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

Related Articles

Real-time Polymerase Chain Reaction:

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: 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). ..

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.

Amplification:

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. ..

Synthesized:

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

Isolation:

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

Quantitative RT-PCR:

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

Article Title: 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: 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: 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). ..

SYBR Green Assay:

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. ..

Reverse Transcription Polymerase Chain Reaction:

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

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.

Polymerase Chain Reaction:

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. ..

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    Roche gene expression real time quantitative reverse transcription pcr qrt pcr analyses
    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by <t>qRT-PCR</t> and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.
    Gene Expression Real Time Quantitative Reverse Transcription Pcr Qrt Pcr Analyses, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene expression real time quantitative reverse transcription pcr qrt pcr analyses/product/Roche
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    Roche qrt pcr analysis total rna
    Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by <t>qRT-PCR.</t> Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p
    Qrt Pcr Analysis Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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