Structured Review

Thermo Fisher blca
Correlation of transcribed ultraconserved regions <t>(T-UCRs)</t> with the host gene CASZ1 in bladder cancer <t>(BlCa)</t> A. Representation of the genomic localization of CASZ1 with respect to 1p36.22 (red) obtained using the UCSC Genome Browser (University of California Santa Cruz). B. Representation of the seven T-UCRs in CASZ1 according to their genomic locations with respect to protein-coding genes ( CASZ1 defined using the RefSeq database). C. The expression levels for all T-UCRs located in CASZ1 were measured using qRT-PCR. RNA was extracted from BlCa tissues obtained from 24 patients (dark gray) and 17 normal bladder epithelium (NBE) samples (gray) (Table 1 , Dataset 1). The bold lines inside the boxes represent the medians. The boxes represent the first (Q1) and the third (Q3) quartiles, and the two whiskers represent the minimum and the maximum values, except for outliers. Circles represent outliers, i.e., values lower than Q1-1.5 (Q3-Q1) or higher than Q3+1.5 (Q3-Q1). D. Representative negative correlation of CASZ1 and T-UCR (uc.2+–uc.7+) expression in patients with BlCa (n=14). qRT-PCR analysis results of CASZ1 expression (abscissa) versus T-UCR expression are shown.
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1) Product Images from "Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis"

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

Journal: Oncotarget

doi: 10.18632/oncotarget.7833

Correlation of transcribed ultraconserved regions (T-UCRs) with the host gene CASZ1 in bladder cancer (BlCa) A. Representation of the genomic localization of CASZ1 with respect to 1p36.22 (red) obtained using the UCSC Genome Browser (University of California Santa Cruz). B. Representation of the seven T-UCRs in CASZ1 according to their genomic locations with respect to protein-coding genes ( CASZ1 defined using the RefSeq database). C. The expression levels for all T-UCRs located in CASZ1 were measured using qRT-PCR. RNA was extracted from BlCa tissues obtained from 24 patients (dark gray) and 17 normal bladder epithelium (NBE) samples (gray) (Table 1 , Dataset 1). The bold lines inside the boxes represent the medians. The boxes represent the first (Q1) and the third (Q3) quartiles, and the two whiskers represent the minimum and the maximum values, except for outliers. Circles represent outliers, i.e., values lower than Q1-1.5 (Q3-Q1) or higher than Q3+1.5 (Q3-Q1). D. Representative negative correlation of CASZ1 and T-UCR (uc.2+–uc.7+) expression in patients with BlCa (n=14). qRT-PCR analysis results of CASZ1 expression (abscissa) versus T-UCR expression are shown.
Figure Legend Snippet: Correlation of transcribed ultraconserved regions (T-UCRs) with the host gene CASZ1 in bladder cancer (BlCa) A. Representation of the genomic localization of CASZ1 with respect to 1p36.22 (red) obtained using the UCSC Genome Browser (University of California Santa Cruz). B. Representation of the seven T-UCRs in CASZ1 according to their genomic locations with respect to protein-coding genes ( CASZ1 defined using the RefSeq database). C. The expression levels for all T-UCRs located in CASZ1 were measured using qRT-PCR. RNA was extracted from BlCa tissues obtained from 24 patients (dark gray) and 17 normal bladder epithelium (NBE) samples (gray) (Table 1 , Dataset 1). The bold lines inside the boxes represent the medians. The boxes represent the first (Q1) and the third (Q3) quartiles, and the two whiskers represent the minimum and the maximum values, except for outliers. Circles represent outliers, i.e., values lower than Q1-1.5 (Q3-Q1) or higher than Q3+1.5 (Q3-Q1). D. Representative negative correlation of CASZ1 and T-UCR (uc.2+–uc.7+) expression in patients with BlCa (n=14). qRT-PCR analysis results of CASZ1 expression (abscissa) versus T-UCR expression are shown.

Techniques Used: Expressing, Quantitative RT-PCR

Transcribed ultraconserved region (T-UCR) expression in human bladder cancer (BlCa) tissues A. Bar plot of the expression of a subset of the investigated T-UCRs (48 of 293) with expression increases greater than 2 fold and expression decreases lower than −2.3 fold in BlCa and normal bladder epithelium (NBE) tissues. B. Bar plot of the expression of a subset of the investigated T-UCRs (48 of 141) with expression increases greater than 1 fold and expression decreases lower than −1.66 fold in BlCa and pericancerous BlCa (PBlCa) tissues. C. Comparison of the fold change in expression of 50 T-UCRs for which two different controls (NBE and PBlCa tissues) were used. The outlying ultraconserved RNA (uc).8+ is shown in red. D. RNA was extracted from 18 BlCa and adjacent PBlCa tissues. Evaluation of uc.8+ expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of uc.8+ is higher in PBlCa than in BlCa tissues. ***P
Figure Legend Snippet: Transcribed ultraconserved region (T-UCR) expression in human bladder cancer (BlCa) tissues A. Bar plot of the expression of a subset of the investigated T-UCRs (48 of 293) with expression increases greater than 2 fold and expression decreases lower than −2.3 fold in BlCa and normal bladder epithelium (NBE) tissues. B. Bar plot of the expression of a subset of the investigated T-UCRs (48 of 141) with expression increases greater than 1 fold and expression decreases lower than −1.66 fold in BlCa and pericancerous BlCa (PBlCa) tissues. C. Comparison of the fold change in expression of 50 T-UCRs for which two different controls (NBE and PBlCa tissues) were used. The outlying ultraconserved RNA (uc).8+ is shown in red. D. RNA was extracted from 18 BlCa and adjacent PBlCa tissues. Evaluation of uc.8+ expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of uc.8+ is higher in PBlCa than in BlCa tissues. ***P

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

2) Product Images from "Curcumin suppresses the progression of laryngeal squamous cell carcinoma through the upregulation of miR-145 and inhibition of the PI3K/Akt/mTOR pathway"

Article Title: Curcumin suppresses the progression of laryngeal squamous cell carcinoma through the upregulation of miR-145 and inhibition of the PI3K/Akt/mTOR pathway

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S159236

Curcumin reversed anti-miR-145-induced enhanced effect on the progression of laryngeal squamous cell carcinoma (LSCC) cells. Notes: ( A ) TU212 and AMC-HN-8 cells were transfected with anti-miR-145 or anti-miR-con, followed by treatment with different concentrations of curcumin (0, 5, 10, 20 and 40 μM). Cell viability was then detected by MTT assay. TU212 and AMC-HN-8 cells were transfected with anti-miR-145 or anti-miR-con, followed by 20 μM curcumin treatment. ( B ) miR-145 expression in treated TU212 and AMC-HN-8 cells was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). ( C ) Cell viability was detected by MTT assay in treated TU212 and AMC-HN-8 cells. ( D and E ) Cell migration and invasion abilities were examined by Transwell migration and invasion assays in treated TU212 and AMC-HN-8 cells. ( F ) Apoptosis of treated TU212 and AMC-HN-8 cells was analyzed by flow cytometry analysis. *.
Figure Legend Snippet: Curcumin reversed anti-miR-145-induced enhanced effect on the progression of laryngeal squamous cell carcinoma (LSCC) cells. Notes: ( A ) TU212 and AMC-HN-8 cells were transfected with anti-miR-145 or anti-miR-con, followed by treatment with different concentrations of curcumin (0, 5, 10, 20 and 40 μM). Cell viability was then detected by MTT assay. TU212 and AMC-HN-8 cells were transfected with anti-miR-145 or anti-miR-con, followed by 20 μM curcumin treatment. ( B ) miR-145 expression in treated TU212 and AMC-HN-8 cells was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). ( C ) Cell viability was detected by MTT assay in treated TU212 and AMC-HN-8 cells. ( D and E ) Cell migration and invasion abilities were examined by Transwell migration and invasion assays in treated TU212 and AMC-HN-8 cells. ( F ) Apoptosis of treated TU212 and AMC-HN-8 cells was analyzed by flow cytometry analysis. *.

Techniques Used: Transfection, MTT Assay, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Migration, Flow Cytometry, Cytometry

The anticancer effects of curcumin on the progression of laryngeal squamous cell carcinoma (LSCC) cells. Notes: TU212 and AMC-HN-8 cells were treated with 0, 5, 10 and 20 μM curcumin for 48 h. ( A ) IC50 value of curcumin in TU212 and AMC-HN-8 cells was detected by MTT assay. ( B ) miR-145 expression was detected by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in curcumin-treated TU212 and AMC-HN-8 cells. ( C ) Cell viability was assessed by MTT assay in TU212 and AMC-HN-8 cells after curcumin treatment. ( D and E ) Migration and invasion abilities were determined by Transwell migration and invasion assays in curcumin-treated TU212 and AMC-HN-8 cells. ( F ) Cell cycle analysis was performed in curcumin-treated TU212 and AMC-HN-8 cells by flow cytometry analysis. ( F ) Apoptotic rate of curcumin-treated TU212 and AMC-HN-8 cells was measured by flow cytometry analysis. *.
Figure Legend Snippet: The anticancer effects of curcumin on the progression of laryngeal squamous cell carcinoma (LSCC) cells. Notes: TU212 and AMC-HN-8 cells were treated with 0, 5, 10 and 20 μM curcumin for 48 h. ( A ) IC50 value of curcumin in TU212 and AMC-HN-8 cells was detected by MTT assay. ( B ) miR-145 expression was detected by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in curcumin-treated TU212 and AMC-HN-8 cells. ( C ) Cell viability was assessed by MTT assay in TU212 and AMC-HN-8 cells after curcumin treatment. ( D and E ) Migration and invasion abilities were determined by Transwell migration and invasion assays in curcumin-treated TU212 and AMC-HN-8 cells. ( F ) Cell cycle analysis was performed in curcumin-treated TU212 and AMC-HN-8 cells by flow cytometry analysis. ( F ) Apoptotic rate of curcumin-treated TU212 and AMC-HN-8 cells was measured by flow cytometry analysis. *.

Techniques Used: MTT Assay, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Migration, Cell Cycle Assay, Flow Cytometry, Cytometry

The effects of miR-145 overexpression on the progression of LSCC. Notes: TU212 and AMC-HN-8 cells were transfected with miR-145 or miR-con for gain-of-function analyses. ( A ) Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis of miR-145 expression in treated TU212 and AMC-HN-8 cells. ( B ) MTT assay was conducted to detect cell proliferation at 24 h, 48 h and 72 h in treated TU212 and AMC-HN-8 cells. ( C ) Flow cytometry analysis was performed to analyze apoptosis of treated TU212 and AMC-HN-8 cells. Transwell migration ( D ) and invasion ( E ) assays were applied to determine the migration and invasion capabilities of treated TU212 and AMC-HN-8 cells; magnification: 200×. *.
Figure Legend Snippet: The effects of miR-145 overexpression on the progression of LSCC. Notes: TU212 and AMC-HN-8 cells were transfected with miR-145 or miR-con for gain-of-function analyses. ( A ) Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis of miR-145 expression in treated TU212 and AMC-HN-8 cells. ( B ) MTT assay was conducted to detect cell proliferation at 24 h, 48 h and 72 h in treated TU212 and AMC-HN-8 cells. ( C ) Flow cytometry analysis was performed to analyze apoptosis of treated TU212 and AMC-HN-8 cells. Transwell migration ( D ) and invasion ( E ) assays were applied to determine the migration and invasion capabilities of treated TU212 and AMC-HN-8 cells; magnification: 200×. *.

Techniques Used: Over Expression, Transfection, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, MTT Assay, Flow Cytometry, Cytometry, Migration

Curcumin exacerbated miR-145-induced inhibition of the PI3K/Akt/mTOR pathway in LSCC cells. Notes: TU212 and AMC-HN-8 cells were treated with miR-145, miR-con, miR-145 + 20 μM curcumin, anti-miR-145, anti-miR-con or anti-miR-145 + 20 μM curcumin. Western blot analysis was conducted to evaluate the protein levels of phosphoinositol 1,3 kinase (PI3K), protein kinase B (Akt), p-Akt, mammalian target of rapamycin (mTOR) and p-mTOR in treated AMC-HN-8 ( A ) and TU212 cells ( B ). *.
Figure Legend Snippet: Curcumin exacerbated miR-145-induced inhibition of the PI3K/Akt/mTOR pathway in LSCC cells. Notes: TU212 and AMC-HN-8 cells were treated with miR-145, miR-con, miR-145 + 20 μM curcumin, anti-miR-145, anti-miR-con or anti-miR-145 + 20 μM curcumin. Western blot analysis was conducted to evaluate the protein levels of phosphoinositol 1,3 kinase (PI3K), protein kinase B (Akt), p-Akt, mammalian target of rapamycin (mTOR) and p-mTOR in treated AMC-HN-8 ( A ) and TU212 cells ( B ). *.

Techniques Used: Inhibition, Western Blot

The expression of miR-145 in laryngeal squamous cell carcinoma (LSCC) tissues and cells. Notes: ( A and B ) The expression of miR-145 in 32 paired LSCC tissues and adjacent normal tissues was quantified by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). ( C ) The expression of miR-145 in LSCC cell lines including TU-177, TU212, AMC-HN-8 and TU686 cells and normal human oral keratinocytes (NHOKs) were evaluated by qRT-PCR. *.
Figure Legend Snippet: The expression of miR-145 in laryngeal squamous cell carcinoma (LSCC) tissues and cells. Notes: ( A and B ) The expression of miR-145 in 32 paired LSCC tissues and adjacent normal tissues was quantified by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). ( C ) The expression of miR-145 in LSCC cell lines including TU-177, TU212, AMC-HN-8 and TU686 cells and normal human oral keratinocytes (NHOKs) were evaluated by qRT-PCR. *.

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

3) Product Images from "Salivary microRNAs as new molecular markers in cleft lip and palate: a new frontier in molecular medicine"

Article Title: Salivary microRNAs as new molecular markers in cleft lip and palate: a new frontier in molecular medicine

Journal: Oncotarget

doi: 10.18632/oncotarget.24838

Scatter plot of the expression of the 29 out of 131 different miRNAs found to be deregulated in the patients The fold change was calculated respect to the average ΔCT value of control group. The error bars indicate the mean ± standard deviation (SD).
Figure Legend Snippet: Scatter plot of the expression of the 29 out of 131 different miRNAs found to be deregulated in the patients The fold change was calculated respect to the average ΔCT value of control group. The error bars indicate the mean ± standard deviation (SD).

Techniques Used: Expressing, Standard Deviation

4) Product Images from "Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish"

Article Title: Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003583

Protein-protein interactions between myotubularin and MTMR12 proteins. (A) GST pull down of MTM1-GST recombinant protein with MTMR12 showed a direct interaction between the two proteins (above). Coomassie blue stained gel (below) showing GST and MTM1-GST protein used in the pull down (B) Co-IP experiments from Cos-1 transfected cells with MTM1 and MTMR12-B10 constructs showing the interaction of the two proteins in the cellular context in-vivo (C) MTMR12 co-immunoprecipitates with myotubularin (using 1G1 monoclonal antibody) in mouse muscle lysates revealed with anti-MTMR12 polyclonal antibody (upper panel) and with the 2827 polyclonal anti-myotubularin (bottom panel). (D) Confocal microscopic immunofluorescence studies of longitudinal frozen sections of skeletal muscle from mouse tibialis anterior muscle with sarcomeric markers. Individual immunostaining with 2827 anti-myotubularin or anti-MTMR12 showed similar striated localization in skeletal muscle (top panel). Double-immunostaining with both proteins showed a co-localization of myotubularin and MTMR12 to Triads and partial co-localization with the ryanodine receptor, RyR1, a sarcoplasmic reticulum marker, but not with α-actinin, a Z-line marker. Scale bar = 10 µm.
Figure Legend Snippet: Protein-protein interactions between myotubularin and MTMR12 proteins. (A) GST pull down of MTM1-GST recombinant protein with MTMR12 showed a direct interaction between the two proteins (above). Coomassie blue stained gel (below) showing GST and MTM1-GST protein used in the pull down (B) Co-IP experiments from Cos-1 transfected cells with MTM1 and MTMR12-B10 constructs showing the interaction of the two proteins in the cellular context in-vivo (C) MTMR12 co-immunoprecipitates with myotubularin (using 1G1 monoclonal antibody) in mouse muscle lysates revealed with anti-MTMR12 polyclonal antibody (upper panel) and with the 2827 polyclonal anti-myotubularin (bottom panel). (D) Confocal microscopic immunofluorescence studies of longitudinal frozen sections of skeletal muscle from mouse tibialis anterior muscle with sarcomeric markers. Individual immunostaining with 2827 anti-myotubularin or anti-MTMR12 showed similar striated localization in skeletal muscle (top panel). Double-immunostaining with both proteins showed a co-localization of myotubularin and MTMR12 to Triads and partial co-localization with the ryanodine receptor, RyR1, a sarcoplasmic reticulum marker, but not with α-actinin, a Z-line marker. Scale bar = 10 µm.

Techniques Used: Recombinant, Staining, Co-Immunoprecipitation Assay, Transfection, Construct, In Vivo, Immunofluorescence, Immunostaining, Double Immunostaining, Marker

Abnormal histology of MTMR12-deficient zebrafish. (A)Toluidine blue stained longitudinal sections of skeletal muscle in control and morphant fish at 3 dpf. In comparison to the control fish, mtmr12 morphants showed disorganized myofibers (arrowhead) with central nucleation (arrow), similar to histological changes observed in the skeletal muscle of mtm1 morphant fish (arrow). Knockdown of both mtm1 as well as mtmr12 results in severe muscle disorganization greater than seen in mtm1 or mtmr12 alone morphants. (B) Centrally nucleated myofibers were quantified. Serial sections from 3–4 different embryos were analyzed and the relative number of centrally nucleated fibers in the middle somites (10–13) were counted. (C) Hematoxylin and Eosin staining of mtmr12 morphant zebrafish at different time points. An increase in sarcomeric disorganization was observed at 3 dpf in comparison to 2 dpf in mtmr12 morphants (arrow) (D) Centrally nucleated myofibers were quantified. Serial sections from 6 different embryos were analyzed and the relative number of centrally nucleated fibers in the middle somites (10–13) were counted. Scale bar = 10 µm.
Figure Legend Snippet: Abnormal histology of MTMR12-deficient zebrafish. (A)Toluidine blue stained longitudinal sections of skeletal muscle in control and morphant fish at 3 dpf. In comparison to the control fish, mtmr12 morphants showed disorganized myofibers (arrowhead) with central nucleation (arrow), similar to histological changes observed in the skeletal muscle of mtm1 morphant fish (arrow). Knockdown of both mtm1 as well as mtmr12 results in severe muscle disorganization greater than seen in mtm1 or mtmr12 alone morphants. (B) Centrally nucleated myofibers were quantified. Serial sections from 3–4 different embryos were analyzed and the relative number of centrally nucleated fibers in the middle somites (10–13) were counted. (C) Hematoxylin and Eosin staining of mtmr12 morphant zebrafish at different time points. An increase in sarcomeric disorganization was observed at 3 dpf in comparison to 2 dpf in mtmr12 morphants (arrow) (D) Centrally nucleated myofibers were quantified. Serial sections from 6 different embryos were analyzed and the relative number of centrally nucleated fibers in the middle somites (10–13) were counted. Scale bar = 10 µm.

Techniques Used: Staining, Fluorescence In Situ Hybridization

Myotubularin-MTMR12 interactions in XLMTM. (A) Schematic diagram of different domains of myotubularin protein displaying representative pathogenic mutations found in XLMTM patients or an artificial inactivating mutation C375S* (GRAM, N terminal lipid or protein interacting domain; RID, putative membrane targeting motif; PTP/DSP, phosphatase domain; SID, protein-protein interacting domain; CC, coiled-coil domain; PDZB, PDZ binding site). (B) Wild-type or mutant MTM1-B10 fusion proteins with indicated missense mutations and wild-type MTMR12-GFP proteins were overexpressed in Cos1 cells. Immunoprecipitation of protein extracts with anti-B10 tag antibody showed that mutations on GRAM or RID domains disrupt the interactions between MTM1 and MTMR12. (C) Western blotting of XLMTM patient myotubes showed that mutants that decrease the stability of myotubularin protein also results in a reduction of MTMR12 levels. Histograms depict the western quantification for panels (B) and (C). Asterisks indicate statistically significant differences from measurements of wild type controls, P≤0.05.
Figure Legend Snippet: Myotubularin-MTMR12 interactions in XLMTM. (A) Schematic diagram of different domains of myotubularin protein displaying representative pathogenic mutations found in XLMTM patients or an artificial inactivating mutation C375S* (GRAM, N terminal lipid or protein interacting domain; RID, putative membrane targeting motif; PTP/DSP, phosphatase domain; SID, protein-protein interacting domain; CC, coiled-coil domain; PDZB, PDZ binding site). (B) Wild-type or mutant MTM1-B10 fusion proteins with indicated missense mutations and wild-type MTMR12-GFP proteins were overexpressed in Cos1 cells. Immunoprecipitation of protein extracts with anti-B10 tag antibody showed that mutations on GRAM or RID domains disrupt the interactions between MTM1 and MTMR12. (C) Western blotting of XLMTM patient myotubes showed that mutants that decrease the stability of myotubularin protein also results in a reduction of MTMR12 levels. Histograms depict the western quantification for panels (B) and (C). Asterisks indicate statistically significant differences from measurements of wild type controls, P≤0.05.

