green PCR Master Mix.

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). .. Two microliters of cDNA was used in 12 μl qPCR reactions with appropriate primers and SYBR Green PCR Master Mix (Applied Biosystems).

Lysis:

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.

Software:

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

SYBR Green Assay:

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

Article Title: 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: Effects of GADL1 overexpression on cell migration and the associated morphological changes
Article Snippet: The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Roche, Switzerland). .. Expressions of GADL1 (PPH22451A), FN1 (PPH00143B), ITGA2 (PPH00625F), ITGAV (PPH00628C), and CCL2 (PPH00192F) were examined with SYBR Green (Qiagen, Germany) using gene-specific primers (all designed by Qiagen) in triplicates.

Article Title: Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027
Article Snippet: .. Primers for qRT-PCR were designed using the online PrimerQuest tool from Integrated DNA Technologies ( http://www.idtdna.com/Scitools/Applications/Primerquest ), and amplification efficiencies for each primer set were determined prior to use. cDNA samples were used as templates for quantitative PCR of rpoA (defined as that of R20291_0096) (primers oLB273/oLB274), tcdA (primers oLB131/oLB132) and tcdB (primers oND32/oND33) using Roche SYBR Green I PCR mix and a Roche LightCycler 480 II thermocycler. ..

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. .. This was carried out using an ABI real-time PCR system 7300 (Applied Biosystems) with Power SYBR Green PCR Master Mix.

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). .. Two microliters of cDNA was used in 12 μl qPCR reactions with appropriate primers and SYBR Green PCR Master Mix (Applied Biosystems).

RNA Extraction:

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

Agarose Gel Electrophoresis:

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). .. For XBP1, mRNA was amplified by PCR and products were separated by electrophoresis through a 2.5% agarose gel and visualized by ethidium bromide staining.

Electrophoresis:

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). .. For XBP1, mRNA was amplified by PCR and products were separated by electrophoresis through a 2.5% agarose gel and visualized by ethidium bromide staining.

Spectrophotometry:

Article Title: Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027
Article Snippet: DNA-free RNA (500 ng), prepared as previously described [ , ], was quantitated by absorbance (A260 and A260 /A280 ratio) using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and subjected to cDNA synthesis using a QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s recommendation. .. Primers for qRT-PCR were designed using the online PrimerQuest tool from Integrated DNA Technologies ( http://www.idtdna.com/Scitools/Applications/Primerquest ), and amplification efficiencies for each primer set were determined prior to use. cDNA samples were used as templates for quantitative PCR of rpoA (defined as that of R20291_0096) (primers oLB273/oLB274), tcdA (primers oLB131/oLB132) and tcdB (primers oND32/oND33) using Roche SYBR Green I PCR mix and a Roche LightCycler 480 II thermocycler.

Staining:

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). .. For XBP1, mRNA was amplified by PCR and products were separated by electrophoresis through a 2.5% agarose gel and visualized by ethidium bromide staining.

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  • 92
    Roche gene expression real time quantitative reverse transcription pcr qrt pcr analyses
    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by <t>qRT-PCR</t> and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.
    Gene Expression Real Time Quantitative Reverse Transcription Pcr Qrt Pcr Analyses, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene expression real time quantitative reverse transcription pcr qrt pcr analyses/product/Roche
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    gene expression real time quantitative reverse transcription pcr qrt pcr analyses - by Bioz Stars, 2020-04
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    92
    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 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr analysis total rna/product/Roche
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
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    86
    Roche qrt pcr experiment quantitative reverse transcription pcr qrt pcr
    Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and <t>qRT-PCR</t> experiments followed using primers for DUSP1 and DUSP5.
    Qrt Pcr Experiment Quantitative Reverse Transcription Pcr Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr experiment quantitative reverse transcription pcr qrt pcr/product/Roche
    Average 86 stars, based on 1 article reviews
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    qrt pcr experiment quantitative reverse transcription pcr qrt pcr - by Bioz Stars, 2020-04
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    86
    Roche qrt pcr roche lightcycler technology
    ASPP2 mRNA expression in acute leukemia. <t>qRT-PCR</t> based mRNA expression levels are displayed after normalizing to a healthy blood donor (set as 1) on a logarithmic scale. Cohort analysis reveals significant lower ASPP2 levels for an acute leukemia population compared to a healthy peripheral blood and bone marrow donor cohort (A). Comparison of prognostic risk groups confirms lower ASPP2 expression levels for the good-risk as well as higher-risk cohort when compared to a healthy donor population – whereas attenuated ASPP2 expression levels are more pronounced and statistically significantly different for the higher-risk cohort (B). Analysis of therapy responders (i.e. achievement of complete remission after one cycle of induction chemotherapy) demonstrates significantly lower ASPP2 levels for the therapy-failure population when compared to the responder cohort (including good-/higher-risk pts.) (C). ROC curve analysis defining the ideal threshold to distinguish a definite non-responding sub-population is shown in figure 1D (i.e. patients with attenuated ASPP2 expression levels ≤0.8 are likely not to respond to induction chemotherapy (with no single falsely positive tested patient at this threshold). P-values are provided as indicated by an asterix. Patient characteristics, including definitions of the prognostic risk groups, are summarized in Table 1 and 2.
    Qrt Pcr Roche Lightcycler Technology, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr roche lightcycler technology/product/Roche
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    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