Techniques Used: Mutagenesis, Binding Assay, Immunoprecipitation, Western Blot

Myotubularin and PtdIns3 P alterations in mtmr12 morphants. (A) Immunofluorescence of control and mtmr12 knockdown fish showed significantly decreased myotubularin staining in mtmr12 knockdown fish in images taken under identical conditions. Immunofluorescence detection of PtdIns3 P showed apparent increases of this myotubularin substrate in mtmr12 morphant embryos as compared to controls. (B) PtdIns3 P levels are increased in mtmr12 , mtm1 and mtm1-mtmr12 morphant zebrafish, * P ≤0.05. Total lipids were extracted from zebrafish at 3 dpf and PtdIns3 P levels were measured using a lipid-protein overlay enzyme-linked immunosorbent assay.
Figure Legend Snippet: Myotubularin and PtdIns3 P alterations in mtmr12 morphants. (A) Immunofluorescence of control and mtmr12 knockdown fish showed significantly decreased myotubularin staining in mtmr12 knockdown fish in images taken under identical conditions. Immunofluorescence detection of PtdIns3 P showed apparent increases of this myotubularin substrate in mtmr12 morphant embryos as compared to controls. (B) PtdIns3 P levels are increased in mtmr12 , mtm1 and mtm1-mtmr12 morphant zebrafish, * P ≤0.05. Total lipids were extracted from zebrafish at 3 dpf and PtdIns3 P levels were measured using a lipid-protein overlay enzyme-linked immunosorbent assay.

Techniques Used: Immunofluorescence, Fluorescence In Situ Hybridization, Staining, Enzyme-linked Immunosorbent Assay

Ultrastructural abnormalities in MTMR12 deficiency. Transmission electron micrographs of skeletal muscles in control and morphant fish. In comparison to wild-type controls (A), mtmr12 morphant fish displayed abnormal triads (C, high magnification, inset). mtm1 morphant muscle also had absent or disorganized triads (B). mtmr12-mtm1 double knockdown morphants displayed exacerbated defects in muscle with many absent Z-lines in addition to disorganized triads (D inset shows high magnification view). Higher magnification examination showed whorled membranous structures in mtmr12 and mtmr12-mtm1 morphants (E–F). (G) Histograms represent quantification of disorganized triads of the single or double morphants. Total number of triads were counted in at least 15 myofibers within each embryo (n = 5 embryos). * P ≤0.05, ns: statistically not significant.
Figure Legend Snippet: Ultrastructural abnormalities in MTMR12 deficiency. Transmission electron micrographs of skeletal muscles in control and morphant fish. In comparison to wild-type controls (A), mtmr12 morphant fish displayed abnormal triads (C, high magnification, inset). mtm1 morphant muscle also had absent or disorganized triads (B). mtmr12-mtm1 double knockdown morphants displayed exacerbated defects in muscle with many absent Z-lines in addition to disorganized triads (D inset shows high magnification view). Higher magnification examination showed whorled membranous structures in mtmr12 and mtmr12-mtm1 morphants (E–F). (G) Histograms represent quantification of disorganized triads of the single or double morphants. Total number of triads were counted in at least 15 myofibers within each embryo (n = 5 embryos). * P ≤0.05, ns: statistically not significant.

Techniques Used: Transmission Assay, Fluorescence In Situ Hybridization

Loss of protein stability in the absence of myotubularin-MTMR12 interactions. (A) Knockdown of mtmr12 resulted in a strong decrease of myotubularin protein in mtmr12 morphant zebrafish at 3 dpf by western blotting. Western blot analysis was done on three independently injected clutches (n in each clutch = 50–75). The histogram at right shows normalized amounts of myotubularin in control and mtmr12 morphant zerbrafish, * P ≤0.01. (B) siRNA-mediated knockdown of Mtmr12 in C2C12 myoblasts leads to decreased protein levels of MTM1. Histograms showed western blot quantification of MTM1 and MTMR12 with reference to α-actinin as a loading control. Data represent mean of 3 independent experiments, * P
Figure Legend Snippet: Loss of protein stability in the absence of myotubularin-MTMR12 interactions. (A) Knockdown of mtmr12 resulted in a strong decrease of myotubularin protein in mtmr12 morphant zebrafish at 3 dpf by western blotting. Western blot analysis was done on three independently injected clutches (n in each clutch = 50–75). The histogram at right shows normalized amounts of myotubularin in control and mtmr12 morphant zerbrafish, * P ≤0.01. (B) siRNA-mediated knockdown of Mtmr12 in C2C12 myoblasts leads to decreased protein levels of MTM1. Histograms showed western blot quantification of MTM1 and MTMR12 with reference to α-actinin as a loading control. Data represent mean of 3 independent experiments, * P

Techniques Used: Western Blot, Injection

Rescue of mtmr12 morphant phenotypes by MTM1 . The ability of human MTM1 or MTMR12 transcripts to rescue abnormalities seen in morphant zebrafish was classified in to phenotypic index of five groups: Normal, mild, moderate, severe and dead, described in the table depending on body length, birefringence and ultrastructure of skeletal muscle. (A) Polarized light microscopy of 3 dpf live embryos showed that birefringence of mtmr12 morphant embryos increased significantly upon overexpression of human MTM1 mRNA. (B) Overexpression of human MTMR12 mRNA in mtm1 morphant fish resulted in a mild rescue of skeletal muscle defects as seen by birefringence of zebrafish embryos. (C) Overexpression of human MTM1 mRNA in mtm1-mtmr12 morphant fish resulted in a moderate rescue of skeletal muscle defects as seen by birefringence of zebrafish embryos. (D) Electron microscopy showed normal skeletal muscle structure of mtmr12 and mtm1-mtmr12 morphant fish rescued with MTM1 mRNA but displayed disorganized triads in mtm1 morphants that were rescued with MTMR12 mRNA. (E) Quantification of the body length and disorganized triads in morphant and rescued fish. Body length was measured in 10–15 embryos in each group. Total number of triads were counted in at least 15 myofibers within each embryo (n = 5 embryos). P≤0.05.
Figure Legend Snippet: Rescue of mtmr12 morphant phenotypes by MTM1 . The ability of human MTM1 or MTMR12 transcripts to rescue abnormalities seen in morphant zebrafish was classified in to phenotypic index of five groups: Normal, mild, moderate, severe and dead, described in the table depending on body length, birefringence and ultrastructure of skeletal muscle. (A) Polarized light microscopy of 3 dpf live embryos showed that birefringence of mtmr12 morphant embryos increased significantly upon overexpression of human MTM1 mRNA. (B) Overexpression of human MTMR12 mRNA in mtm1 morphant fish resulted in a mild rescue of skeletal muscle defects as seen by birefringence of zebrafish embryos. (C) Overexpression of human MTM1 mRNA in mtm1-mtmr12 morphant fish resulted in a moderate rescue of skeletal muscle defects as seen by birefringence of zebrafish embryos. (D) Electron microscopy showed normal skeletal muscle structure of mtmr12 and mtm1-mtmr12 morphant fish rescued with MTM1 mRNA but displayed disorganized triads in mtm1 morphants that were rescued with MTMR12 mRNA. (E) Quantification of the body length and disorganized triads in morphant and rescued fish. Body length was measured in 10–15 embryos in each group. Total number of triads were counted in at least 15 myofibers within each embryo (n = 5 embryos). P≤0.05.

Techniques Used: Light Microscopy, Over Expression, Fluorescence In Situ Hybridization, Electron Microscopy

Expression patterns and morpholino-based knockdown of mtmr12 in developing zebrafish. (A) Whole mount in-situ hybridization detected ubiquitous expression of mtmr12 and mtm1 transcripts in zebrafish embryos at 1 dpf (above). Below is RT-PCR analysis of mtm1 and mtmr12 expression during zebrafish development using RNA extracts from whole zebrafish embryos at indicated developmental timepoints. (B) Synergistic expression level of Mtm1 and Mtmr12 transcripts and protein at indicated time points of C2C12 differentiation (0–9 days) monitored by RT-quantitative PCR (corresponding histogram, *P≤0.05) and by western blot analysis (right panel). (C) Live embryos at 3 dpf injected with control, mtmr12 , mtm1 or both mtmr12 and mtm1 morpholinos in normal (left) and polarized lights (right). mtmr12 morphant fish showed a dorsal curvature in skeletal muscle and reduced birefringence in polarized light similar to mtm1 morphant embryos. mtmr12 morphant fish also exhibited pericardial edema (arrow). mtmr12-mtm1 double knockdown fish exhibited smaller size and reduced birefringence relative to mtm1 or mtmr12 alone morphant fish. (D) mtmr12 mRNA levels in mtmr12 morphant zebrafish following injection of two different amounts of morpholino (indicated below, upper panel). In mtm1 morphant fish, no residual myotubularin was observed showing that mtm1 morpholinos are completely penetrant to the limits of detection for western blotting. (E) Over-expression of human MTMR12 mRNA rescued small body length and skeletal muscle abnormalities observed in mtmr12 morphant embryos. (F) Quantification of the chorion hatching at 60 hpf. The number of embryos was quantified in three independent clutches (number of embryos in each clutch = 90–120). (G) Quantification of touch evoke response at 3 dpf (n = 5–8 embryos were assayed in each morpholino group).* P ≤0.01.
Figure Legend Snippet: Expression patterns and morpholino-based knockdown of mtmr12 in developing zebrafish. (A) Whole mount in-situ hybridization detected ubiquitous expression of mtmr12 and mtm1 transcripts in zebrafish embryos at 1 dpf (above). Below is RT-PCR analysis of mtm1 and mtmr12 expression during zebrafish development using RNA extracts from whole zebrafish embryos at indicated developmental timepoints. (B) Synergistic expression level of Mtm1 and Mtmr12 transcripts and protein at indicated time points of C2C12 differentiation (0–9 days) monitored by RT-quantitative PCR (corresponding histogram, *P≤0.05) and by western blot analysis (right panel). (C) Live embryos at 3 dpf injected with control, mtmr12 , mtm1 or both mtmr12 and mtm1 morpholinos in normal (left) and polarized lights (right). mtmr12 morphant fish showed a dorsal curvature in skeletal muscle and reduced birefringence in polarized light similar to mtm1 morphant embryos. mtmr12 morphant fish also exhibited pericardial edema (arrow). mtmr12-mtm1 double knockdown fish exhibited smaller size and reduced birefringence relative to mtm1 or mtmr12 alone morphant fish. (D) mtmr12 mRNA levels in mtmr12 morphant zebrafish following injection of two different amounts of morpholino (indicated below, upper panel). In mtm1 morphant fish, no residual myotubularin was observed showing that mtm1 morpholinos are completely penetrant to the limits of detection for western blotting. (E) Over-expression of human MTMR12 mRNA rescued small body length and skeletal muscle abnormalities observed in mtmr12 morphant embryos. (F) Quantification of the chorion hatching at 60 hpf. The number of embryos was quantified in three independent clutches (number of embryos in each clutch = 90–120). (G) Quantification of touch evoke response at 3 dpf (n = 5–8 embryos were assayed in each morpholino group).* P ≤0.01.

Techniques Used: Expressing, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Injection, Fluorescence In Situ Hybridization, Over Expression

MTM1-MTMR12 interactions in normal and disease states. Under normal conditions, MTM1 and MTMR12 interact in skeletal muscle and regulate skeletal muscle architecture and function. Loss of function mutations of MTM1 (red cross) in skeletal muscle are associated with centronuclear myopathy and with a secondary reduction in MTMR12 levels. In centronuclear myopathy, disease causing missense mutations (red circle) that disrupt interactions between MTM1 and MTMR12 result in decreased stability of myotubularin causing myotubular myopathy associated with reduced levels of MTMR12. Loss of MTMR12 in zebrafish and mammalian cells, results in decreased levels of myotubularin resulting in pathological changes similar to centronuclear myopathy.
Figure Legend Snippet: MTM1-MTMR12 interactions in normal and disease states. Under normal conditions, MTM1 and MTMR12 interact in skeletal muscle and regulate skeletal muscle architecture and function. Loss of function mutations of MTM1 (red cross) in skeletal muscle are associated with centronuclear myopathy and with a secondary reduction in MTMR12 levels. In centronuclear myopathy, disease causing missense mutations (red circle) that disrupt interactions between MTM1 and MTMR12 result in decreased stability of myotubularin causing myotubular myopathy associated with reduced levels of MTMR12. Loss of MTMR12 in zebrafish and mammalian cells, results in decreased levels of myotubularin resulting in pathological changes similar to centronuclear myopathy.

Techniques Used:

5) Product Images from "6-Shogaol Suppresses 2-Amino-1-Methyl-6-Phenylimidazo [4,5-b] Pyridine (PhIP)-Induced Human 786-O Renal Cell Carcinoma Osteoclastogenic Activity and Metastatic Potential"

Article Title: 6-Shogaol Suppresses 2-Amino-1-Methyl-6-Phenylimidazo [4,5-b] Pyridine (PhIP)-Induced Human 786-O Renal Cell Carcinoma Osteoclastogenic Activity and Metastatic Potential

Journal: Nutrients

doi: 10.3390/nu11102306

PhIP increased IL-8 production through PTHrP-mediated mechanism in 786-O. ( A ) The levels of IL-8 levels in medium of 786-O cells after vehicle control or PhIP (20 μM) treatment for 24 h. ( B ) The efficiency of PTHrP siRNA was determined via qRT-PCR. ( C ) The levels of IL-8 levels in siRNA-transfected 786-O cells. Each value is the mean ± SD of three independent experiments. * P
Figure Legend Snippet: PhIP increased IL-8 production through PTHrP-mediated mechanism in 786-O. ( A ) The levels of IL-8 levels in medium of 786-O cells after vehicle control or PhIP (20 μM) treatment for 24 h. ( B ) The efficiency of PTHrP siRNA was determined via qRT-PCR. ( C ) The levels of IL-8 levels in siRNA-transfected 786-O cells. Each value is the mean ± SD of three independent experiments. * P

Techniques Used: Quantitative RT-PCR, Transfection

6) Product Images from "CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation"

Article Title: CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation

Journal: Scientific Reports

doi: 10.1038/s41598-018-21506-7

CXCL12-Treated Cells Demonstrate Significant Up-Regulation of Transcripts Encoding COPII Vesicle Proteins. ( A ) Table of GO terms associated with specific genes encoding proteins that function in ER to Golgi transport that were significantly up-regulated by CXCL12, but not TGFβ, treatment. All of these genes encode proteins that comprise multi-subunit RING-finger type 3 Cullin-RBX E3 structures which, upon the addition of KLHL12, forms COPII vesicles essential for procollagen secretion. The gene Accession numbers and Symbols are as indicated. LogFC (log fold change) and FC (fold change) refer to CXCL12 > TGF β after normalization to vehicle; logCPM = log counts per million; FDR = false discovery rate. ( B , C ) Graphs demonstrating averages of triplicate measures of normalized (to vehicle) fold expression of CUL3 ( B ) and KLHL12 ( C ) in CXCL12- or TGF β -treated cells by qRT-PCR analysis confirm that both genes were more robustly transcriptionally induced by CXCL12 than TGF β , particularly at 8 hours treatment (* p
Figure Legend Snippet: CXCL12-Treated Cells Demonstrate Significant Up-Regulation of Transcripts Encoding COPII Vesicle Proteins. ( A ) Table of GO terms associated with specific genes encoding proteins that function in ER to Golgi transport that were significantly up-regulated by CXCL12, but not TGFβ, treatment. All of these genes encode proteins that comprise multi-subunit RING-finger type 3 Cullin-RBX E3 structures which, upon the addition of KLHL12, forms COPII vesicles essential for procollagen secretion. The gene Accession numbers and Symbols are as indicated. LogFC (log fold change) and FC (fold change) refer to CXCL12 > TGF β after normalization to vehicle; logCPM = log counts per million; FDR = false discovery rate. ( B , C ) Graphs demonstrating averages of triplicate measures of normalized (to vehicle) fold expression of CUL3 ( B ) and KLHL12 ( C ) in CXCL12- or TGF β -treated cells by qRT-PCR analysis confirm that both genes were more robustly transcriptionally induced by CXCL12 than TGF β , particularly at 8 hours treatment (* p

Techniques Used: Expressing, Quantitative RT-PCR

7) Product Images from "Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses"

Article Title: Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

Journal: Nature Communications

doi: 10.1038/s41467-018-03772-1

Analytical performance comparison between Trioplex assay multiplex and the ZIKV singleplex format assay using small volume and large volume RNA extraction. Normal human serum or urine was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of every dilution were extracted using the MagNA Pure 96 instrument (Roche) and tested by Trioplex assay multiplex or ZIKV singleplex format assay on the ABI 7500 Fast Dx or the QuantStudio Dx instruments. a Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum at each dilution on the ABI 7500 Fast Dx instrument. A linear regression was plotted for multiplex with small volume protocol (Sv) (0.2 mL) (black straight line), singleplex assay with small volume protocol (gray straight line), multiplex with large volume protocol (Lv) (1 mL) (black dashed line). and singleplex assay with large volume protocol (gray dashed line). b Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum or urine at each dilution on the QuantStudio Dx instrument. A linear regression was plotted for multiplex with small volume protocol serum (Sv) (black straight line), multiplex with large volume protocol serum (Lv) (gray straight line), multiplex with small volume protocol urine (Lv) (black dashed line), and multiplex with large volume protocol urine (gray dashed line). Error bars represent GCE/mL standard deviation. The CT values for every dilution replicate in serum tested was plotted for c small volume and d large volume extractions
Figure Legend Snippet: Analytical performance comparison between Trioplex assay multiplex and the ZIKV singleplex format assay using small volume and large volume RNA extraction. Normal human serum or urine was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of every dilution were extracted using the MagNA Pure 96 instrument (Roche) and tested by Trioplex assay multiplex or ZIKV singleplex format assay on the ABI 7500 Fast Dx or the QuantStudio Dx instruments. a Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum at each dilution on the ABI 7500 Fast Dx instrument. A linear regression was plotted for multiplex with small volume protocol (Sv) (0.2 mL) (black straight line), singleplex assay with small volume protocol (gray straight line), multiplex with large volume protocol (Lv) (1 mL) (black dashed line). and singleplex assay with large volume protocol (gray dashed line). b Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum or urine at each dilution on the QuantStudio Dx instrument. A linear regression was plotted for multiplex with small volume protocol serum (Sv) (black straight line), multiplex with large volume protocol serum (Lv) (gray straight line), multiplex with small volume protocol urine (Lv) (black dashed line), and multiplex with large volume protocol urine (gray dashed line). Error bars represent GCE/mL standard deviation. The CT values for every dilution replicate in serum tested was plotted for c small volume and d large volume extractions

Techniques Used: Multiplex Assay, RNA Extraction, Polymerase Chain Reaction, Singleplex Assay, Standard Deviation

Clinical performance of the Trioplex assay across specimen types. Clinical specimens collected concurrently from 373 cases with previous Zika determination in the acute stage were tested. RNA was extracted with the MagNA Pure 96 small volume external lysis protocol from 373 case-paired serum, 373 urine, and 345 whole blood-EDTA specimens and tested with the Trioplex assay in multiplex format in the ABI 7500 Fast Dx instrument. a Correlation of CT values between case-matching serum and urine specimens; R 2 = 0.36 p
Figure Legend Snippet: Clinical performance of the Trioplex assay across specimen types. Clinical specimens collected concurrently from 373 cases with previous Zika determination in the acute stage were tested. RNA was extracted with the MagNA Pure 96 small volume external lysis protocol from 373 case-paired serum, 373 urine, and 345 whole blood-EDTA specimens and tested with the Trioplex assay in multiplex format in the ABI 7500 Fast Dx instrument. a Correlation of CT values between case-matching serum and urine specimens; R 2 = 0.36 p

Techniques Used: Lysis, Multiplex Assay

8) Product Images from "MiR-429 Regulated by Endothelial Monocyte Activating Polypeptide-II (EMAP-II) Influences Blood-Tumor Barrier Permeability by Inhibiting the Expressions of ZO-1, Occludin and Claudin-5"

Article Title: MiR-429 Regulated by Endothelial Monocyte Activating Polypeptide-II (EMAP-II) Influences Blood-Tumor Barrier Permeability by Inhibiting the Expressions of ZO-1, Occludin and Claudin-5

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00035

The variety of miR-429 expression and the effect of EMAP-II on the expression of miR-429. (A) The relative expressions of miR-429 were tested in ECs and GECs from cell lines by qRT-PCR. All data represent mean ± SD ( n = 5, each). ∗∗ P
Figure Legend Snippet: The variety of miR-429 expression and the effect of EMAP-II on the expression of miR-429. (A) The relative expressions of miR-429 were tested in ECs and GECs from cell lines by qRT-PCR. All data represent mean ± SD ( n = 5, each). ∗∗ P

Techniques Used: Expressing, Quantitative RT-PCR

p70S6K influencing BTB permeability and the expression of ZO-1, occludin and claudin-5. (A) The mRNA expressions of p70S6K with the EMAP-II influence by qRT-PCR. All data represent mean ± SD ( n = 5, each). ∗∗ P
Figure Legend Snippet: p70S6K influencing BTB permeability and the expression of ZO-1, occludin and claudin-5. (A) The mRNA expressions of p70S6K with the EMAP-II influence by qRT-PCR. All data represent mean ± SD ( n = 5, each). ∗∗ P

Techniques Used: Permeability, Expressing, Quantitative RT-PCR

miR-429 influencing the expression of ZO-1, occludin and claudin-5. (A) The mRNA expressions of ZO-1, occludin and claudin-5 were tested by qRT-PCR of miR-429 overexpression and silencing. All data represent mean ± SD ( n = 5, each). ∗∗ P
Figure Legend Snippet: miR-429 influencing the expression of ZO-1, occludin and claudin-5. (A) The mRNA expressions of ZO-1, occludin and claudin-5 were tested by qRT-PCR of miR-429 overexpression and silencing. All data represent mean ± SD ( n = 5, each). ∗∗ P

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

miR-429 regulating p70S6K. (A) The mRNA expressions of p70S6K of miR-429 overexpression and silencing were tested by qRT-PCR. All data represent mean ± SD ( n = 5, each). ∗∗ P
Figure Legend Snippet: miR-429 regulating p70S6K. (A) The mRNA expressions of p70S6K of miR-429 overexpression and silencing were tested by qRT-PCR. All data represent mean ± SD ( n = 5, each). ∗∗ P

Techniques Used: Over Expression, Quantitative RT-PCR

9) Product Images from "Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses"

Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

Journal: Memórias do Instituto Oswaldo Cruz

doi: 10.1590/0074-02760160062

: reverse transcription real-time polymerase chain reaction (RT-qPCR) with serial dilutions of the chimeric in vitro transcribed RNA containing both Mayaro (MAYV) and Oropouche (OROV) targets. Amplification plots for MAYV (A) and OROV (B), and linear regression for MAYV (C) and OROV (D) for ten-fold, 8-log, dilutions from 20 to 2E+08 copies, in duplicate. PCR efficiency in the multiplex assay was calculated with StepOnePlus Software v2.2 to be 98.642% [slope: -3.355, R2: 1] for MAYV, and 99.181% [slope: -3.341, R2: 1] for OROV. Ct = cycle threshold. Fluorescence values were exported to MS Excel and plotted using GraphPad Prism 6.0.
Figure Legend Snippet: : reverse transcription real-time polymerase chain reaction (RT-qPCR) with serial dilutions of the chimeric in vitro transcribed RNA containing both Mayaro (MAYV) and Oropouche (OROV) targets. Amplification plots for MAYV (A) and OROV (B), and linear regression for MAYV (C) and OROV (D) for ten-fold, 8-log, dilutions from 20 to 2E+08 copies, in duplicate. PCR efficiency in the multiplex assay was calculated with StepOnePlus Software v2.2 to be 98.642% [slope: -3.355, R2: 1] for MAYV, and 99.181% [slope: -3.341, R2: 1] for OROV. Ct = cycle threshold. Fluorescence values were exported to MS Excel and plotted using GraphPad Prism 6.0.

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, In Vitro, Amplification, Polymerase Chain Reaction, Multiplex Assay, Software, Fluorescence, Mass Spectrometry

10) Product Images from "Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR"

Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-1158-5_5

Representative qRT-PCR amplification and melting curve plots for the human IFN-γ- and CD8-specific amplicons. ( a ) CD8 and IFN-γ were amplified from cDNA of a normal HLA-A2+ human donor through qRT-PCR using the StepOnePlus™ Real-Time
Figure Legend Snippet: Representative qRT-PCR amplification and melting curve plots for the human IFN-γ- and CD8-specific amplicons. ( a ) CD8 and IFN-γ were amplified from cDNA of a normal HLA-A2+ human donor through qRT-PCR using the StepOnePlus™ Real-Time

Techniques Used: Quantitative RT-PCR, Amplification

11) Product Images from "Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR"

Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-1158-5_5

Representative qRT-PCR amplification and melting curve plots for the human IFN-γ- and CD8-specific amplicons. ( a ) CD8 and IFN-γ were amplified from cDNA of a normal HLA-A2+ human donor through qRT-PCR using the StepOnePlus™ Real-Time
Figure Legend Snippet: Representative qRT-PCR amplification and melting curve plots for the human IFN-γ- and CD8-specific amplicons. ( a ) CD8 and IFN-γ were amplified from cDNA of a normal HLA-A2+ human donor through qRT-PCR using the StepOnePlus™ Real-Time

Techniques Used: Quantitative RT-PCR, Amplification

12) Product Images from "An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation"

Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation

Journal: Oncotarget

doi: 10.18632/oncotarget.7502

Analysis of mRNA and protein levels for pRb and E2F1 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of pRb and E2F1 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of pRb and E2F1 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P
Figure Legend Snippet: Analysis of mRNA and protein levels for pRb and E2F1 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of pRb and E2F1 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of pRb and E2F1 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

Techniques Used: Infection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Analysis of mRNA and protein levels for p53, Mdm2, CyclinD1 and Cdk6 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of p53, Mdm2, CyclinD1 and Cdk6 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of p53, Mdm2, CyclinD1 and Cdk6 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P
Figure Legend Snippet: Analysis of mRNA and protein levels for p53, Mdm2, CyclinD1 and Cdk6 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of p53, Mdm2, CyclinD1 and Cdk6 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of p53, Mdm2, CyclinD1 and Cdk6 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

Techniques Used: Infection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

13) Product Images from "Evidence against a Beneficial Effect of Irisin in Humans"

Article Title: Evidence against a Beneficial Effect of Irisin in Humans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073680

Gene expression analysis of human adipocytes after incubation with BMP7, FNDC5 and irisin. Isolated preadipocytes from human subcutaneous AT of different donors were differentiated in the presence of 50/ml BMP7, 200 ng/ml FNDC5 (Abnova), and 60 ng/ml irisin (Phoenix). Gene expression of 40 genes, related to adipocyte differentiation (A) and brite differentiation (B), was assessed by a microfluidic card TaqMan gene expression assay; n ≥4, *p
Figure Legend Snippet: Gene expression analysis of human adipocytes after incubation with BMP7, FNDC5 and irisin. Isolated preadipocytes from human subcutaneous AT of different donors were differentiated in the presence of 50/ml BMP7, 200 ng/ml FNDC5 (Abnova), and 60 ng/ml irisin (Phoenix). Gene expression of 40 genes, related to adipocyte differentiation (A) and brite differentiation (B), was assessed by a microfluidic card TaqMan gene expression assay; n ≥4, *p

Techniques Used: Expressing, Incubation, Isolation

FNDC5 mRNA level is not contraction-regulated in skeletal muscle cells and is not increased by endurance or strength training in humans. (A) and (B) Primary human skeletal muscle cells were differentiated in αMEM containing 2% (vol./vol.) horse serum, followed by overnight starvation, and subjected to EPS for 24 h in serum-free medium (1 Hz, 2 ms, 11.5 V). Relative gene expression of PGC1α , FNDC5 (A), MYH1 , 2 , and 7 (B) was measured by quantitative real-time PCR (qRT-PCR). All expression data were normalized to actin; n = 5 (A), n = 10 (B); **p
Figure Legend Snippet: FNDC5 mRNA level is not contraction-regulated in skeletal muscle cells and is not increased by endurance or strength training in humans. (A) and (B) Primary human skeletal muscle cells were differentiated in αMEM containing 2% (vol./vol.) horse serum, followed by overnight starvation, and subjected to EPS for 24 h in serum-free medium (1 Hz, 2 ms, 11.5 V). Relative gene expression of PGC1α , FNDC5 (A), MYH1 , 2 , and 7 (B) was measured by quantitative real-time PCR (qRT-PCR). All expression data were normalized to actin; n = 5 (A), n = 10 (B); **p

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

BMP7 activates the brite fat gene program in human adipocytes, but not FNDC5 and irisin. Isolated preadipocytes from human subcutaneous preadipocytes of different donors were differentiated in the presence of 50/ml BMP7, 200 ng/ml FNDC5 (Abnova), 200 ng/ml FNDC5 (Phoenix) and 60 ng/ml irisin (Phoenix). (A) Relative gene expression of PPARγ, UCP1, TCF21 and ZIC1 was measured by qRT-PCR after 12–14 days of differentiation. All expression data were normalized to actin; n≥4; ***p
Figure Legend Snippet: BMP7 activates the brite fat gene program in human adipocytes, but not FNDC5 and irisin. Isolated preadipocytes from human subcutaneous preadipocytes of different donors were differentiated in the presence of 50/ml BMP7, 200 ng/ml FNDC5 (Abnova), 200 ng/ml FNDC5 (Phoenix) and 60 ng/ml irisin (Phoenix). (A) Relative gene expression of PPARγ, UCP1, TCF21 and ZIC1 was measured by qRT-PCR after 12–14 days of differentiation. All expression data were normalized to actin; n≥4; ***p

Techniques Used: Isolation, Expressing, Quantitative RT-PCR

The human FNDC5 gene differs from other species by a mutation in the start codon. (A) Multiple alignment of the exon 1 sequences: the conserved partial Kozak ATG start sequence of FNDC5 is bold and red. The mutated ATG to ATA in human is bold and blue. There is no other ATG present in exon 1. (B) Multiple sequence alignment of FNDC5 proteins of different species including two human versions. FNDC5_human_o: sequence published by Boström et al.; FNDC5_human_c: current version in Uniprot; red M = start methionines including the potential downstream human start site; light blue = irisin sequence; blue I = mutated start site claimed to be a non canonical start site; purple LRL = sequence shown in UniProt (Q8NAU1_old) as MRR. The underlined sequence indicates the transmembrane part of the protein. Green sequence = peptide used for the generation of the Abcam FNDC5 antibody.
Figure Legend Snippet: The human FNDC5 gene differs from other species by a mutation in the start codon. (A) Multiple alignment of the exon 1 sequences: the conserved partial Kozak ATG start sequence of FNDC5 is bold and red. The mutated ATG to ATA in human is bold and blue. There is no other ATG present in exon 1. (B) Multiple sequence alignment of FNDC5 proteins of different species including two human versions. FNDC5_human_o: sequence published by Boström et al.; FNDC5_human_c: current version in Uniprot; red M = start methionines including the potential downstream human start site; light blue = irisin sequence; blue I = mutated start site claimed to be a non canonical start site; purple LRL = sequence shown in UniProt (Q8NAU1_old) as MRR. The underlined sequence indicates the transmembrane part of the protein. Green sequence = peptide used for the generation of the Abcam FNDC5 antibody.

Techniques Used: Mutagenesis, Sequencing

Human FNDC5 with an ATA start codon is translated into full-length protein only at very low abundance. (A) Schematic representation of the predicted FNDC5 protein structures. Using the first ATG/ATA as start codon of human FNDC5 tagged with GFP would result in full-length FNDC5 protein (a). The use of downstream ATG as start codon would result in truncated FNDC5 protein isoforms (b and c). Murine FNDC5 with ATG as start codon tagged with GFP (d) or without GFP (e). (B) Expression of FNDC5 in HEK293 cells. Cells transfected with constructs containing human FNDC5-GFP gene with ATA and ATG as start codon as well as mouse FNDC5-GFP gene were analyzed 24 h after transfection. Cell lysates were analyzed by immunodetection using antibodies against irisin/FNDC5 and GFP. Cell lysates were treated with PNGaseF to deglycosylate proteins. (C) Supernatants of primary human and C2C12 myotubes were collected for 24 h in serum-free medium and concentrated 60fold using centrifugal filter devices. Irisin protein levels in concentrated supernatants were measured using EIA kit. Medium alone showed no cross-reactivity with the kit; n = 5, *** p
Figure Legend Snippet: Human FNDC5 with an ATA start codon is translated into full-length protein only at very low abundance. (A) Schematic representation of the predicted FNDC5 protein structures. Using the first ATG/ATA as start codon of human FNDC5 tagged with GFP would result in full-length FNDC5 protein (a). The use of downstream ATG as start codon would result in truncated FNDC5 protein isoforms (b and c). Murine FNDC5 with ATG as start codon tagged with GFP (d) or without GFP (e). (B) Expression of FNDC5 in HEK293 cells. Cells transfected with constructs containing human FNDC5-GFP gene with ATA and ATG as start codon as well as mouse FNDC5-GFP gene were analyzed 24 h after transfection. Cell lysates were analyzed by immunodetection using antibodies against irisin/FNDC5 and GFP. Cell lysates were treated with PNGaseF to deglycosylate proteins. (C) Supernatants of primary human and C2C12 myotubes were collected for 24 h in serum-free medium and concentrated 60fold using centrifugal filter devices. Irisin protein levels in concentrated supernatants were measured using EIA kit. Medium alone showed no cross-reactivity with the kit; n = 5, *** p

Techniques Used: Expressing, Transfection, Construct, Immunodetection, Enzyme-linked Immunosorbent Assay

14) Product Images from "Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity"

Article Title: Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003698

Signaling-induced changes in H3K27me3 level are not required for those in transcriptional activity. ( A ) Time line for transfection with control (Ctrl) or Suz12 siRNAs, treatment with 4HT or ethanol (EtOH) vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (B) through (D). ( B ) Immunoblot analysis of Suz12 (arrow) and α-tubulin in the cytosolic fraction as well as of H3K27me3 and total H3 in the chromatin fraction. ( C ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 for the regions of Itgb5 , Adcy7 , and Smad6 indicated in Figure 4A . Data are means ± SE from two independent experiments. ( D ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from three independent experiments. ( E ) Time line for transfection with control or Suz12 siRNAs, treatment with 4HT or ethanol vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (F) and (G). ( F ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 at Itgb5 , Adcy7 , and Smad6 . Data are means ± SE from two independent experiments. ( G ) RT-qPCR analysis of relative Suz12 , Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments.
Figure Legend Snippet: Signaling-induced changes in H3K27me3 level are not required for those in transcriptional activity. ( A ) Time line for transfection with control (Ctrl) or Suz12 siRNAs, treatment with 4HT or ethanol (EtOH) vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (B) through (D). ( B ) Immunoblot analysis of Suz12 (arrow) and α-tubulin in the cytosolic fraction as well as of H3K27me3 and total H3 in the chromatin fraction. ( C ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 for the regions of Itgb5 , Adcy7 , and Smad6 indicated in Figure 4A . Data are means ± SE from two independent experiments. ( D ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from three independent experiments. ( E ) Time line for transfection with control or Suz12 siRNAs, treatment with 4HT or ethanol vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (F) and (G). ( F ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 at Itgb5 , Adcy7 , and Smad6 . Data are means ± SE from two independent experiments. ( G ) RT-qPCR analysis of relative Suz12 , Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments.

Techniques Used: Activity Assay, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

15) Product Images from "Characterization of a Toxoplasma effector uncovers an alternative GSK3/β-catenin-regulatory pathway of inflammation"

Article Title: Characterization of a Toxoplasma effector uncovers an alternative GSK3/β-catenin-regulatory pathway of inflammation

Journal: eLife

doi: 10.7554/eLife.39887

GRA18 activity is dependent on its interactions with GSK3 and PP2A-B56. ( A–B ) RAW264.7 cells were transfected with mCherry vector control or the FH-GRA18 expression vectors pcDNA-FH-GRA18 FL (GRA18 FL ), pcDNA-FH-GRA18 Nt (GRA18 Nt ), and pcDNA-FH-GRA18 Ct (GRA18 Ct ). At 18 hr after transfection, cells were harvested and ( A ) whole cell extracts were analyzed by immunoblot using the indicated antibodies, or ( B ) quantitative chemokine expression was determined by qRT-PCR as in Figure 6 . Asterisks indicate P -values (p
Figure Legend Snippet: GRA18 activity is dependent on its interactions with GSK3 and PP2A-B56. ( A–B ) RAW264.7 cells were transfected with mCherry vector control or the FH-GRA18 expression vectors pcDNA-FH-GRA18 FL (GRA18 FL ), pcDNA-FH-GRA18 Nt (GRA18 Nt ), and pcDNA-FH-GRA18 Ct (GRA18 Ct ). At 18 hr after transfection, cells were harvested and ( A ) whole cell extracts were analyzed by immunoblot using the indicated antibodies, or ( B ) quantitative chemokine expression was determined by qRT-PCR as in Figure 6 . Asterisks indicate P -values (p

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

GRA18 promotes Ccl17, Ccl22 and Ccl24 chemokines expression in a β-catenin-dependent fashion. ( A ) Schematic diagram of gRNAs targeting the Ctnnb1 locus. The Protospacer Adjacent Motif (PAM) sequence is lined and highlighted in red; the targeting sequences is shown in green. Bi-directional arrow indicates the Cas9 cleavage site. DNA sequences from the wild-type and mutated RAW264.7-derived cell lines were analyzed by DNA sequencing; the identified deletions are indicated with (-). No mutations in control samples were observed. ( B ) Immunoblot analysis of β-catenin in parental RAW264.7 and the Ctnnb1 -/- mutant confirmed the absence of β-catenin expression. As positive controls, cells were treated with 3 mM of BIO or 2 mM of CHIR GSK3 inhibitors for 12 hr. TBP was used as loading control. ( C–E ) RAW264.7 (WT) and β-catenin-deficient ( Ctnnb1 -/- ) RAW264.7-derived cell lines were transfected with mCherry vector control (pcDNA-mCherry-HF) or the FH-GRA18 II expression vector (pcDNA-FH-GRA18 FL ). At 18 hr after transfection, cells were either ( C ) fixed for IFA using anti-HA (red) and anti-β-catenin (green) antibodies or ( D ) cells were harvested and analyzed by immunoblot using the indicated antibodies. Anti-HA was used to detect FH-GRA18 and mCherry-HF. In ( E ) and ( F ) transcripts for Ccl17, Ccl22, Ccl24 , and Ifnb1 were quantified by qPCR and normalized using Tbp . Data are mean value ± s.d. of three replicates. The P -values were calculated using two-tailed unpaired Student’s t -test or one-way ANOVA with Bonferroni posttests analysis of variance; *p
Figure Legend Snippet: GRA18 promotes Ccl17, Ccl22 and Ccl24 chemokines expression in a β-catenin-dependent fashion. ( A ) Schematic diagram of gRNAs targeting the Ctnnb1 locus. The Protospacer Adjacent Motif (PAM) sequence is lined and highlighted in red; the targeting sequences is shown in green. Bi-directional arrow indicates the Cas9 cleavage site. DNA sequences from the wild-type and mutated RAW264.7-derived cell lines were analyzed by DNA sequencing; the identified deletions are indicated with (-). No mutations in control samples were observed. ( B ) Immunoblot analysis of β-catenin in parental RAW264.7 and the Ctnnb1 -/- mutant confirmed the absence of β-catenin expression. As positive controls, cells were treated with 3 mM of BIO or 2 mM of CHIR GSK3 inhibitors for 12 hr. TBP was used as loading control. ( C–E ) RAW264.7 (WT) and β-catenin-deficient ( Ctnnb1 -/- ) RAW264.7-derived cell lines were transfected with mCherry vector control (pcDNA-mCherry-HF) or the FH-GRA18 II expression vector (pcDNA-FH-GRA18 FL ). At 18 hr after transfection, cells were either ( C ) fixed for IFA using anti-HA (red) and anti-β-catenin (green) antibodies or ( D ) cells were harvested and analyzed by immunoblot using the indicated antibodies. Anti-HA was used to detect FH-GRA18 and mCherry-HF. In ( E ) and ( F ) transcripts for Ccl17, Ccl22, Ccl24 , and Ifnb1 were quantified by qPCR and normalized using Tbp . Data are mean value ± s.d. of three replicates. The P -values were calculated using two-tailed unpaired Student’s t -test or one-way ANOVA with Bonferroni posttests analysis of variance; *p

Techniques Used: Expressing, Sequencing, Derivative Assay, DNA Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Immunofluorescence, Real-time Polymerase Chain Reaction, Two Tailed Test

16) Product Images from "Functional and biochemical characterization of a T cell-associated anti-apoptotic protein, GIMAP6"

Article Title: Functional and biochemical characterization of a T cell-associated anti-apoptotic protein, GIMAP6

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.768689

Exogenous GIMAP6 displays anti-apoptotic effects in Huh-7 stable transfectant cells. A , the GIMAP6 expression level in Huh-7 derivative cells was assessed by immunoblot analysis. After transfection with a recombinant pcDNA3.1(+) vector carrying the GIMAP6 sequence, stably expressing single colonies were picked under G418 selection (500 μg/ml) for 2 weeks. Three drug-resistant clones ( Neg , TF1 , and TF2 ) were analyzed for GIMAP6 expression, and these clones were found to show substantial variation in protein levels by immunoblot analysis. B and C , the effect of GIMAP6 on OA-mediated cell apoptosis. B , cells were treated with OA for 24 h, and the level of immunofluorescence staining was analyzed. The cleaved form of caspase-3 (Cell Signaling Technology, 9661S) was used as an apoptotic marker ( green ), and DAPI ( blue ) was used as a counterstain as well as a total cell dye. C , the GIMAP6-negative cell line ( Neg ) was found to have a relatively higher proportion of caspase-3-positive cells compared with the GIMAP6-expressing cell lines ( TF1 and TF2 ). The data represent the average of two independent experiments (mean ± S.D.). ***, p
Figure Legend Snippet: Exogenous GIMAP6 displays anti-apoptotic effects in Huh-7 stable transfectant cells. A , the GIMAP6 expression level in Huh-7 derivative cells was assessed by immunoblot analysis. After transfection with a recombinant pcDNA3.1(+) vector carrying the GIMAP6 sequence, stably expressing single colonies were picked under G418 selection (500 μg/ml) for 2 weeks. Three drug-resistant clones ( Neg , TF1 , and TF2 ) were analyzed for GIMAP6 expression, and these clones were found to show substantial variation in protein levels by immunoblot analysis. B and C , the effect of GIMAP6 on OA-mediated cell apoptosis. B , cells were treated with OA for 24 h, and the level of immunofluorescence staining was analyzed. The cleaved form of caspase-3 (Cell Signaling Technology, 9661S) was used as an apoptotic marker ( green ), and DAPI ( blue ) was used as a counterstain as well as a total cell dye. C , the GIMAP6-negative cell line ( Neg ) was found to have a relatively higher proportion of caspase-3-positive cells compared with the GIMAP6-expressing cell lines ( TF1 and TF2 ). The data represent the average of two independent experiments (mean ± S.D.). ***, p