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

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Frontiers in Plant Science

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

    doi: 10.3389/fpls.2018.01259

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

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

    Techniques: Expressing, Quantitative RT-PCR

    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

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

    Journal: Molecular Cancer

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

    doi: 10.1186/1476-4598-11-35

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

    Article Snippet: qRT-PCR experiment Quantitative Reverse Transcription-PCR (qRT-PCR) experiments were performed using The LightCycler® 2.0 Instrument (Roche Diagnostics GmbH, Mannheim, Germany) and QuantiTect SYBR Green RT-PCR Kit (Qiagen, GmbH, Germany).

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

    ASPP2 mRNA expression in acute leukemia. qRT-PCR based mRNA expression levels are displayed after normalizing to a healthy blood donor (set as 1) on a logarithmic scale. Cohort analysis reveals significant lower ASPP2 levels for an acute leukemia population compared to a healthy peripheral blood and bone marrow donor cohort (A). Comparison of prognostic risk groups confirms lower ASPP2 expression levels for the good-risk as well as higher-risk cohort when compared to a healthy donor population – whereas attenuated ASPP2 expression levels are more pronounced and statistically significantly different for the higher-risk cohort (B). Analysis of therapy responders (i.e. achievement of complete remission after one cycle of induction chemotherapy) demonstrates significantly lower ASPP2 levels for the therapy-failure population when compared to the responder cohort (including good-/higher-risk pts.) (C). ROC curve analysis defining the ideal threshold to distinguish a definite non-responding sub-population is shown in figure 1D (i.e. patients with attenuated ASPP2 expression levels ≤0.8 are likely not to respond to induction chemotherapy (with no single falsely positive tested patient at this threshold). P-values are provided as indicated by an asterix. Patient characteristics, including definitions of the prognostic risk groups, are summarized in Table 1 and 2.

    Journal: PLoS ONE

    Article Title: Attenuated Expression of Apoptosis Stimulating Protein of p53-2 (ASPP2) in Human Acute Leukemia Is Associated with Therapy Failure

    doi: 10.1371/journal.pone.0080193

    Figure Lengend Snippet: ASPP2 mRNA expression in acute leukemia. qRT-PCR based mRNA expression levels are displayed after normalizing to a healthy blood donor (set as 1) on a logarithmic scale. Cohort analysis reveals significant lower ASPP2 levels for an acute leukemia population compared to a healthy peripheral blood and bone marrow donor cohort (A). Comparison of prognostic risk groups confirms lower ASPP2 expression levels for the good-risk as well as higher-risk cohort when compared to a healthy donor population – whereas attenuated ASPP2 expression levels are more pronounced and statistically significantly different for the higher-risk cohort (B). Analysis of therapy responders (i.e. achievement of complete remission after one cycle of induction chemotherapy) demonstrates significantly lower ASPP2 levels for the therapy-failure population when compared to the responder cohort (including good-/higher-risk pts.) (C). ROC curve analysis defining the ideal threshold to distinguish a definite non-responding sub-population is shown in figure 1D (i.e. patients with attenuated ASPP2 expression levels ≤0.8 are likely not to respond to induction chemotherapy (with no single falsely positive tested patient at this threshold). P-values are provided as indicated by an asterix. Patient characteristics, including definitions of the prognostic risk groups, are summarized in Table 1 and 2.

    Article Snippet: ASPP2 mRNA expression levels, relative to GAPD as the housekeeping gene, were determined by qRT-PCR Roche® LightCycler Technology (Roche, Basel, Switzerland).

    Techniques: Expressing, Quantitative RT-PCR