Techniques Used: Transfection, Expressing, Recombinant, Plasmid Preparation, Sequencing, Stable Transfection, Selection, Clone Assay, Immunofluorescence, Staining, Marker

Increased IL-2 secretion and CD25 and CD154 surface expression by GIMAP6 knockdown in primary CD3+ T cells after activation with PMA/ionomycin. A , GIMAP6 RNA expression in primary CD3+ T cells after transfection of siRNA; these were analyzed by quantitative PCR assay. CD3+ T cells were enriched from PBMCs, which were obtained from two healthy donors, and knockdown of GIMAP6 was performed by electroporation with either negative control ( Neg ) or siRNA oligonucleotides against GIMAP6 ( G6-si599 and G6-si600 ). To monitor the efficiency of GIMAP6 knockdown, transfected cells were harvested at 48 h, and total RNA was extracted for the quantitative RT-PCR assay. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. B , the effect of GIMAP6 expression on P/I-induced IL-2 secretion. Cells were incubated with either P/I or DMSO (as a control) for 72 h. P/I indicates that the cells were treated with both PMA (10 ng/ml) and ionomycin (1 μg/ml). Media were harvested at the indicated times (24, 48, and 72 h). The data represent the average of two independent experiments with triplicate measurements (mean ± S.D.). C , the effect of GIMAP6 expression on the induction of surface antigens. Transfected primary CD3+ T cells were stimulated with either P/I or an equivalent concentration of DMSO for 72 h. Expression of CD25 and CD154 was analyzed using flow cytometry. The gray histograms show the profiles of P/I-treated cells, whereas the white histograms show those of DMSO-treated cells. The percentage in the top right corner of each panel indicates the increment of positive cells. The results were analyzed and quantified by FlowJo 7.6.1 and are shown in D . The data represent the average of two independent experiments with triplicate measurements (mean ± S.D.). ***, p
Figure Legend Snippet: Increased IL-2 secretion and CD25 and CD154 surface expression by GIMAP6 knockdown in primary CD3+ T cells after activation with PMA/ionomycin. A , GIMAP6 RNA expression in primary CD3+ T cells after transfection of siRNA; these were analyzed by quantitative PCR assay. CD3+ T cells were enriched from PBMCs, which were obtained from two healthy donors, and knockdown of GIMAP6 was performed by electroporation with either negative control ( Neg ) or siRNA oligonucleotides against GIMAP6 ( G6-si599 and G6-si600 ). To monitor the efficiency of GIMAP6 knockdown, transfected cells were harvested at 48 h, and total RNA was extracted for the quantitative RT-PCR assay. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. B , the effect of GIMAP6 expression on P/I-induced IL-2 secretion. Cells were incubated with either P/I or DMSO (as a control) for 72 h. P/I indicates that the cells were treated with both PMA (10 ng/ml) and ionomycin (1 μg/ml). Media were harvested at the indicated times (24, 48, and 72 h). The data represent the average of two independent experiments with triplicate measurements (mean ± S.D.). C , the effect of GIMAP6 expression on the induction of surface antigens. Transfected primary CD3+ T cells were stimulated with either P/I or an equivalent concentration of DMSO for 72 h. Expression of CD25 and CD154 was analyzed using flow cytometry. The gray histograms show the profiles of P/I-treated cells, whereas the white histograms show those of DMSO-treated cells. The percentage in the top right corner of each panel indicates the increment of positive cells. The results were analyzed and quantified by FlowJo 7.6.1 and are shown in D . The data represent the average of two independent experiments with triplicate measurements (mean ± S.D.). ***, p

Techniques Used: Expressing, Activation Assay, RNA Expression, Transfection, Real-time Polymerase Chain Reaction, Electroporation, Negative Control, Quantitative RT-PCR, Standard Deviation, Incubation, Concentration Assay, Flow Cytometry, Cytometry

17) Product Images from "LRRK2 Expression Is Deregulated in Fibroblasts and Neurons from Parkinson Patients with Mutations in PINK1"

Article Title: LRRK2 Expression Is Deregulated in Fibroblasts and Neurons from Parkinson Patients with Mutations in PINK1

Journal: Molecular Neurobiology

doi: 10.1007/s12035-016-0303-7

Over-expression of wild-type PINK1 downregulates LRRK2 . a PINK1 RNA levels analyzed by quantitative RT-PCR. Average increase by qPCR was not different across groups (31 ± 4.5-fold increase over mock GFP transfection). b A representative image of an agarose gel electrophoresis showing the expression of the 499 bp band corresponding to the exons 6 to 8 of PINK1 in the PINK1 -exon7/del samples (PDP1 and PDP3; boxed ) after electroporation with PINK1.GFP. c RNA levels analyzed by quantitative RT-PCR in fibroblasts after PINK1 over-expression. All data are expressed as fold change over mock (GFP) transfected samples. Scatter plot graphics of two to three independent experiments in control ( N = 2), carrier ( N = 2), and mutant ( N = 2) fibroblasts. One-way ANOVA. * p
Figure Legend Snippet: Over-expression of wild-type PINK1 downregulates LRRK2 . a PINK1 RNA levels analyzed by quantitative RT-PCR. Average increase by qPCR was not different across groups (31 ± 4.5-fold increase over mock GFP transfection). b A representative image of an agarose gel electrophoresis showing the expression of the 499 bp band corresponding to the exons 6 to 8 of PINK1 in the PINK1 -exon7/del samples (PDP1 and PDP3; boxed ) after electroporation with PINK1.GFP. c RNA levels analyzed by quantitative RT-PCR in fibroblasts after PINK1 over-expression. All data are expressed as fold change over mock (GFP) transfected samples. Scatter plot graphics of two to three independent experiments in control ( N = 2), carrier ( N = 2), and mutant ( N = 2) fibroblasts. One-way ANOVA. * p

Techniques Used: Over Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Transfection, Agarose Gel Electrophoresis, Expressing, Electroporation, Mutagenesis

18) Product Images from "MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis"

Article Title: MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis

Journal: Gut

doi: 10.1136/gutjnl-2011-301846

Predicted miR-200c target genes and validation in colorectal cancer (CRC) cell lines. (A–C) Overexpression of miR-200c-suppressed mRNA (quantitative real-time (qRT)-PCR) and protein expression (western blotting) of ZEB1, ETS1 and FLT1 , respectively.
Figure Legend Snippet: Predicted miR-200c target genes and validation in colorectal cancer (CRC) cell lines. (A–C) Overexpression of miR-200c-suppressed mRNA (quantitative real-time (qRT)-PCR) and protein expression (western blotting) of ZEB1, ETS1 and FLT1 , respectively.

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

Methylation analysis of miR-200b/429 and miR-200c/141 clusters. (A) Schematic illustration of miR-200b, -200c, -141 and -429 genomic loci. Blue arrows represent miRNA mapping locations; orange bars indicate a CpG island or CpG-rich regions; green bars
Figure Legend Snippet: Methylation analysis of miR-200b/429 and miR-200c/141 clusters. (A) Schematic illustration of miR-200b, -200c, -141 and -429 genomic loci. Blue arrows represent miRNA mapping locations; orange bars indicate a CpG island or CpG-rich regions; green bars

Techniques Used: Methylation

The expression of miR-200c in primary colorectal cancer and liver metastasis tissue specimens. (A) Quantitative expression analysis of miR-200c by quantitative real-time (qRT) PCR in primary CRC (PC) with and without metastasis (**p
Figure Legend Snippet: The expression of miR-200c in primary colorectal cancer and liver metastasis tissue specimens. (A) Quantitative expression analysis of miR-200c by quantitative real-time (qRT) PCR in primary CRC (PC) with and without metastasis (**p

Techniques Used: Expressing, Quantitative RT-PCR

Functional analysis of miR-200c. (A) Expression of miR-200c following transfection with pre-miR-200c was confirmed by TaqMan real-time PCR. (B–E) Data from various assays following transfection with pre-miR-200c in colorectal cancer (CRC) cell
Figure Legend Snippet: Functional analysis of miR-200c. (A) Expression of miR-200c following transfection with pre-miR-200c was confirmed by TaqMan real-time PCR. (B–E) Data from various assays following transfection with pre-miR-200c in colorectal cancer (CRC) cell

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

19) Product Images from "MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis"

Article Title: MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis

Journal: Gut

doi: 10.1136/gutjnl-2011-301846

Predicted miR-200c target genes and validation in colorectal cancer (CRC) cell lines. (A–C) Overexpression of miR-200c-suppressed mRNA (quantitative real-time (qRT)-PCR) and protein expression (western blotting) of ZEB1, ETS1 and FLT1 , respectively.
Figure Legend Snippet: Predicted miR-200c target genes and validation in colorectal cancer (CRC) cell lines. (A–C) Overexpression of miR-200c-suppressed mRNA (quantitative real-time (qRT)-PCR) and protein expression (western blotting) of ZEB1, ETS1 and FLT1 , respectively.

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

Methylation analysis of miR-200b/429 and miR-200c/141 clusters. (A) Schematic illustration of miR-200b, -200c, -141 and -429 genomic loci. Blue arrows represent miRNA mapping locations; orange bars indicate a CpG island or CpG-rich regions; green bars
Figure Legend Snippet: Methylation analysis of miR-200b/429 and miR-200c/141 clusters. (A) Schematic illustration of miR-200b, -200c, -141 and -429 genomic loci. Blue arrows represent miRNA mapping locations; orange bars indicate a CpG island or CpG-rich regions; green bars

Techniques Used: Methylation

The expression of miR-200c in primary colorectal cancer and liver metastasis tissue specimens. (A) Quantitative expression analysis of miR-200c by quantitative real-time (qRT) PCR in primary CRC (PC) with and without metastasis (**p
Figure Legend Snippet: The expression of miR-200c in primary colorectal cancer and liver metastasis tissue specimens. (A) Quantitative expression analysis of miR-200c by quantitative real-time (qRT) PCR in primary CRC (PC) with and without metastasis (**p

Techniques Used: Expressing, Quantitative RT-PCR

Functional analysis of miR-200c. (A) Expression of miR-200c following transfection with pre-miR-200c was confirmed by TaqMan real-time PCR. (B–E) Data from various assays following transfection with pre-miR-200c in colorectal cancer (CRC) cell
Figure Legend Snippet: Functional analysis of miR-200c. (A) Expression of miR-200c following transfection with pre-miR-200c was confirmed by TaqMan real-time PCR. (B–E) Data from various assays following transfection with pre-miR-200c in colorectal cancer (CRC) cell

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

20) Product Images from "MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis"

Article Title: MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis

Journal: Gut

doi: 10.1136/gutjnl-2011-301846

Predicted miR-200c target genes and validation in colorectal cancer (CRC) cell lines. (A–C) Overexpression of miR-200c-suppressed mRNA (quantitative real-time (qRT)-PCR) and protein expression (western blotting) of ZEB1, ETS1 and FLT1 , respectively.
Figure Legend Snippet: Predicted miR-200c target genes and validation in colorectal cancer (CRC) cell lines. (A–C) Overexpression of miR-200c-suppressed mRNA (quantitative real-time (qRT)-PCR) and protein expression (western blotting) of ZEB1, ETS1 and FLT1 , respectively.

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

Methylation analysis of miR-200b/429 and miR-200c/141 clusters. (A) Schematic illustration of miR-200b, -200c, -141 and -429 genomic loci. Blue arrows represent miRNA mapping locations; orange bars indicate a CpG island or CpG-rich regions; green bars
Figure Legend Snippet: Methylation analysis of miR-200b/429 and miR-200c/141 clusters. (A) Schematic illustration of miR-200b, -200c, -141 and -429 genomic loci. Blue arrows represent miRNA mapping locations; orange bars indicate a CpG island or CpG-rich regions; green bars

Techniques Used: Methylation

The expression of miR-200c in primary colorectal cancer and liver metastasis tissue specimens. (A) Quantitative expression analysis of miR-200c by quantitative real-time (qRT) PCR in primary CRC (PC) with and without metastasis (**p
Figure Legend Snippet: The expression of miR-200c in primary colorectal cancer and liver metastasis tissue specimens. (A) Quantitative expression analysis of miR-200c by quantitative real-time (qRT) PCR in primary CRC (PC) with and without metastasis (**p

Techniques Used: Expressing, Quantitative RT-PCR

Functional analysis of miR-200c. (A) Expression of miR-200c following transfection with pre-miR-200c was confirmed by TaqMan real-time PCR. (B–E) Data from various assays following transfection with pre-miR-200c in colorectal cancer (CRC) cell
Figure Legend Snippet: Functional analysis of miR-200c. (A) Expression of miR-200c following transfection with pre-miR-200c was confirmed by TaqMan real-time PCR. (B–E) Data from various assays following transfection with pre-miR-200c in colorectal cancer (CRC) cell

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

21) Product Images from "Overexpression of microRNA-132 enhances the radiosensitivity of cervical cancer cells by down-regulating Bmi-1"

Article Title: Overexpression of microRNA-132 enhances the radiosensitivity of cervical cancer cells by down-regulating Bmi-1

Journal: Oncotarget

doi: 10.18632/oncotarget.20358

Effects of miR-132 on the radiosensitivity of Hela cell-induced tumors in nude mice (A) Growth curve for transplanted tumors. After treatment with miRNA Agomir beginning on the 22 nd day, tumor growth was slower in the miR-132-agomir + 8 Gy and siBmi-1 + 8Gy groups than in the NC-agomir + 8 Gy group; this difference increased as treatment continued. (B and C) On the 35 th day after tumor transplantation, mice were sacrificed and tumors were removed, weighed, and photographed. Tumor weights were higher in the NC-agomir + 8 Gy group than in the miR-132-agomir + 8Gy and siBmi-1 + 8 Gy groups. (D) qRT-PCR performed using a small amount of transplanted tissue revealed that miR-132 expression in the miR-132-agomir + 8 Gy group was elevated compared to the NC-agomir + 8 Gy group. * P
Figure Legend Snippet: Effects of miR-132 on the radiosensitivity of Hela cell-induced tumors in nude mice (A) Growth curve for transplanted tumors. After treatment with miRNA Agomir beginning on the 22 nd day, tumor growth was slower in the miR-132-agomir + 8 Gy and siBmi-1 + 8Gy groups than in the NC-agomir + 8 Gy group; this difference increased as treatment continued. (B and C) On the 35 th day after tumor transplantation, mice were sacrificed and tumors were removed, weighed, and photographed. Tumor weights were higher in the NC-agomir + 8 Gy group than in the miR-132-agomir + 8Gy and siBmi-1 + 8 Gy groups. (D) qRT-PCR performed using a small amount of transplanted tissue revealed that miR-132 expression in the miR-132-agomir + 8 Gy group was elevated compared to the NC-agomir + 8 Gy group. * P

Techniques Used: Mouse Assay, Transplantation Assay, Quantitative RT-PCR, Expressing

Comparison of miR-132 and Bmi-1 expression in radiotherapy-sensitive and insensitive patients (A) miR-132 expression and Bmi-1 mRNA expression were detected using qRT-PCR. (B) correlation analysis of miRNA-132 and Bmi-1 mRNA expression in CC tissues. (C) Bmi-1 protein expression was detected using western blots. (D) the gray value of Bmi-1 protein band. * P
Figure Legend Snippet: Comparison of miR-132 and Bmi-1 expression in radiotherapy-sensitive and insensitive patients (A) miR-132 expression and Bmi-1 mRNA expression were detected using qRT-PCR. (B) correlation analysis of miRNA-132 and Bmi-1 mRNA expression in CC tissues. (C) Bmi-1 protein expression was detected using western blots. (D) the gray value of Bmi-1 protein band. * P

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

22) Product Images from "Resveratrol and pterostilbene epigenetically restore PTEN expression by targeting oncomiRs of the miR-17 family in prostate cancer"

Article Title: Resveratrol and pterostilbene epigenetically restore PTEN expression by targeting oncomiRs of the miR-17 family in prostate cancer

Journal: Oncotarget

doi:

Resveratrol and pterostilbene rescue PTEN inhibition by oncomiRs -17, -20a and -106b A. Schematic representation of the predicted target sites of miRs-17, -20a and -106b in the 3′UTR of PTEN mRNA. The miRNA seed sequence is shared by all three miRs and shown in red. Mutated nucleotides in the 3′UTR are shown in bold. B, C. Resveratrol and pterostilbene oppose PTEN 3′UTR targeting. Relative luciferase activity in DU145 cells co-transfected with wt PTEN 3′UTR along with either pre-miR-17, -20a or 106b and treated with resveratrol (B) or pterostrilbene (C) D. Co-transfections with mutated 3′UTR did not show any inhibitory effect on luciferase activity. MiR-negative#1 was used as a negative control (miR-NC). Values are normalized to Renilla luciferase activity and relative to 3′UTR/EV (Empty Vector) ratio which is set at 1. Data represent the mean ± SEM from four independent experiments. Comparisons between non-transfected and miR transfected samples (#) and vehicle and compound-treated samples (*) are shown. # p
Figure Legend Snippet: Resveratrol and pterostilbene rescue PTEN inhibition by oncomiRs -17, -20a and -106b A. Schematic representation of the predicted target sites of miRs-17, -20a and -106b in the 3′UTR of PTEN mRNA. The miRNA seed sequence is shared by all three miRs and shown in red. Mutated nucleotides in the 3′UTR are shown in bold. B, C. Resveratrol and pterostilbene oppose PTEN 3′UTR targeting. Relative luciferase activity in DU145 cells co-transfected with wt PTEN 3′UTR along with either pre-miR-17, -20a or 106b and treated with resveratrol (B) or pterostrilbene (C) D. Co-transfections with mutated 3′UTR did not show any inhibitory effect on luciferase activity. MiR-negative#1 was used as a negative control (miR-NC). Values are normalized to Renilla luciferase activity and relative to 3′UTR/EV (Empty Vector) ratio which is set at 1. Data represent the mean ± SEM from four independent experiments. Comparisons between non-transfected and miR transfected samples (#) and vehicle and compound-treated samples (*) are shown. # p

Techniques Used: Inhibition, Sequencing, Luciferase, Activity Assay, Transfection, Negative Control, Plasmid Preparation

Pterostilbene induces PTEN expression and apoptosis, inhibits tumor cell proliferation and downregulates circulating tumor-derived oncomiRs in vivo A. Representative H E and IHC images of PTEN, Ki-67 (proliferation); cleaved caspase-3 and M30 (apoptosis) staining in EV and miR-17/106a MIMIC xenografts upon pterostilbene treatment (magnification x100). B. Percent quantitation of Ki-67 and cleaved Caspase-3 staining is shown ( n = 3 per group). C. Pterostilbene downregulated oncomiRs in serum of xenograft mice. Quantitative RT-PCR analysis of circulating levels of miRs-17-5p and -106a-5p in sera ( n = 3 per group) from EV MIMIC and miR-17/106a MIMIC xenograft mice. Data represents the mean ± SEM of three independent experiments. Comparisons between EV and miR-overexpressing tumors (#) and vehicle and Pter-treated samples (*) is depicted. # p
Figure Legend Snippet: Pterostilbene induces PTEN expression and apoptosis, inhibits tumor cell proliferation and downregulates circulating tumor-derived oncomiRs in vivo A. Representative H E and IHC images of PTEN, Ki-67 (proliferation); cleaved caspase-3 and M30 (apoptosis) staining in EV and miR-17/106a MIMIC xenografts upon pterostilbene treatment (magnification x100). B. Percent quantitation of Ki-67 and cleaved Caspase-3 staining is shown ( n = 3 per group). C. Pterostilbene downregulated oncomiRs in serum of xenograft mice. Quantitative RT-PCR analysis of circulating levels of miRs-17-5p and -106a-5p in sera ( n = 3 per group) from EV MIMIC and miR-17/106a MIMIC xenograft mice. Data represents the mean ± SEM of three independent experiments. Comparisons between EV and miR-overexpressing tumors (#) and vehicle and Pter-treated samples (*) is depicted. # p

Techniques Used: Expressing, Derivative Assay, In Vivo, Immunohistochemistry, Staining, Quantitation Assay, Mouse Assay, Quantitative RT-PCR

Establishment and characterization of DU145 cells stably overexpressing miR-17/106a A. PTEN mRNA (left) and protein (right) were significantly decreased in miR-17/106a MIMIC compared to EV MIMIC cells. PTEN mRNA expression was detected by real time PCR and protein was detected by western blot. B. Resveratrol inhibits relative abundance of miRs-17 and -106a in EV and miR-17/106a MIMIC cells as detected by real time PCR. C, D. Resveratrol and pterostilbene enhanced expression of PTEN mRNA (C) and protein (D) in EV MIMIC and miR-17/106a MIMIC cells. Fold change in expression of miRNAs and mRNA was calculated by the 2 −ΔΔCt method. Data represent the mean ± SEM from at least three independent experiments. Quantitation of blots was performed using Image J software. Comparisons between non-transfected and miR transfected samples (#) and vehicle and compound-treated samples (*) are shown. # p
Figure Legend Snippet: Establishment and characterization of DU145 cells stably overexpressing miR-17/106a A. PTEN mRNA (left) and protein (right) were significantly decreased in miR-17/106a MIMIC compared to EV MIMIC cells. PTEN mRNA expression was detected by real time PCR and protein was detected by western blot. B. Resveratrol inhibits relative abundance of miRs-17 and -106a in EV and miR-17/106a MIMIC cells as detected by real time PCR. C, D. Resveratrol and pterostilbene enhanced expression of PTEN mRNA (C) and protein (D) in EV MIMIC and miR-17/106a MIMIC cells. Fold change in expression of miRNAs and mRNA was calculated by the 2 −ΔΔCt method. Data represent the mean ± SEM from at least three independent experiments. Quantitation of blots was performed using Image J software. Comparisons between non-transfected and miR transfected samples (#) and vehicle and compound-treated samples (*) are shown. # p

Techniques Used: Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay, Software, Transfection

23) Product Images from "Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf"

Article Title: Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf

Journal: Nature Communications

doi: 10.1038/s41467-018-07853-z

Aberrantly upregulated p19Arf antagonizes T FH differentiation and survival. a Diagram showing the structure of WT and mutant forms of p19Arf, with their capacity of interacting with Mdm2 and Bcl6 summarized. b p19Arf and Bcl6 interaction capacity. WT or mutant p19Arf was co-transfected with WT Bcl6 expression vector into 293T cells. Cell lysates were immunoprecipitated with anti-Bcl6 and then immunoblotted with anti-p19Arf. Data are representative of two experiments. c Impact of p19Arf on T FH differentiation and survival. In vivo primed WT Smarta CD4 + T cells were infected with EV -GFP retrovirus or that expressing WT or mutant p19Arf, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi (corresponding to day 5 after initial priming), CD45.2 + CD4 + T cells were detected for GFP expression (top panels), and GFP + cells were analyzed for AnnexinV positivity (middle) or CXCR5 + SLAM lo T FH cells (bottom panels). d Interplay of p19Arf and Bcl6 in T FH differentiation. WT Smarta CD4 + T cells were transduced with EV- GFP or ArfΔ14 retrovirus in combination with EV -mCherry or Bcl6 retrovirus, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi, GFP + mCherry + CD45.2 + CD4 + T cells were analyzed for frequency of CXCR5 + SLAM lo T FH cells. e – f Effect of genetically ablating p19Arf on T FH and B cell responses to protein immunization. WT, Ezh2 –/– , or Ezh2 –/– Arf –/– CD45.2 + Smarta CD4 + T cells were adoptively transferred into CD45.1 + Bcl6 –/– recipients followed by GP61-KLH immunization. On day 5 post-immunization, CXCR5 + T FH cells were detected in the draining LNs ( e ), and on day 8, KLH-specific IgG was detected in the sera by ELISA ( f ). Data are in ( c – f ) are means ± s.d. from ≥2 experiments. * p
Figure Legend Snippet: Aberrantly upregulated p19Arf antagonizes T FH differentiation and survival. a Diagram showing the structure of WT and mutant forms of p19Arf, with their capacity of interacting with Mdm2 and Bcl6 summarized. b p19Arf and Bcl6 interaction capacity. WT or mutant p19Arf was co-transfected with WT Bcl6 expression vector into 293T cells. Cell lysates were immunoprecipitated with anti-Bcl6 and then immunoblotted with anti-p19Arf. Data are representative of two experiments. c Impact of p19Arf on T FH differentiation and survival. In vivo primed WT Smarta CD4 + T cells were infected with EV -GFP retrovirus or that expressing WT or mutant p19Arf, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi (corresponding to day 5 after initial priming), CD45.2 + CD4 + T cells were detected for GFP expression (top panels), and GFP + cells were analyzed for AnnexinV positivity (middle) or CXCR5 + SLAM lo T FH cells (bottom panels). d Interplay of p19Arf and Bcl6 in T FH differentiation. WT Smarta CD4 + T cells were transduced with EV- GFP or ArfΔ14 retrovirus in combination with EV -mCherry or Bcl6 retrovirus, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi, GFP + mCherry + CD45.2 + CD4 + T cells were analyzed for frequency of CXCR5 + SLAM lo T FH cells. e – f Effect of genetically ablating p19Arf on T FH and B cell responses to protein immunization. WT, Ezh2 –/– , or Ezh2 –/– Arf –/– CD45.2 + Smarta CD4 + T cells were adoptively transferred into CD45.1 + Bcl6 –/– recipients followed by GP61-KLH immunization. On day 5 post-immunization, CXCR5 + T FH cells were detected in the draining LNs ( e ), and on day 8, KLH-specific IgG was detected in the sera by ELISA ( f ). Data are in ( c – f ) are means ± s.d. from ≥2 experiments. * p

Techniques Used: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, In Vivo, Infection, Adoptive Transfer Assay, Transduction, Enzyme-linked Immunosorbent Assay

Ezh2 acts upstream of Bcl6 induction to promote T FH differentiation. a – b Impact of Ezh2 deficiency on monoclonal CD4 + T cell responses. CD45.2 + Smarta CD4 + T cells from WT or Ezh2 –/– Smarta-Tg mice were adoptively transferred into congenic mice, followed by infection with LCMV-Arm. On 4 dpi, CXCR5 + SLAM lo ( a ) and CXCR5 + Bcl6 + T FH cells ( b ) were detected in recipient spleens. Contour plots are representative of ≥2 experiments, and cumulative data on frequency and numbers of each subset are means ± s.d. (each dot represents a mouse). c Detection of Bcl6 transcripts in T FH cells. CD45.2 + CXCR5 + SLAM lo T FH cells were sorted from the recipient spleens on 4 dpi as in ( a ), and Bcl6 transcript was detected by quantitative RT-PCR. d Detection of Ezh2 binding to the Bcl6 promoter. WT CD45.2 + T FH and T H 1 cells were sorted from recipient spleens on 5 dpi , and together with WT naïve CD4 + T cells, were analyzed by ChIP with anti-Ezh2 antibody or control IgG. Enriched Ezh2 binding at the TSSs of Bcl6 or Hprt1 genes was determined by ChIP-qPCR. Data in c and d are means ± s.d. from ≥2 experiments. e – f Impact of forced expression of Bcl6 on T FH and antibody responses. WT or Ezh2 –/– Smarta CD4 + T cells were primed in vivo for 24 h and infected with empty vector ( EV )-mCherry or Bcl6 -mCherry retrovirus. Transduced Smarta CD4 + T cells were adoptively transferred into congenic mice, followed by LCMV-Arm infection ( e ), or into Bcl6 –/– recipients, followed by KLH-GP61 immunization ( f ). In e , mCherry + CXCR5 + SLAM lo T FH cells were detected in the recipient spleens on 3 dpi (corresponding to day 5 after initial priming), with representative contour plots and cumulative data on frequency and numbers of mCherry + CXCR5 + SLAM lo T FH cells shown. In f , KLH-specific IgG was detected in the recipient sera on day 8 post-immunization. All cumulative data are means ± s.d. * p
Figure Legend Snippet: Ezh2 acts upstream of Bcl6 induction to promote T FH differentiation. a – b Impact of Ezh2 deficiency on monoclonal CD4 + T cell responses. CD45.2 + Smarta CD4 + T cells from WT or Ezh2 –/– Smarta-Tg mice were adoptively transferred into congenic mice, followed by infection with LCMV-Arm. On 4 dpi, CXCR5 + SLAM lo ( a ) and CXCR5 + Bcl6 + T FH cells ( b ) were detected in recipient spleens. Contour plots are representative of ≥2 experiments, and cumulative data on frequency and numbers of each subset are means ± s.d. (each dot represents a mouse). c Detection of Bcl6 transcripts in T FH cells. CD45.2 + CXCR5 + SLAM lo T FH cells were sorted from the recipient spleens on 4 dpi as in ( a ), and Bcl6 transcript was detected by quantitative RT-PCR. d Detection of Ezh2 binding to the Bcl6 promoter. WT CD45.2 + T FH and T H 1 cells were sorted from recipient spleens on 5 dpi , and together with WT naïve CD4 + T cells, were analyzed by ChIP with anti-Ezh2 antibody or control IgG. Enriched Ezh2 binding at the TSSs of Bcl6 or Hprt1 genes was determined by ChIP-qPCR. Data in c and d are means ± s.d. from ≥2 experiments. e – f Impact of forced expression of Bcl6 on T FH and antibody responses. WT or Ezh2 –/– Smarta CD4 + T cells were primed in vivo for 24 h and infected with empty vector ( EV )-mCherry or Bcl6 -mCherry retrovirus. Transduced Smarta CD4 + T cells were adoptively transferred into congenic mice, followed by LCMV-Arm infection ( e ), or into Bcl6 –/– recipients, followed by KLH-GP61 immunization ( f ). In e , mCherry + CXCR5 + SLAM lo T FH cells were detected in the recipient spleens on 3 dpi (corresponding to day 5 after initial priming), with representative contour plots and cumulative data on frequency and numbers of mCherry + CXCR5 + SLAM lo T FH cells shown. In f , KLH-specific IgG was detected in the recipient sera on day 8 post-immunization. All cumulative data are means ± s.d. * p

Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, In Vivo, Plasmid Preparation

24) Product Images from "Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency"

Article Title: Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2016.8

Intramuscular (i.m.) injections of plasmid (PT2/C-fluc) complexed with nonfrozen (nF) or freeze-thawed (F) LF2000 . ( a ) Mean luminescence readouts of different treatments + SD; n = 4; * P
Figure Legend Snippet: Intramuscular (i.m.) injections of plasmid (PT2/C-fluc) complexed with nonfrozen (nF) or freeze-thawed (F) LF2000 . ( a ) Mean luminescence readouts of different treatments + SD; n = 4; * P

Techniques Used: Plasmid Preparation

Evaluation of nonfrozen and freeze-thawed LF2000 plasmid transfection efficiency and toxicity by flow cytometry analysis and WST-1 assay . ( a ) Representative contour plots showing the % of GFP+ and PI+ cells together with the fluorescence intensity after GFP plasmid transfection using nonfrozen and freeze-thawed LF2000 in N2a cells. ( b–d ) GFP expression, cell viability, and median fluorescence intensity values for the dose-titrated plasmid ( n = 2; 24-well format; triplicates). ( e,f ) WST-1 toxicity assay 24 hours post-transfection with luc-plasmid in C2C12 ( e ) and N2a ( f ) cell-lines ( n = 2; 96-well format; quintuplicates). Statistical significance was evaluated by a two-tailed Student's t -test (n.s., not significant; *** P
Figure Legend Snippet: Evaluation of nonfrozen and freeze-thawed LF2000 plasmid transfection efficiency and toxicity by flow cytometry analysis and WST-1 assay . ( a ) Representative contour plots showing the % of GFP+ and PI+ cells together with the fluorescence intensity after GFP plasmid transfection using nonfrozen and freeze-thawed LF2000 in N2a cells. ( b–d ) GFP expression, cell viability, and median fluorescence intensity values for the dose-titrated plasmid ( n = 2; 24-well format; triplicates). ( e,f ) WST-1 toxicity assay 24 hours post-transfection with luc-plasmid in C2C12 ( e ) and N2a ( f ) cell-lines ( n = 2; 96-well format; quintuplicates). Statistical significance was evaluated by a two-tailed Student's t -test (n.s., not significant; *** P

Techniques Used: Plasmid Preparation, Transfection, Flow Cytometry, Cytometry, WST-1 Assay, Fluorescence, Expressing, Two Tailed Test

Transfection of splice-correcting U7 snRNA minicircle plasmid and splice-correcting oligonucleotides . ( a ) Luciferase expression of HeLa Luc705 cells after transfection with the indicated amount of plasmid in 96-well format. Luciferase activity was measured from cell lysates and normalized to total protein content. Mean values + s.d. from seven replicates are shown. ( b ) U7 asLuc705 snRNA levels after transfection, as measured by RT-qPCR. Cells were transfected in 24-well format. After RNA extraction, U7 asLuc705 snRNA expression was measured by qPCR and normalized to that of RNU24. Mean + SD from three replicates are shown. ( c ) Mean fold increase in luciferase signal 24 hours post-transfection of HeLa Luc705 cells with a 18-mer 2'- O- Me-PS splice-correcting oligonucleotide ( n = 2) ( d ) Representative fluorescence microscopy images of N2a cells 24 hours post-transfection with 100 nmol/l FAM-labeled 18-mer oligonucleotide using nonfrozen or freeze-thawed LF2000 (additional time points in Supplementary Figure S2 . Results in ( a ) and ( c ) are presented as fold increase over untreated cells. In all cases, statistical significance was evaluated by a two-tailed Student's t -test (n.s., not significant; * P
Figure Legend Snippet: Transfection of splice-correcting U7 snRNA minicircle plasmid and splice-correcting oligonucleotides . ( a ) Luciferase expression of HeLa Luc705 cells after transfection with the indicated amount of plasmid in 96-well format. Luciferase activity was measured from cell lysates and normalized to total protein content. Mean values + s.d. from seven replicates are shown. ( b ) U7 asLuc705 snRNA levels after transfection, as measured by RT-qPCR. Cells were transfected in 24-well format. After RNA extraction, U7 asLuc705 snRNA expression was measured by qPCR and normalized to that of RNU24. Mean + SD from three replicates are shown. ( c ) Mean fold increase in luciferase signal 24 hours post-transfection of HeLa Luc705 cells with a 18-mer 2'- O- Me-PS splice-correcting oligonucleotide ( n = 2) ( d ) Representative fluorescence microscopy images of N2a cells 24 hours post-transfection with 100 nmol/l FAM-labeled 18-mer oligonucleotide using nonfrozen or freeze-thawed LF2000 (additional time points in Supplementary Figure S2 . Results in ( a ) and ( c ) are presented as fold increase over untreated cells. In all cases, statistical significance was evaluated by a two-tailed Student's t -test (n.s., not significant; * P

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Expressing, Activity Assay, Quantitative RT-PCR, RNA Extraction, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy, Labeling, Two Tailed Test

Representative experiments comparing luc-plasmid transfection with nonfrozen versus freeze-thawed LF2000 in the following cell-lines: HEK293T ( a ), N2a ( b ), C2C12 myoblasts ( c ), C2C12 myotubes ( d ), hTERT MSCs ( e ), spinal muscular atrophy fibroblasts ( f ), and HepG2 ( g ). ( c–g ) Relative luminescence units (RLU) are depicted in log-scale. Each graph shows a representative experiment (mean + SD) in triplicates (24-well format; a,b,f , and g ) or quintuplicates (96-well format; c–e ).
Figure Legend Snippet: Representative experiments comparing luc-plasmid transfection with nonfrozen versus freeze-thawed LF2000 in the following cell-lines: HEK293T ( a ), N2a ( b ), C2C12 myoblasts ( c ), C2C12 myotubes ( d ), hTERT MSCs ( e ), spinal muscular atrophy fibroblasts ( f ), and HepG2 ( g ). ( c–g ) Relative luminescence units (RLU) are depicted in log-scale. Each graph shows a representative experiment (mean + SD) in triplicates (24-well format; a,b,f , and g ) or quintuplicates (96-well format; c–e ).

Techniques Used: Plasmid Preparation, Transfection

Analysis of nonfrozen (nF) and freeze-thawed (F) LF2000 lipoplexes by nanoparticle tracking analysis (NTA) ( a ), density gradient centrifugation ( b ), transmission electron microscopy (TEM) ( c ), TIRF ( d ), and TIRF-M ( e ). ( a ) NTA analysis of LF2000 alone or complexed with luc-plasmid at 21 °C. A significant difference ( P
Figure Legend Snippet: Analysis of nonfrozen (nF) and freeze-thawed (F) LF2000 lipoplexes by nanoparticle tracking analysis (NTA) ( a ), density gradient centrifugation ( b ), transmission electron microscopy (TEM) ( c ), TIRF ( d ), and TIRF-M ( e ). ( a ) NTA analysis of LF2000 alone or complexed with luc-plasmid at 21 °C. A significant difference ( P

Techniques Used: Gradient Centrifugation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Plasmid Preparation

25) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

Journal: Cell

doi: 10.1016/j.cell.2017.11.008

Deletion of YY1 Binding Sites Causes Loss of Enhancer-Promoter Interactions (A) Model depicting CRISPR/Cas9-mediated deletion of a YY1 binding motif in the regulatory region of a gene. (B and C) CRISPR/Cas9-mediated deletion of YY1 binding motifs in the regulatory regions of two genes, Raf1 (B) and Etv4 ), and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. .
Figure Legend Snippet: Deletion of YY1 Binding Sites Causes Loss of Enhancer-Promoter Interactions (A) Model depicting CRISPR/Cas9-mediated deletion of a YY1 binding motif in the regulatory region of a gene. (B and C) CRISPR/Cas9-mediated deletion of YY1 binding motifs in the regulatory regions of two genes, Raf1 (B) and Etv4 ), and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. .

Techniques Used: Binding Assay, CRISPR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

26) Product Images from "Self-assembled HCV core virus-like particles targeted and inhibited tumor cell migration and invasion"

Article Title: Self-assembled HCV core virus-like particles targeted and inhibited tumor cell migration and invasion

Journal: Nanoscale Research Letters

doi: 10.1186/1556-276X-8-401

Transcription and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) RT-PCR results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay.
Figure Legend Snippet: Transcription and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) RT-PCR results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay.

Techniques Used: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Infection, Synthesized, Quantitative RT-PCR, SYBR Green Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Marker, SDS Page, Western Blot

27) Product Images from "Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis"

Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

Journal: Molecular and cellular neurosciences

doi: 10.1016/j.mcn.2015.03.009

( A ) In-house QRT-PCR confirmation of the most significantly downregulated Complex I gene, Ndusf2 , confirms 4-month array data (5xFAD values expressed as fold-2 month wild type values, +/- SEM, n=5. ** p≤0.01). Reduced Ndusf2 expression correlates
Figure Legend Snippet: ( A ) In-house QRT-PCR confirmation of the most significantly downregulated Complex I gene, Ndusf2 , confirms 4-month array data (5xFAD values expressed as fold-2 month wild type values, +/- SEM, n=5. ** p≤0.01). Reduced Ndusf2 expression correlates

Techniques Used: Quantitative RT-PCR, Expressing

( A ) Increased aspa expression in 5xFAD brains at 2 months of age relative to wild type as assessed by QRT-PCR. Aspa expression presented as fold-wild type 2 month levels for each group, mean +/- SEM, n=5. Immunhistochemical evidence of increases in ASPA
Figure Legend Snippet: ( A ) Increased aspa expression in 5xFAD brains at 2 months of age relative to wild type as assessed by QRT-PCR. Aspa expression presented as fold-wild type 2 month levels for each group, mean +/- SEM, n=5. Immunhistochemical evidence of increases in ASPA

Techniques Used: Expressing, Quantitative RT-PCR

Whole brain homogenates analyzed by HPLC show a significant reduction in NAA from 2-4 months of age in 5xFAD brains ( 1A ; mean +/- SEM shown for each genotype at each age, n=7). QRT-PCR analysis of Nat8L expression 2-4 months of age showing a significant
Figure Legend Snippet: Whole brain homogenates analyzed by HPLC show a significant reduction in NAA from 2-4 months of age in 5xFAD brains ( 1A ; mean +/- SEM shown for each genotype at each age, n=7). QRT-PCR analysis of Nat8L expression 2-4 months of age showing a significant

Techniques Used: High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing

28) Product Images from "The Inhibitory Effect of Ojeoksan on Early and Advanced Atherosclerosis"

Article Title: The Inhibitory Effect of Ojeoksan on Early and Advanced Atherosclerosis

Journal: Nutrients

doi: 10.3390/nu10091256

Effects of OJS on miR-10a and miR-126 3p expression in the aorta of ApoE −/− mice. mRNA expression of adhesion molecules determined by real-time Reverse Transcription-PCR (RT-qPCR) analysis ( A ). Levels of miRNA determined by real-time RT-qPCR ( B ). Data are presented as means ± S.E. ** p
Figure Legend Snippet: Effects of OJS on miR-10a and miR-126 3p expression in the aorta of ApoE −/− mice. mRNA expression of adhesion molecules determined by real-time Reverse Transcription-PCR (RT-qPCR) analysis ( A ). Levels of miRNA determined by real-time RT-qPCR ( B ). Data are presented as means ± S.E. ** p

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

29) Product Images from "Development of gold nanoparticles biosensor for ultrasensitive diagnosis of foot and mouth disease virus"

Article Title: Development of gold nanoparticles biosensor for ultrasensitive diagnosis of foot and mouth disease virus

Journal: Journal of Nanobiotechnology

doi: 10.1186/s12951-018-0374-x

The amplification plot of FMDV isolates with classical rRT-PCR reagents and AuNPs-FMDV biosensor: the amplification plot is showing amplification of FMDV isolates with classical rRT-PCR reagents (yellow and violet) curves with CT values 7.03 and 15.9 and with AuNPs-FMDV biosensor (red and blue) curves with CT values 4.09 and 12.93
Figure Legend Snippet: The amplification plot of FMDV isolates with classical rRT-PCR reagents and AuNPs-FMDV biosensor: the amplification plot is showing amplification of FMDV isolates with classical rRT-PCR reagents (yellow and violet) curves with CT values 7.03 and 15.9 and with AuNPs-FMDV biosensor (red and blue) curves with CT values 4.09 and 12.93

Techniques Used: Amplification, Quantitative RT-PCR

30) Product Images from "Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells"

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-9-267

Validation of methylated targets in LNcaP and DU145 cells . A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of
Figure Legend Snippet: Validation of methylated targets in LNcaP and DU145 cells . A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of

Techniques Used: Methylation, Modification, Mass Spectrometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay

31) Product Images from "Differential Expression of Granulopoiesis Related Genes in Neutrophil Subsets Distinguished by Membrane Expression of CD177"

Article Title: Differential Expression of Granulopoiesis Related Genes in Neutrophil Subsets Distinguished by Membrane Expression of CD177

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099671

Expression of mRNA levels of graule proteins measured by taqman RT-PCR. Expression levels of mRNA of on left Y-axis defensin α1, defensin α3 (DEFA1, 3), and on right y-axis CD177, PR3 (PRTN3), and defensin α4 (DEFA4), BPI, and lipocalin 2 (LCN2) were measured by q-PCR. Expression levels of GP-related genes in CD177 + and CD177 − subsets sorted from healthy donors (n = 7) are depicted. *, P
Figure Legend Snippet: Expression of mRNA levels of graule proteins measured by taqman RT-PCR. Expression levels of mRNA of on left Y-axis defensin α1, defensin α3 (DEFA1, 3), and on right y-axis CD177, PR3 (PRTN3), and defensin α4 (DEFA4), BPI, and lipocalin 2 (LCN2) were measured by q-PCR. Expression levels of GP-related genes in CD177 + and CD177 − subsets sorted from healthy donors (n = 7) are depicted. *, P

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

32) Product Images from "Role of PU.1 in MHC Class II Expression via CIITA Transcription in Plasmacytoid Dendritic Cells"

Article Title: Role of PU.1 in MHC Class II Expression via CIITA Transcription in Plasmacytoid Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0154094

Effect of GM-CSF stimulation on the expression of PU.1, CIITA, and MHC class II, and on the recruitment of PU.1 to the pIII. A-H. Quantitative real-time PCR analysis of the mRNA expression of PU.1 (A, E), MHC class II (B, F), total CIITA (C, G), and CIITA driven by pIII (D, H) in mouse splenic pDCs (A-D) or CAL-1 cells (E-H) with (+) or without (-) GM-CSF stimulation. Mouse pDCs and CAL-1 cells were stimulated with 20 ng/ml mGM-CSF for 24h and 10 ng/ml hGM-CSF for 72h, respectively. I. The amount of PU.1 binding to the pIII in GM-CSF-stimulated CAL-1 cells (+) or control cells (-) was determined by a ChIP assay. Binding level of PU.1 (open bar) is expressed as fold change against that of control IgG (closed bar). In E-I, data represent means ± SEMs of three independent experiments performed with duplicate samples. In A-E, the results are expressed as means ± SEMs of triplicate samples, and similar result was obtained in another experiment. *, p
Figure Legend Snippet: Effect of GM-CSF stimulation on the expression of PU.1, CIITA, and MHC class II, and on the recruitment of PU.1 to the pIII. A-H. Quantitative real-time PCR analysis of the mRNA expression of PU.1 (A, E), MHC class II (B, F), total CIITA (C, G), and CIITA driven by pIII (D, H) in mouse splenic pDCs (A-D) or CAL-1 cells (E-H) with (+) or without (-) GM-CSF stimulation. Mouse pDCs and CAL-1 cells were stimulated with 20 ng/ml mGM-CSF for 24h and 10 ng/ml hGM-CSF for 72h, respectively. I. The amount of PU.1 binding to the pIII in GM-CSF-stimulated CAL-1 cells (+) or control cells (-) was determined by a ChIP assay. Binding level of PU.1 (open bar) is expressed as fold change against that of control IgG (closed bar). In E-I, data represent means ± SEMs of three independent experiments performed with duplicate samples. In A-E, the results are expressed as means ± SEMs of triplicate samples, and similar result was obtained in another experiment. *, p

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

PU.1 knockdown cancels GM-CSF-mediated increase of mRNAs. A-D. Quantitative real-time PCR analysis of the mRNA expression of PU.1 (A), HLA-DRα (B), total CIITA (C), and CIITA driven by pIII (D) in PU.1 siRNA (PU.1)- or control siRNA (cont.)-introduced CAL-1 cells. Cells were incubated for 24h after siRNA transfection and cultured for additional 72h with GM-CSF stimulation. The results are expressed as means ± SEMs of three independent experiments. Each experiment was performed with duplicate samples. *, p
Figure Legend Snippet: PU.1 knockdown cancels GM-CSF-mediated increase of mRNAs. A-D. Quantitative real-time PCR analysis of the mRNA expression of PU.1 (A), HLA-DRα (B), total CIITA (C), and CIITA driven by pIII (D) in PU.1 siRNA (PU.1)- or control siRNA (cont.)-introduced CAL-1 cells. Cells were incubated for 24h after siRNA transfection and cultured for additional 72h with GM-CSF stimulation. The results are expressed as means ± SEMs of three independent experiments. Each experiment was performed with duplicate samples. *, p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Incubation, Transfection, Cell Culture

33) Product Images from "Expression of Glycogen synthase kinase 3-? (GSK3-?) gene in azoospermic men"

Article Title: Expression of Glycogen synthase kinase 3-? (GSK3-?) gene in azoospermic men

Journal: Iranian Journal of Reproductive Medicine

doi:

Real time-PCR for GSK3-β; Total RNA was extracted; the standard reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit. Subsequent real time PCR was done with Power SYBR Green real time master mix and Stepone real time PCR (Applied Biosystems, Carlsbad, U.S.A.). GAPDH primer was used as a housekeeping gene. Each reaction was performed in triplicate. Expression of GSK3-β was down-regulated in non-obstructive azoospermia (3.10±0.19) compared with normal (7.12±0.39) and obstructive azoospermia (6.32±0.42) groups. The difference was significant (p=0.001)
Figure Legend Snippet: Real time-PCR for GSK3-β; Total RNA was extracted; the standard reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit. Subsequent real time PCR was done with Power SYBR Green real time master mix and Stepone real time PCR (Applied Biosystems, Carlsbad, U.S.A.). GAPDH primer was used as a housekeeping gene. Each reaction was performed in triplicate. Expression of GSK3-β was down-regulated in non-obstructive azoospermia (3.10±0.19) compared with normal (7.12±0.39) and obstructive azoospermia (6.32±0.42) groups. The difference was significant (p=0.001)

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

34) Product Images from "Combinatorial Action of miRNAs Regulates Transcriptional and Post-Transcriptional Gene Silencing following invivo PNS Injury"

Article Title: Combinatorial Action of miRNAs Regulates Transcriptional and Post-Transcriptional Gene Silencing following invivo PNS Injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039674

miRNAs induce post-transcriptional gene silencing through association with Ago-2 in functional complexes. (A). Protein expression of Ago-1, Ago-2 and Dicer in sciatic nerves before and after nerve injury to confirm the expression of the miRNA processing machinery proteins. Actin was used as a loading control. (B) Cytoplasmic lysates isolated from control sciatic nerves and injured distal segments (10 nerves each) were immunoprecipitated with Ago-2 antibody (Cell Signaling, USA) or IgG control. A portion (1/3 rd ) of the sample was used for each of the analysis and the experiment was repeated twice. Input, No-Antibody (No AB) and Ago-2 immunoprecipitated protein was analyzed by western blotting with Ago-2 antibody, which shows enrichment of Ago-2 in the 24 hour post-injury samples. (C). For RNA-IPs (RIPs), mRNAs that were co-immunoprecipitated with Ago-2 in vivo, were reverse transcribed using oligo-dT primer and genes of interest were PCR amplified using gene-specific primers (Krox-20 = 1274 bp, C-Jun = 689 bp, Nanog = 753 bp, QKI-6 = 1345 bp, Sox-2 = 958 bp and ID-2 = 588 bp). (D). microRNAs that were co-immunoprecipitated with Ago-2 in complex with their targeted mRNAs, were reverse-transcribed with microRNA specific RT-primers using Multiscribe RT kit. Ago-2 associated microRNAs were detected by real time qPCR with miRNA specific Taqman probe/primer sets. Data were normalized to input and an internal control. Fold difference (2 -ΔΔCT ) in the association of individual microRNAs with Ago-2 protein between injured and control nerves was plotted as log 2 median ratio and error is expressed as standard deviation.
Figure Legend Snippet: miRNAs induce post-transcriptional gene silencing through association with Ago-2 in functional complexes. (A). Protein expression of Ago-1, Ago-2 and Dicer in sciatic nerves before and after nerve injury to confirm the expression of the miRNA processing machinery proteins. Actin was used as a loading control. (B) Cytoplasmic lysates isolated from control sciatic nerves and injured distal segments (10 nerves each) were immunoprecipitated with Ago-2 antibody (Cell Signaling, USA) or IgG control. A portion (1/3 rd ) of the sample was used for each of the analysis and the experiment was repeated twice. Input, No-Antibody (No AB) and Ago-2 immunoprecipitated protein was analyzed by western blotting with Ago-2 antibody, which shows enrichment of Ago-2 in the 24 hour post-injury samples. (C). For RNA-IPs (RIPs), mRNAs that were co-immunoprecipitated with Ago-2 in vivo, were reverse transcribed using oligo-dT primer and genes of interest were PCR amplified using gene-specific primers (Krox-20 = 1274 bp, C-Jun = 689 bp, Nanog = 753 bp, QKI-6 = 1345 bp, Sox-2 = 958 bp and ID-2 = 588 bp). (D). microRNAs that were co-immunoprecipitated with Ago-2 in complex with their targeted mRNAs, were reverse-transcribed with microRNA specific RT-primers using Multiscribe RT kit. Ago-2 associated microRNAs were detected by real time qPCR with miRNA specific Taqman probe/primer sets. Data were normalized to input and an internal control. Fold difference (2 -ΔΔCT ) in the association of individual microRNAs with Ago-2 protein between injured and control nerves was plotted as log 2 median ratio and error is expressed as standard deviation.

Techniques Used: Functional Assay, Expressing, Isolation, Immunoprecipitation, Western Blot, In Vivo, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Standard Deviation

35) Product Images from "Antioxidant effects of fucoxanthin rich powder in rats fed with high fat diet"

Article Title: Antioxidant effects of fucoxanthin rich powder in rats fed with high fat diet

Journal: Nutrition Research and Practice

doi: 10.4162/nrp.2013.7.6.475

Effect of fucoxanthin rich powder on mRNA expression of transcription factor and enzymes related to antioxidant system in the liver of rats. Total RNA was isolated using TRI-reagenet, and cDNA was synthesized using 3 ug of total RNA with SuperScript II reverse transcriptase. Realtime PCR was performed using SYBR green and standard procedures to assess the mRNA expression of the primer in liver samples obtained from each group. An Applied Biosystem StepOne softwere v2.1 was used. Each bar represents the mean ± S.E of three independent experiments. Different letters above each bar indicate significant differences among the groups at α = 0.05 as determined by Duncan's multiple range tests. HO-1: heme oxygenase (decycling) 1, Nrf2: nuclear factor, erythroid derived 2, like 2, NQO-1: NAD(P)H quinone oxidoreductase 1.
Figure Legend Snippet: Effect of fucoxanthin rich powder on mRNA expression of transcription factor and enzymes related to antioxidant system in the liver of rats. Total RNA was isolated using TRI-reagenet, and cDNA was synthesized using 3 ug of total RNA with SuperScript II reverse transcriptase. Realtime PCR was performed using SYBR green and standard procedures to assess the mRNA expression of the primer in liver samples obtained from each group. An Applied Biosystem StepOne softwere v2.1 was used. Each bar represents the mean ± S.E of three independent experiments. Different letters above each bar indicate significant differences among the groups at α = 0.05 as determined by Duncan's multiple range tests. HO-1: heme oxygenase (decycling) 1, Nrf2: nuclear factor, erythroid derived 2, like 2, NQO-1: NAD(P)H quinone oxidoreductase 1.

Techniques Used: Expressing, Isolation, Synthesized, Polymerase Chain Reaction, SYBR Green Assay, Derivative Assay

36) Product Images from "Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia"

Article Title: Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia

Journal: Nature Communications

doi: 10.1038/s41467-017-00479-7

Deletion of MCS-R2 using CRISPR/Cas9 genome editing in human CD34+ cells. a Schematic of sgRNA target sites. Four sgRNAs (Cr1, Cr2, Cr9, and Cr10) were designed to target the 5′ end of the MCS-R2 core element, whereas three sgRNAs (Cr7, Cr8, and Cr12) target the 3′ end. b Representative flow cytometry plots showing GFP expression and forward scatter (FS) after gating for live cells in non-transfection control (NTC), Cas9 control (C9), and CRISPR/Cas9 plasmid pair-transfected cells. Orange : GFP negative, blue : GFP positive (low), and green : GFP positive (high). Mean and SD of the percentage of cells within each region is indicated ( n = 3). Gating strategy is shown in Supplementary Fig. 10a . c Representative gel electrophoresis image of genomic DNA extracted from cells targeted by four CRISPR/Cas9 plasmid pairs analyzed by PCR. d Gene editing deletion induction efficiency as measured independently by percentages of mutated alleles determined by band size in end-point PCR and subsequent Sanger sequence analysis and by determining inverse of proportions of amplicons inside: outside deletion (amplicons of same length) by multiplexed droplet digital PCR; mean values are presented and error bars represent SD ( n = 3). e Characterization of deletion break points by sequencing (co-ordinates from Hum Mar 2006 (NCBI36/hg18) assembly). f α-and β-globin gene expression normalized to the expression of RPL13A and α/β-globin mRNA ratios relative to Cas9 control (C9) analyzed by qPCR; error bars represent SD ( n = 3); * P
Figure Legend Snippet: Deletion of MCS-R2 using CRISPR/Cas9 genome editing in human CD34+ cells. a Schematic of sgRNA target sites. Four sgRNAs (Cr1, Cr2, Cr9, and Cr10) were designed to target the 5′ end of the MCS-R2 core element, whereas three sgRNAs (Cr7, Cr8, and Cr12) target the 3′ end. b Representative flow cytometry plots showing GFP expression and forward scatter (FS) after gating for live cells in non-transfection control (NTC), Cas9 control (C9), and CRISPR/Cas9 plasmid pair-transfected cells. Orange : GFP negative, blue : GFP positive (low), and green : GFP positive (high). Mean and SD of the percentage of cells within each region is indicated ( n = 3). Gating strategy is shown in Supplementary Fig. 10a . c Representative gel electrophoresis image of genomic DNA extracted from cells targeted by four CRISPR/Cas9 plasmid pairs analyzed by PCR. d Gene editing deletion induction efficiency as measured independently by percentages of mutated alleles determined by band size in end-point PCR and subsequent Sanger sequence analysis and by determining inverse of proportions of amplicons inside: outside deletion (amplicons of same length) by multiplexed droplet digital PCR; mean values are presented and error bars represent SD ( n = 3). e Characterization of deletion break points by sequencing (co-ordinates from Hum Mar 2006 (NCBI36/hg18) assembly). f α-and β-globin gene expression normalized to the expression of RPL13A and α/β-globin mRNA ratios relative to Cas9 control (C9) analyzed by qPCR; error bars represent SD ( n = 3); * P

Techniques Used: CRISPR, Flow Cytometry, Cytometry, Expressing, Transfection, Plasmid Preparation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Digital PCR, Real-time Polymerase Chain Reaction

37) Product Images from "Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro"

Article Title: Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro

Journal: Journal of Orthopaedic Surgery and Research

doi: 10.1186/s13018-018-0800-7

Effect of alcohol on primary cultured MSC integrin β1 mRNA and protein expression. Primary rat MSC were cultured in media alone or media plus 50 mM ethanol for 24 h. Cells were harvested and used for a mRNA or b total protein isolation as described. Int β1 mRNA levels were assessed by qRT-PCR as described. Int β1 protein levels were assessed by western blot analysis as described. a mRNA: media vs EtOH p = 0.0021. b Protein: media vs EtOH p = 0.0030. Each experiment was repeated at least three times utilizing unique primary MSC cultures. * p
Figure Legend Snippet: Effect of alcohol on primary cultured MSC integrin β1 mRNA and protein expression. Primary rat MSC were cultured in media alone or media plus 50 mM ethanol for 24 h. Cells were harvested and used for a mRNA or b total protein isolation as described. Int β1 mRNA levels were assessed by qRT-PCR as described. Int β1 protein levels were assessed by western blot analysis as described. a mRNA: media vs EtOH p = 0.0021. b Protein: media vs EtOH p = 0.0030. Each experiment was repeated at least three times utilizing unique primary MSC cultures. * p

Techniques Used: Cell Culture, Expressing, Isolation, Quantitative RT-PCR, Western Blot

38) Product Images from "STING-associated vasculopathy develops independently of IRF3 in mice"

Article Title: STING-associated vasculopathy develops independently of IRF3 in mice

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20171351

STING N153S triggers perivascular pulmonary inflammation, myeloid cell expansion, and T cell cytopenia independently of IRF3. (A) Expression of ISGs in STING N153S, STING knockout ( Sting −/− ), and Trex1 −/− MEFs as well as STING N153S and Sting −/− spleens. Each data point represents a unique ISG as fold increase compared with a WT littermate control. Data were assembled using ISG expression values from RNA-sequencing. (B) qRT-PCR analysis of ISGs in WT, STING N153S, Sting −/− MEFs. mRNA expression is normalized to WT. (C) ISG expression in two healthy control (HC) and SAVI patient skin fibroblasts. Data represent a selection of ISGs assembled from RNA-sequencing. (D) qRT-PCR analysis of ISGs in skin fibroblasts. Data are from two independent experiments with two technical replicates per ISG, normalized to HC. (E) Hematoxylin and eosin images of lungs from Irf3 −/− and Irf3 −/− STING N153S mice (4–6 mo of age). Pulmonary vessels are indicated (v) with black arrows highlighting perivascular immune cell infiltration. Pulmonary vessels are indicated (v) with arrows. Bar, 100 µm. (F) Quantitation of perivascular lung lesions in STING N153S ( n = 7) and Irf3 −/− N153S ( n = 4) mice. Results in F represent the mean ± SEM of data collected and analyzed in two independent experiments. (G) ISG mRNA expression in Irf3 −/− and Irf3 −/− N153S MEFs normalized to gene expression in Irf3 −/− MEFs. Data represent the mean ± SEM from two independent experiments with n = 3 biological replicates per genotype. (H–J) Representative dot plots (H) and quantification (I and J) of splenic myeloid cell populations from WT, STING N153S, Irf3 −/− , and Irf3 −/− STING N153S mice. Data represent the mean of two independent experiments with n = 4 or 5 mice per genotype. (K and L) Percentage (K) and total number (L) of CD8 + splenocytes from WT, STING N153S, Irf3 −/− , and Irf3 −/− STING N153S mice. Data were pooled from two independent experiments with n = 4 or 5 mice per genotype. ns, not significant; *, P
Figure Legend Snippet: STING N153S triggers perivascular pulmonary inflammation, myeloid cell expansion, and T cell cytopenia independently of IRF3. (A) Expression of ISGs in STING N153S, STING knockout ( Sting −/− ), and Trex1 −/− MEFs as well as STING N153S and Sting −/− spleens. Each data point represents a unique ISG as fold increase compared with a WT littermate control. Data were assembled using ISG expression values from RNA-sequencing. (B) qRT-PCR analysis of ISGs in WT, STING N153S, Sting −/− MEFs. mRNA expression is normalized to WT. (C) ISG expression in two healthy control (HC) and SAVI patient skin fibroblasts. Data represent a selection of ISGs assembled from RNA-sequencing. (D) qRT-PCR analysis of ISGs in skin fibroblasts. Data are from two independent experiments with two technical replicates per ISG, normalized to HC. (E) Hematoxylin and eosin images of lungs from Irf3 −/− and Irf3 −/− STING N153S mice (4–6 mo of age). Pulmonary vessels are indicated (v) with black arrows highlighting perivascular immune cell infiltration. Pulmonary vessels are indicated (v) with arrows. Bar, 100 µm. (F) Quantitation of perivascular lung lesions in STING N153S ( n = 7) and Irf3 −/− N153S ( n = 4) mice. Results in F represent the mean ± SEM of data collected and analyzed in two independent experiments. (G) ISG mRNA expression in Irf3 −/− and Irf3 −/− N153S MEFs normalized to gene expression in Irf3 −/− MEFs. Data represent the mean ± SEM from two independent experiments with n = 3 biological replicates per genotype. (H–J) Representative dot plots (H) and quantification (I and J) of splenic myeloid cell populations from WT, STING N153S, Irf3 −/− , and Irf3 −/− STING N153S mice. Data represent the mean of two independent experiments with n = 4 or 5 mice per genotype. (K and L) Percentage (K) and total number (L) of CD8 + splenocytes from WT, STING N153S, Irf3 −/− , and Irf3 −/− STING N153S mice. Data were pooled from two independent experiments with n = 4 or 5 mice per genotype. ns, not significant; *, P

Techniques Used: Expressing, Knock-Out, RNA Sequencing Assay, Quantitative RT-PCR, Selection, Mouse Assay, Quantitation Assay

39) Product Images from "Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways"

Article Title: Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-18-74

Cpc attenuated α-MSH-stimulated melanogenesis and elevated the abundance of intracellular cAMP . Cells were pretreated with 20 nM α-MSH for 30 mins, and then treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Tyrosinase activity (black) and melanin content (grey) were measured. (B) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. (C) The cAMP concentration was measured by enzyme immunoassay at assigned time intervals (10, 30, 60 min) after Cpc treatment. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P
Figure Legend Snippet: Cpc attenuated α-MSH-stimulated melanogenesis and elevated the abundance of intracellular cAMP . Cells were pretreated with 20 nM α-MSH for 30 mins, and then treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Tyrosinase activity (black) and melanin content (grey) were measured. (B) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. (C) The cAMP concentration was measured by enzyme immunoassay at assigned time intervals (10, 30, 60 min) after Cpc treatment. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P

Techniques Used: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

Effect of Cpc on cAMP/MAPK/ERK pathway and MITF expression at protein and mRNA levels . Immunoblot analysis was performed with cell extract proteins treated with (A) Cpc (0.1 mg/mL) at assigned time intervals for ERK1/2 (control (black); CPC-treated (grey)), and (B) different Cpc concentration (0.05, 0.1, 0.2 mg/mL) at 540 min for MEK. (C) Cell extract proteins at assigned time intervals treated with Cpc (0.1 mg/mL) were examined by Immunoblot analysis for MITF using β-actin as internal standards (control (black); CPC-treated (grey)). (D) Different levels of Cpc (0.05, 0.1, 0.2 mg/mL) treated MITF mRNA were analyzed by Q-PCR at 540 min. (E) Immunoblot analysis treated with Cpc (0.1 mg/mL), PD98059 (PD, 20 μM), and Cpc+PD at 72 hrs were performed for the evaluation of MITF and tyrosinase expression (MITF (black); tyrosinase (grey)). Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P
Figure Legend Snippet: Effect of Cpc on cAMP/MAPK/ERK pathway and MITF expression at protein and mRNA levels . Immunoblot analysis was performed with cell extract proteins treated with (A) Cpc (0.1 mg/mL) at assigned time intervals for ERK1/2 (control (black); CPC-treated (grey)), and (B) different Cpc concentration (0.05, 0.1, 0.2 mg/mL) at 540 min for MEK. (C) Cell extract proteins at assigned time intervals treated with Cpc (0.1 mg/mL) were examined by Immunoblot analysis for MITF using β-actin as internal standards (control (black); CPC-treated (grey)). (D) Different levels of Cpc (0.05, 0.1, 0.2 mg/mL) treated MITF mRNA were analyzed by Q-PCR at 540 min. (E) Immunoblot analysis treated with Cpc (0.1 mg/mL), PD98059 (PD, 20 μM), and Cpc+PD at 72 hrs were performed for the evaluation of MITF and tyrosinase expression (MITF (black); tyrosinase (grey)). Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P

Techniques Used: Expressing, Concentration Assay, Polymerase Chain Reaction

Effect of Cpc on viability of B16F10 melanoma cell, tyrosinase activity and melanin contents . Cells were treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Cell viability was determined by MTT assay as described in Materials and Methods. (B) Tyrosinase activity (black) and melanin content (grey) were measured. (C) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P
Figure Legend Snippet: Effect of Cpc on viability of B16F10 melanoma cell, tyrosinase activity and melanin contents . Cells were treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Cell viability was determined by MTT assay as described in Materials and Methods. (B) Tyrosinase activity (black) and melanin content (grey) were measured. (C) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P

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

40) Product Images from "KPNA2 promotes metabolic reprogramming in glioblastomas by regulation of c-myc"

Article Title: KPNA2 promotes metabolic reprogramming in glioblastomas by regulation of c-myc

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0861-9

KPNA2 promoted the glycolytic metabolism in the glioblastoma cells. a Levels of KPNA2 were analyzed by qRT-PCR and immunoblotting in the U87 glioblastoma cells transfected with the lentiviruses expressing small hairpin RNAs of KPNA2 (shKPNA2), wild-type KPNA2 (KPNA2), or a vector with scrambled nonspecific shRNAs(SC). NC is for the original U87 cells that without any handles, GAPDH served as a loading control. b mRNA levels and c enzymatic activities of HK2, PKM2 and PFK1 were determined in the U87 cells transfected with KPNA2-shRNA(siKPNA2), scrambled shRNA(SC) or wild-type KPNA2(KPNA2). Each bar represented the mean ± s.d. from three independent experiments. * P
Figure Legend Snippet: KPNA2 promoted the glycolytic metabolism in the glioblastoma cells. a Levels of KPNA2 were analyzed by qRT-PCR and immunoblotting in the U87 glioblastoma cells transfected with the lentiviruses expressing small hairpin RNAs of KPNA2 (shKPNA2), wild-type KPNA2 (KPNA2), or a vector with scrambled nonspecific shRNAs(SC). NC is for the original U87 cells that without any handles, GAPDH served as a loading control. b mRNA levels and c enzymatic activities of HK2, PKM2 and PFK1 were determined in the U87 cells transfected with KPNA2-shRNA(siKPNA2), scrambled shRNA(SC) or wild-type KPNA2(KPNA2). Each bar represented the mean ± s.d. from three independent experiments. * P

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

41) Product Images from "Involvement of Interleukin-6 and Androgen Receptor Signaling in Pancreatic Cancer"

Article Title: Involvement of Interleukin-6 and Androgen Receptor Signaling in Pancreatic Cancer

Journal: Genes & Cancer

doi: 10.1177/1947601910383417

Migration of pancreatic cancer cell lines was investigated using a wound-healing scratch assay ( A-E ). Knockdown of AR suppressed migration of pancreatic cancer cells in the presence of IL-6 and DHT ( F-H ). KP-2 ( A ), SUIT-2 ( B ), Panc-1 ( C ), AsPC-1 ( D ), and MIAPaCa-2 ( E ) cells were subjected to wound-healing scratch assay. Cells were incubated with or without IL-6 (100 ng/mL) and/or DHT (10 nM) for approximately 24 hours. The migration thus observed was presented as a ratio of migration, considering migration in untreated control as 1. Scion images were used for analysis. The error bars represent mean ± SD of 3 independent experiments. Results of semiquantitative RT-PCR analysis for AR and GAPDH mRNAs are shown in the lower part below each graph ( A-E ). KP-2 cells were transfected with 10 nM AR siRNA (si-AR) or 10 nM control siRNA (si-C) (Thermo Fisher Scientific Dharmacon), subjected to wound-healing scratch assay, and incubated with IL-6 (100 ng/mL) and DHT (10 nM) for 24 hours. ( F ) Light microscopy pictures show KP-2 cells transfected with 10 nM si-C or 10 nM si-AR and incubated with 100 ng/mL IL-6 and 10 nM DHT at 0 hours (left panel) and 24 hours later (right panels). ( G ) The migration observed was presented as a ratio of migration, considering migration in transfection with si-C as 1. The error bars represent mean ± SD of 3 independent experiments. ( H ) Knockdown of AR expression in KP-2 cells. Whole cell lysates from KP-2 cells after si-C or si-AR treatment were immunoblotted with antibodies against AR or GAPDH.
Figure Legend Snippet: Migration of pancreatic cancer cell lines was investigated using a wound-healing scratch assay ( A-E ). Knockdown of AR suppressed migration of pancreatic cancer cells in the presence of IL-6 and DHT ( F-H ). KP-2 ( A ), SUIT-2 ( B ), Panc-1 ( C ), AsPC-1 ( D ), and MIAPaCa-2 ( E ) cells were subjected to wound-healing scratch assay. Cells were incubated with or without IL-6 (100 ng/mL) and/or DHT (10 nM) for approximately 24 hours. The migration thus observed was presented as a ratio of migration, considering migration in untreated control as 1. Scion images were used for analysis. The error bars represent mean ± SD of 3 independent experiments. Results of semiquantitative RT-PCR analysis for AR and GAPDH mRNAs are shown in the lower part below each graph ( A-E ). KP-2 cells were transfected with 10 nM AR siRNA (si-AR) or 10 nM control siRNA (si-C) (Thermo Fisher Scientific Dharmacon), subjected to wound-healing scratch assay, and incubated with IL-6 (100 ng/mL) and DHT (10 nM) for 24 hours. ( F ) Light microscopy pictures show KP-2 cells transfected with 10 nM si-C or 10 nM si-AR and incubated with 100 ng/mL IL-6 and 10 nM DHT at 0 hours (left panel) and 24 hours later (right panels). ( G ) The migration observed was presented as a ratio of migration, considering migration in transfection with si-C as 1. The error bars represent mean ± SD of 3 independent experiments. ( H ) Knockdown of AR expression in KP-2 cells. Whole cell lysates from KP-2 cells after si-C or si-AR treatment were immunoblotted with antibodies against AR or GAPDH.

Techniques Used: Migration, Wound Healing Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Transfection, Light Microscopy, Expressing

AR and IL-6R expression in pancreatic cancer cell lines. ( A ) Relative expression of AR mRNA. RNAs from MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2 cells, and LNCaP (AR-positive) cells were subjected to real-time RT-PCR for AR and GAPDH gene expression. AR/GAPDH mRNA in MIAPaCa-2 was set as 1-fold. The results were based on the relative expression level of the housekeeping gene GAPDH as an internal control. ( B ) IL-6Rα, GP130, AR, and GAPDH protein expression. Total cell lysates (5 µg) of MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2, and LNCaP were subjected to 10% SDS-PAGE for immunoblotting with specific antibodies against IL-6Rα (Santa Cruz Biotechnology), GP130 (Cell Signaling Technology), AR (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology).
Figure Legend Snippet: AR and IL-6R expression in pancreatic cancer cell lines. ( A ) Relative expression of AR mRNA. RNAs from MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2 cells, and LNCaP (AR-positive) cells were subjected to real-time RT-PCR for AR and GAPDH gene expression. AR/GAPDH mRNA in MIAPaCa-2 was set as 1-fold. The results were based on the relative expression level of the housekeeping gene GAPDH as an internal control. ( B ) IL-6Rα, GP130, AR, and GAPDH protein expression. Total cell lysates (5 µg) of MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2, and LNCaP were subjected to 10% SDS-PAGE for immunoblotting with specific antibodies against IL-6Rα (Santa Cruz Biotechnology), GP130 (Cell Signaling Technology), AR (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology).

Techniques Used: Expressing, Quantitative RT-PCR, SDS Page

42) Product Images from "Insight into the role of PIKK family members and NF-кB in DNAdamage-induced senescence and senescence-associated secretory phenotype of colon cancer cells"

Article Title: Insight into the role of PIKK family members and NF-кB in DNAdamage-induced senescence and senescence-associated secretory phenotype of colon cancer cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-017-0069-5

The impact of simultaneous ATM, ATR, and DNA-PKc downregulation on senescence-associated secretory phenotype RT-PCR analysis of expression of selected senescence-associated secretory phenotype genes ( IL-8 , VEGF , RANTES ) in cells transfected either with negative siRNA or with combination of siRNAs targeting ATM , ATR , and PRKDC , treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Normalized relative expression levels are shown (mean value of three independent experiments). Bars represent standard deviation
Figure Legend Snippet: The impact of simultaneous ATM, ATR, and DNA-PKc downregulation on senescence-associated secretory phenotype RT-PCR analysis of expression of selected senescence-associated secretory phenotype genes ( IL-8 , VEGF , RANTES ) in cells transfected either with negative siRNA or with combination of siRNAs targeting ATM , ATR , and PRKDC , treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Normalized relative expression levels are shown (mean value of three independent experiments). Bars represent standard deviation

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Standard Deviation

The influence of downregulation of gene RELA (encoding NF-κB p65 subunit) on senescence of HCT116 p53+/+ and p53−/− cells HCT116 p53+/+ and p53−/− cells were transfected with negative siRNA or RELA siRNA (30 nM). Two days after transfection, the cells were treated with doxorubicin (100 nM) for 5 days. a Whole cell extracts were prepared at indicated time points after treatment with doxorubicin. The level of p65 and p21 proteins was estimated by Western blotting; GAPDH was used as a loading control. b ELISA analysis of of IL-8 secreted by HCT116 p53+/+ (left graph) and p53−/− cells (right graph) transfected with negative siRNA or with siRNA targeting RELA on days 1 and 5 of doxorubicin treatment; mean value of three independent experiments. Error bars represent standard deviation. c RT-PCR analysis of expression of IL-8 in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. d RT-PCR analysis of expression of selected NF-κB-regulated genes ( RANTES and GROα ) in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. e SA-β-Gal activity measured by flow cytometry. Representative histograms illustrating C 12 FDG fluorescence in HCT116 p53+/+ (left) and p53−/− (right) cells
Figure Legend Snippet: The influence of downregulation of gene RELA (encoding NF-κB p65 subunit) on senescence of HCT116 p53+/+ and p53−/− cells HCT116 p53+/+ and p53−/− cells were transfected with negative siRNA or RELA siRNA (30 nM). Two days after transfection, the cells were treated with doxorubicin (100 nM) for 5 days. a Whole cell extracts were prepared at indicated time points after treatment with doxorubicin. The level of p65 and p21 proteins was estimated by Western blotting; GAPDH was used as a loading control. b ELISA analysis of of IL-8 secreted by HCT116 p53+/+ (left graph) and p53−/− cells (right graph) transfected with negative siRNA or with siRNA targeting RELA on days 1 and 5 of doxorubicin treatment; mean value of three independent experiments. Error bars represent standard deviation. c RT-PCR analysis of expression of IL-8 in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. d RT-PCR analysis of expression of selected NF-κB-regulated genes ( RANTES and GROα ) in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. e SA-β-Gal activity measured by flow cytometry. Representative histograms illustrating C 12 FDG fluorescence in HCT116 p53+/+ (left) and p53−/− (right) cells

Techniques Used: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Expressing, Activity Assay, Flow Cytometry, Cytometry, Fluorescence

43) Product Images from "A dosage-dependent pleiotropic role of Dicer in prostate cancer growth and metastasis"

Article Title: A dosage-dependent pleiotropic role of Dicer in prostate cancer growth and metastasis

Journal: Oncogene

doi: 10.1038/onc.2013.281

Disrupting Dicer activity inhibits disease progression in the Pten null mouse model for prostate cancer (A) Quantitative PCR analyses of ratio of exon24/exon21 in genomic DNAs from 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. (B) qRT-PCR analysis of 6 representative microRNAs in 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. *: p
Figure Legend Snippet: Disrupting Dicer activity inhibits disease progression in the Pten null mouse model for prostate cancer (A) Quantitative PCR analyses of ratio of exon24/exon21 in genomic DNAs from 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. (B) qRT-PCR analysis of 6 representative microRNAs in 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. *: p

Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Mouse Assay, Quantitative RT-PCR

44) Product Images from "Unraveling the regulatory connections between two controllers of breast cancer cell fate"

Article Title: Unraveling the regulatory connections between two controllers of breast cancer cell fate

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku360

ERα–GATA3 network in breast cancer cells. The signs of autoregulatory and cross-regulatory connections in the ERα-GATA3 network are unknown and can be either positive (+), negative (−) or null ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\emptyset$\end{document} ).
Figure Legend Snippet: ERα–GATA3 network in breast cancer cells. The signs of autoregulatory and cross-regulatory connections in the ERα-GATA3 network are unknown and can be either positive (+), negative (−) or null ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\emptyset$\end{document} ).

Techniques Used:

Model fitting constrains the topology with ERα negative autoregulation such that its strength becomes negligible (A) Strength of ERα autoregulation in the best fit case for the three possible autoregulation topologies—negative, null and positive. ( B) Frequency histograms of ERα autoregulation strengths for topologies with negative (solid) and positive (open) autoregulation.
Figure Legend Snippet: Model fitting constrains the topology with ERα negative autoregulation such that its strength becomes negligible (A) Strength of ERα autoregulation in the best fit case for the three possible autoregulation topologies—negative, null and positive. ( B) Frequency histograms of ERα autoregulation strengths for topologies with negative (solid) and positive (open) autoregulation.

Techniques Used:

ERα is not autoregulated. (A) Computationally generated dose–response curves depicting the change in steady-state ERα mRNA levels with increasing concentrations of ICI for topologies with positive (dark gray) and without (black) autoregulation. For each topology the dose–response curves for the best 50 parameter sets are plotted. ( B) ERα mRNA levels in the T47D cell line after 24 h incubation with different concentrations of ICI ( n = 3). Note that only the 0.1 nM measurement is significantly different ( P
Figure Legend Snippet: ERα is not autoregulated. (A) Computationally generated dose–response curves depicting the change in steady-state ERα mRNA levels with increasing concentrations of ICI for topologies with positive (dark gray) and without (black) autoregulation. For each topology the dose–response curves for the best 50 parameter sets are plotted. ( B) ERα mRNA levels in the T47D cell line after 24 h incubation with different concentrations of ICI ( n = 3). Note that only the 0.1 nM measurement is significantly different ( P

Techniques Used: Generated, Incubation

GATA3 positively regulates its own expression. ( A ) Protein depletion and protein recovery time courses monitored by western blotting (representative blot, n = 3 replicates). (B and D) Best model fits for topologies with negative ( B ), positive ( D ) or without ( C ) ERα autoregulation. Complete topologies with GATA3 autoregulation and cross-regulations are shown at the top of each column. Regular and blunt arrowheads depict positive and negative regulation. The open and solid circles in each panel indicate experimentally-measured ERα and GATA3 concentrations, obtained from panel (A) by western blot quantification. The continuous lines indicate model fits for protein depletion (top half) and recovery (bottom half) experiments. E is the error value of the fit. Model parameters for the three topologies in panels B–D are provided in Table S1.
Figure Legend Snippet: GATA3 positively regulates its own expression. ( A ) Protein depletion and protein recovery time courses monitored by western blotting (representative blot, n = 3 replicates). (B and D) Best model fits for topologies with negative ( B ), positive ( D ) or without ( C ) ERα autoregulation. Complete topologies with GATA3 autoregulation and cross-regulations are shown at the top of each column. Regular and blunt arrowheads depict positive and negative regulation. The open and solid circles in each panel indicate experimentally-measured ERα and GATA3 concentrations, obtained from panel (A) by western blot quantification. The continuous lines indicate model fits for protein depletion (top half) and recovery (bottom half) experiments. E is the error value of the fit. Model parameters for the three topologies in panels B–D are provided in Table S1.

Techniques Used: Expressing, Western Blot

ERα and GATA3 asymmetrically regulate each other in ERα-positive breast cancer. (A ) western blots depicting ERα and GATA3 protein levels in T47D cells that were transfected with control, ERα or GATA3 siRNA. Vinculin was used as the loading control. ( B) qRT-PCR data depicting ERα and GATA3 mRNA levels in T47D cells after siRNA transfection. Error bars represent standard error ( n = 3). ( C and D) western blots and qRT-PCR data ( n = 3) for MCF-7 cells after siRNA transfection. Band signals were quantitated and indicated in numbers (normalized by control). Student's t -test was used for statistical analysis. * P
Figure Legend Snippet: ERα and GATA3 asymmetrically regulate each other in ERα-positive breast cancer. (A ) western blots depicting ERα and GATA3 protein levels in T47D cells that were transfected with control, ERα or GATA3 siRNA. Vinculin was used as the loading control. ( B) qRT-PCR data depicting ERα and GATA3 mRNA levels in T47D cells after siRNA transfection. Error bars represent standard error ( n = 3). ( C and D) western blots and qRT-PCR data ( n = 3) for MCF-7 cells after siRNA transfection. Band signals were quantitated and indicated in numbers (normalized by control). Student's t -test was used for statistical analysis. * P

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

ERα−GATA3 network identified in breast cancer cells. The ERα–GATA3 network consists of an overall negative feedback loop and GATA3 positive autoregulation. The signs of regulatory interactions are indicated as positive (+), negative (−) or null ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\emptyset$\end{document} ).
Figure Legend Snippet: ERα−GATA3 network identified in breast cancer cells. The ERα–GATA3 network consists of an overall negative feedback loop and GATA3 positive autoregulation. The signs of regulatory interactions are indicated as positive (+), negative (−) or null ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\emptyset$\end{document} ).

Techniques Used:

Model with only intrinsic noise predicts that negative feedback suppresses noise in ERα and GATA3 levels. (A and B) Noise in ERα and GATA3 levels in T47D cells after transfection with control, ERα or GATA3 siRNA for 48 h (dark gray bars). In the model, noise was first matched to the levels observed experimentally with control siRNA and later the model was used to predict noise for ERα or GATA3 siRNA treatment ( C and D). WT bars are the same as for control siRNA in panels A and B. The model predicts that either ERα or GATA3 noise levels increase in the mutants without negative feedback. Error bars represent standard error ( n = 3).
Figure Legend Snippet: Model with only intrinsic noise predicts that negative feedback suppresses noise in ERα and GATA3 levels. (A and B) Noise in ERα and GATA3 levels in T47D cells after transfection with control, ERα or GATA3 siRNA for 48 h (dark gray bars). In the model, noise was first matched to the levels observed experimentally with control siRNA and later the model was used to predict noise for ERα or GATA3 siRNA treatment ( C and D). WT bars are the same as for control siRNA in panels A and B. The model predicts that either ERα or GATA3 noise levels increase in the mutants without negative feedback. Error bars represent standard error ( n = 3).

Techniques Used: Transfection

45) Product Images from "Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish"

Article Title: Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003583

Expression patterns and morpholino-based knockdown of mtmr12 in developing zebrafish. (A) Whole mount in-situ hybridization detected ubiquitous expression of mtmr12 and mtm1 transcripts in zebrafish embryos at 1 dpf (above). Below is RT-PCR analysis of mtm1 and mtmr12 expression during zebrafish development using RNA extracts from whole zebrafish embryos at indicated developmental timepoints. (B) Synergistic expression level of Mtm1 and Mtmr12 transcripts and protein at indicated time points of C2C12 differentiation (0–9 days) monitored by RT-quantitative PCR (corresponding histogram, *P≤0.05) and by western blot analysis (right panel). (C) Live embryos at 3 dpf injected with control, mtmr12 , mtm1 or both mtmr12 and mtm1 morpholinos in normal (left) and polarized lights (right). mtmr12 morphant fish showed a dorsal curvature in skeletal muscle and reduced birefringence in polarized light similar to mtm1 morphant embryos. mtmr12 morphant fish also exhibited pericardial edema (arrow). mtmr12-mtm1 double knockdown fish exhibited smaller size and reduced birefringence relative to mtm1 or mtmr12 alone morphant fish. (D) mtmr12 mRNA levels in mtmr12 morphant zebrafish following injection of two different amounts of morpholino (indicated below, upper panel). In mtm1 morphant fish, no residual myotubularin was observed showing that mtm1 morpholinos are completely penetrant to the limits of detection for western blotting. (E) Over-expression of human MTMR12 mRNA rescued small body length and skeletal muscle abnormalities observed in mtmr12 morphant embryos. (F) Quantification of the chorion hatching at 60 hpf. The number of embryos was quantified in three independent clutches (number of embryos in each clutch = 90–120). (G) Quantification of touch evoke response at 3 dpf (n = 5–8 embryos were assayed in each morpholino group).* P ≤0.01.
Figure Legend Snippet: Expression patterns and morpholino-based knockdown of mtmr12 in developing zebrafish. (A) Whole mount in-situ hybridization detected ubiquitous expression of mtmr12 and mtm1 transcripts in zebrafish embryos at 1 dpf (above). Below is RT-PCR analysis of mtm1 and mtmr12 expression during zebrafish development using RNA extracts from whole zebrafish embryos at indicated developmental timepoints. (B) Synergistic expression level of Mtm1 and Mtmr12 transcripts and protein at indicated time points of C2C12 differentiation (0–9 days) monitored by RT-quantitative PCR (corresponding histogram, *P≤0.05) and by western blot analysis (right panel). (C) Live embryos at 3 dpf injected with control, mtmr12 , mtm1 or both mtmr12 and mtm1 morpholinos in normal (left) and polarized lights (right). mtmr12 morphant fish showed a dorsal curvature in skeletal muscle and reduced birefringence in polarized light similar to mtm1 morphant embryos. mtmr12 morphant fish also exhibited pericardial edema (arrow). mtmr12-mtm1 double knockdown fish exhibited smaller size and reduced birefringence relative to mtm1 or mtmr12 alone morphant fish. (D) mtmr12 mRNA levels in mtmr12 morphant zebrafish following injection of two different amounts of morpholino (indicated below, upper panel). In mtm1 morphant fish, no residual myotubularin was observed showing that mtm1 morpholinos are completely penetrant to the limits of detection for western blotting. (E) Over-expression of human MTMR12 mRNA rescued small body length and skeletal muscle abnormalities observed in mtmr12 morphant embryos. (F) Quantification of the chorion hatching at 60 hpf. The number of embryos was quantified in three independent clutches (number of embryos in each clutch = 90–120). (G) Quantification of touch evoke response at 3 dpf (n = 5–8 embryos were assayed in each morpholino group).* P ≤0.01.

Techniques Used: Expressing, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Injection, Fluorescence In Situ Hybridization, Over Expression

46) Product Images from "Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses"

Article Title: Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

Journal: Nature Communications

doi: 10.1038/s41467-018-03772-1

Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in serum and urine. a Normal human serum or b urine pooled from healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of each dilution were extracted using the small volume protocol (0.2 mL) and were tested with the Trioplex assay on the ABI7500 Fast Dx instrument. The same serum ( c ) and urine ( d ) dilutions were tested on the QuantStudio Dx instrument. Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)
Figure Legend Snippet: Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in serum and urine. a Normal human serum or b urine pooled from healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of each dilution were extracted using the small volume protocol (0.2 mL) and were tested with the Trioplex assay on the ABI7500 Fast Dx instrument. The same serum ( c ) and urine ( d ) dilutions were tested on the QuantStudio Dx instrument. Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)

Techniques Used: RNA Extraction, Standard Deviation

Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in whole blood (EDTA). A pool of whole blood donated by healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD) and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicas of every dilution were extracted using small volume protocol (0.2 mL) and tested with Trioplex assay on the ABI7500 Fast Dx instrument ( a ) or in the QuantStudio Dx instrument ( b ). Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)
Figure Legend Snippet: Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in whole blood (EDTA). A pool of whole blood donated by healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD) and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicas of every dilution were extracted using small volume protocol (0.2 mL) and tested with Trioplex assay on the ABI7500 Fast Dx instrument ( a ) or in the QuantStudio Dx instrument ( b ). Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)

Techniques Used: RNA Extraction, Standard Deviation

47) Product Images from "Combinatorial Action of miRNAs Regulates Transcriptional and Post-Transcriptional Gene Silencing following invivo PNS Injury"

Article Title: Combinatorial Action of miRNAs Regulates Transcriptional and Post-Transcriptional Gene Silencing following invivo PNS Injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039674

miRNAs induce post-transcriptional gene silencing through association with Ago-2 in functional complexes. (A). Protein expression of Ago-1, Ago-2 and Dicer in sciatic nerves before and after nerve injury to confirm the expression of the miRNA processing machinery proteins. Actin was used as a loading control. (B) Cytoplasmic lysates isolated from control sciatic nerves and injured distal segments (10 nerves each) were immunoprecipitated with Ago-2 antibody (Cell Signaling, USA) or IgG control. A portion (1/3 rd ) of the sample was used for each of the analysis and the experiment was repeated twice. Input, No-Antibody (No AB) and Ago-2 immunoprecipitated protein was analyzed by western blotting with Ago-2 antibody, which shows enrichment of Ago-2 in the 24 hour post-injury samples. (C). For RNA-IPs (RIPs), mRNAs that were co-immunoprecipitated with Ago-2 in vivo, were reverse transcribed using oligo-dT primer and genes of interest were PCR amplified using gene-specific primers (Krox-20 = 1274 bp, C-Jun = 689 bp, Nanog = 753 bp, QKI-6 = 1345 bp, Sox-2 = 958 bp and ID-2 = 588 bp). (D). microRNAs that were co-immunoprecipitated with Ago-2 in complex with their targeted mRNAs, were reverse-transcribed with microRNA specific RT-primers using Multiscribe RT kit. Ago-2 associated microRNAs were detected by real time qPCR with miRNA specific Taqman probe/primer sets. Data were normalized to input and an internal control. Fold difference (2 -ΔΔCT ) in the association of individual microRNAs with Ago-2 protein between injured and control nerves was plotted as log 2 median ratio and error is expressed as standard deviation.
Figure Legend Snippet: miRNAs induce post-transcriptional gene silencing through association with Ago-2 in functional complexes. (A). Protein expression of Ago-1, Ago-2 and Dicer in sciatic nerves before and after nerve injury to confirm the expression of the miRNA processing machinery proteins. Actin was used as a loading control. (B) Cytoplasmic lysates isolated from control sciatic nerves and injured distal segments (10 nerves each) were immunoprecipitated with Ago-2 antibody (Cell Signaling, USA) or IgG control. A portion (1/3 rd ) of the sample was used for each of the analysis and the experiment was repeated twice. Input, No-Antibody (No AB) and Ago-2 immunoprecipitated protein was analyzed by western blotting with Ago-2 antibody, which shows enrichment of Ago-2 in the 24 hour post-injury samples. (C). For RNA-IPs (RIPs), mRNAs that were co-immunoprecipitated with Ago-2 in vivo, were reverse transcribed using oligo-dT primer and genes of interest were PCR amplified using gene-specific primers (Krox-20 = 1274 bp, C-Jun = 689 bp, Nanog = 753 bp, QKI-6 = 1345 bp, Sox-2 = 958 bp and ID-2 = 588 bp). (D). microRNAs that were co-immunoprecipitated with Ago-2 in complex with their targeted mRNAs, were reverse-transcribed with microRNA specific RT-primers using Multiscribe RT kit. Ago-2 associated microRNAs were detected by real time qPCR with miRNA specific Taqman probe/primer sets. Data were normalized to input and an internal control. Fold difference (2 -ΔΔCT ) in the association of individual microRNAs with Ago-2 protein between injured and control nerves was plotted as log 2 median ratio and error is expressed as standard deviation.

Techniques Used: Functional Assay, Expressing, Isolation, Immunoprecipitation, Western Blot, In Vivo, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Standard Deviation

48) Product Images from "Loss of MTUS1/ATIP expression is associated with adverse outcome in advanced bladder carcinomas: data from a retrospective study"

Article Title: Loss of MTUS1/ATIP expression is associated with adverse outcome in advanced bladder carcinomas: data from a retrospective study

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-214

mRNA and protein expression of MTUS1 in cell lines. A . Relative quantification results after qRT-PCR indicate that MTUS1 is expressed in all cell lines investigated. Prostate cancer cell line LNCaP, used as positive control, shows the highest expression, HCV29, RT112 and J82 the lowest. B . Westernblot results with anti- MTUS1 antibody. Detection of several ATIP isoforms could be observed at 60, 80 and 140 kDa.
Figure Legend Snippet: mRNA and protein expression of MTUS1 in cell lines. A . Relative quantification results after qRT-PCR indicate that MTUS1 is expressed in all cell lines investigated. Prostate cancer cell line LNCaP, used as positive control, shows the highest expression, HCV29, RT112 and J82 the lowest. B . Westernblot results with anti- MTUS1 antibody. Detection of several ATIP isoforms could be observed at 60, 80 and 140 kDa.

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

49) Product Images from "Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2"

Article Title: Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.00967

Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.
Figure Legend Snippet: Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.

Techniques Used: Mutagenesis, Expressing, Polymerase Chain Reaction, Hybridization, CTG Assay, Labeling, Quantitative RT-PCR

50) Product Images from "Auto-regulation of miRNA biogenesis by let-7 and Argonaute"

Article Title: Auto-regulation of miRNA biogenesis by let-7 and Argonaute

Journal: Nature

doi: 10.1038/nature11134

The ALG-1 binding site in pri-let-7 regulates expression of let-7 a , Detection of the indicated transcripts by RIP from two independent transgenic strains. b , Ratio of the levels of pri-let-7 inΔalg-1 versus WT determined by qRT-PCR and normalised to 18S rRNA. c , Northern analysis of RNA from WT orΔalg-1 pri-let-7 transgenes in let-7(mn112) . d , Ratio of the levels of let-7 transcripts expressed fromΔalg-1 versus WT pri-let-7 transgenes in let-7(mn112) determined by qRT-PCR and normalized to 18S rRNA. e – f , Levels of pre- or mature let-7 relative to 5.8S rRNA expressed from WT or Δalg-1 pri-let-7 transgenes in let-7(mn112) quantified from c .
Figure Legend Snippet: The ALG-1 binding site in pri-let-7 regulates expression of let-7 a , Detection of the indicated transcripts by RIP from two independent transgenic strains. b , Ratio of the levels of pri-let-7 inΔalg-1 versus WT determined by qRT-PCR and normalised to 18S rRNA. c , Northern analysis of RNA from WT orΔalg-1 pri-let-7 transgenes in let-7(mn112) . d , Ratio of the levels of let-7 transcripts expressed fromΔalg-1 versus WT pri-let-7 transgenes in let-7(mn112) determined by qRT-PCR and normalized to 18S rRNA. e – f , Levels of pre- or mature let-7 relative to 5.8S rRNA expressed from WT or Δalg-1 pri-let-7 transgenes in let-7(mn112) quantified from c .

Techniques Used: Binding Assay, Expressing, Transgenic Assay, Quantitative RT-PCR, Northern Blot

Association of ALG-1 with pri-let-7 in nuclear fractions a , Detection in whole cell (W), nuclear (N) or cytoplasmic (C) fractions of the indicated proteins or RNAs (mean ± s.e.m., n = 3). b , Levels of the indicated transcripts relative to their respective inputs analysed by RIP and detected by qRT-PCR (mean ± s.e.m., n = 2). c , Ratio of mature let-7 from xpo-1 relative to control RNAi after normalization to 18S rRNA (mean ± s.e.m., n = 7, *, P
Figure Legend Snippet: Association of ALG-1 with pri-let-7 in nuclear fractions a , Detection in whole cell (W), nuclear (N) or cytoplasmic (C) fractions of the indicated proteins or RNAs (mean ± s.e.m., n = 3). b , Levels of the indicated transcripts relative to their respective inputs analysed by RIP and detected by qRT-PCR (mean ± s.e.m., n = 2). c , Ratio of mature let-7 from xpo-1 relative to control RNAi after normalization to 18S rRNA (mean ± s.e.m., n = 7, *, P

Techniques Used: Quantitative RT-PCR

51) Product Images from "Blood Metallothionein Transcript as a Biomarker for Metal Sensitivity: Low Blood Metallothionein Transcripts in Arsenicosis Patients from Guizhou, China"

Article Title: Blood Metallothionein Transcript as a Biomarker for Metal Sensitivity: Low Blood Metallothionein Transcripts in Arsenicosis Patients from Guizhou, China

Journal: Environmental Health Perspectives

doi: 10.1289/ehp.10035

The sex difference in human MT-1A and MT-2A expression in blood cells of arsenicosis patients (26 males and 22 females) and healthy subjects (23 males and 25 females) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE.
Figure Legend Snippet: The sex difference in human MT-1A and MT-2A expression in blood cells of arsenicosis patients (26 males and 22 females) and healthy subjects (23 males and 25 females) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE.

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

The expression of human MT isoforms in buccal cells of arsenic-exposed patients ( n = 44) and healthy subjects ( n = 12) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE. *Significantly different from controls at p
Figure Legend Snippet: The expression of human MT isoforms in buccal cells of arsenic-exposed patients ( n = 44) and healthy subjects ( n = 12) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE. *Significantly different from controls at p

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

The expression of human MT isoforms in blood cells of arsenic-exposed patients ( n = 48) and healthy subjects ( n = 48) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE. *Significantly different from healthy controls at p
Figure Legend Snippet: The expression of human MT isoforms in blood cells of arsenic-exposed patients ( n = 48) and healthy subjects ( n = 48) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE. *Significantly different from healthy controls at p

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

52) Product Images from "A Numb–Mdm2 fuzzy complex reveals an isoform-specific involvement of Numb in breast cancer"

Article Title: A Numb–Mdm2 fuzzy complex reveals an isoform-specific involvement of Numb in breast cancer

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201709092

Role of Numb isoforms in the regulation of Mdm2 activity. (A) Numb inhibits Mdm2-dependent ubiquitination of p53 in the cytoplasm. Mdm2 and p53 shuttle between the nucleus and the cytoplasm ( Roth et al., 1998 ; Tao and Levine, 1999 ), whereas the bulk of Numb is cytosolic ( Santolini et al., 2000 ). However, p53 ubiquitination/degradation occurs both at the cytoplasmic and at the nuclear level ( Joseph et al., 2003 ). We therefore overexpressed in H1299 cells two p53 variants with subcellular localizations restricted to the cytoplasm (p53-ΔNLS) or to the nucleus (p53-ΔNES). The p53-ΔNLS construct carries a nonfunctional p53 nuclear localization signal (with mutations of Lys305, Arg306, Lys319, Lys320, and Lys321 to Ala), and the p53-ΔNES construct carries a nonfunctional p53 nuclear export signal (with mutations of Met340, Leu344, Leu348, and Leu350 to Ala). Cells were also cotransfected with Mdm2 and Numb-1 to test the ability of the latter to inhibit in vivo the Mdm2-mediated ubiquitination of p53-WT or of its mutants. The ubiquitination pattern of the different p53 variants revealed that Numb-1 affects p53 ubiquitination only in the cytoplasm, whereas p53 ubiquitination at the nuclear level was unperturbed. Left: Mdm2-mediated p53 in vivo ubiquitination in H1299 cells transfected with the indicated plasmids and increasing concentrations of FLAG-tagged Numb isoform-1 (see also the Biochemical studies section of Materials and methods). GFP was transfected as an internal normalizer for efficiency of transfection. Lysates were subjected to IB as indicated. Right: the cellular localization of the p53 mutants was monitored by IF with anti-p53. Nuclei were counterstained with DAPI. Bar, 10 µM. (B) Mdm2-mediated p53 in vivo ubiquitination in H1299 cells transfected as indicated on the top (see also the Biochemical studies section of Materials and methods). GFP was transfected as an internal normalizer for efficiency of transfection. Lysates were immunoblotted as indicated. (C) p53 in vitro ubiquitination assay with the components and the Numb species indicated on top fused to a thioredoxin scaffold (Trx; see also the Biochemical studies section of Materials and methods). RG7112 is a small molecule belonging to the Nutlin family of Mdm2 inhibitors (designed to occupy the p53-binding pocket of Mdm2) used as control. Molecular masses are given in kilodaltons.
Figure Legend Snippet: Role of Numb isoforms in the regulation of Mdm2 activity. (A) Numb inhibits Mdm2-dependent ubiquitination of p53 in the cytoplasm. Mdm2 and p53 shuttle between the nucleus and the cytoplasm ( Roth et al., 1998 ; Tao and Levine, 1999 ), whereas the bulk of Numb is cytosolic ( Santolini et al., 2000 ). However, p53 ubiquitination/degradation occurs both at the cytoplasmic and at the nuclear level ( Joseph et al., 2003 ). We therefore overexpressed in H1299 cells two p53 variants with subcellular localizations restricted to the cytoplasm (p53-ΔNLS) or to the nucleus (p53-ΔNES). The p53-ΔNLS construct carries a nonfunctional p53 nuclear localization signal (with mutations of Lys305, Arg306, Lys319, Lys320, and Lys321 to Ala), and the p53-ΔNES construct carries a nonfunctional p53 nuclear export signal (with mutations of Met340, Leu344, Leu348, and Leu350 to Ala). Cells were also cotransfected with Mdm2 and Numb-1 to test the ability of the latter to inhibit in vivo the Mdm2-mediated ubiquitination of p53-WT or of its mutants. The ubiquitination pattern of the different p53 variants revealed that Numb-1 affects p53 ubiquitination only in the cytoplasm, whereas p53 ubiquitination at the nuclear level was unperturbed. Left: Mdm2-mediated p53 in vivo ubiquitination in H1299 cells transfected with the indicated plasmids and increasing concentrations of FLAG-tagged Numb isoform-1 (see also the Biochemical studies section of Materials and methods). GFP was transfected as an internal normalizer for efficiency of transfection. Lysates were subjected to IB as indicated. Right: the cellular localization of the p53 mutants was monitored by IF with anti-p53. Nuclei were counterstained with DAPI. Bar, 10 µM. (B) Mdm2-mediated p53 in vivo ubiquitination in H1299 cells transfected as indicated on the top (see also the Biochemical studies section of Materials and methods). GFP was transfected as an internal normalizer for efficiency of transfection. Lysates were immunoblotted as indicated. (C) p53 in vitro ubiquitination assay with the components and the Numb species indicated on top fused to a thioredoxin scaffold (Trx; see also the Biochemical studies section of Materials and methods). RG7112 is a small molecule belonging to the Nutlin family of Mdm2 inhibitors (designed to occupy the p53-binding pocket of Mdm2) used as control. Molecular masses are given in kilodaltons.

Techniques Used: Activity Assay, Construct, In Vivo, Transfection, In Vitro, Ubiquitin Assay, Binding Assay

The PTBi–Mdm2 interaction requires the Ex3-coded sequence. (A) Schematic representations of the four human Numb isoforms. (B) HEK293 cells were cotransfected with Mdm2 and the indicated FLAG-tagged Numb constructs. Anti-FLAG IPs were analyzed in IB as indicated. β-Adaptin was used as a positive control for binding to full-length Numb-1/3. (C) SEC elution profiles of the Numb PTBi –Mdm2 216–302 complex and of individual subunits with corresponding Coomassie-stained SDS-PAGEs of the peak fractions. Species were injected in a Superdex-200 column at a 300-µM concentration. Mdm2 216–302 in isolation eluted around the 44-kD molecular weight marker as the Numb PTBi –Mdm2 216–302 complex because of its unstructured conformation (see also Fig. S3, A and B). Because of the highly dynamic nature of the interaction, the Numb–Mdm2 complex partially dissociated during the SEC run. Mdm2 216–302 stained poorly, likely as a result of its acidic composition. (D) FP measurements of binding affinity between rhodamine-labeled Mdm2 216–302 and Numb isoform-1 full-length, Numb-PTBi, and Numb-PTBo. The data ( n = 3; means ± SD) were fitted to a curve as described in the Fluorescence polarization (FP) section of Materials and methods. Molecular masses are given in kilodaltons.
Figure Legend Snippet: The PTBi–Mdm2 interaction requires the Ex3-coded sequence. (A) Schematic representations of the four human Numb isoforms. (B) HEK293 cells were cotransfected with Mdm2 and the indicated FLAG-tagged Numb constructs. Anti-FLAG IPs were analyzed in IB as indicated. β-Adaptin was used as a positive control for binding to full-length Numb-1/3. (C) SEC elution profiles of the Numb PTBi –Mdm2 216–302 complex and of individual subunits with corresponding Coomassie-stained SDS-PAGEs of the peak fractions. Species were injected in a Superdex-200 column at a 300-µM concentration. Mdm2 216–302 in isolation eluted around the 44-kD molecular weight marker as the Numb PTBi –Mdm2 216–302 complex because of its unstructured conformation (see also Fig. S3, A and B). Because of the highly dynamic nature of the interaction, the Numb–Mdm2 complex partially dissociated during the SEC run. Mdm2 216–302 stained poorly, likely as a result of its acidic composition. (D) FP measurements of binding affinity between rhodamine-labeled Mdm2 216–302 and Numb isoform-1 full-length, Numb-PTBi, and Numb-PTBo. The data ( n = 3; means ± SD) were fitted to a curve as described in the Fluorescence polarization (FP) section of Materials and methods. Molecular masses are given in kilodaltons.

Techniques Used: Sequencing, Construct, Positive Control, Binding Assay, Size-exclusion Chromatography, Staining, Injection, Concentration Assay, Isolation, Molecular Weight, Marker, Labeling, Fluorescence

Role of Numb isoforms in the regulation of p53 levels. (A) HEK293 cells were transfected with the indicated FLAG-tagged Numb isoforms and Mdm2. IP (anti-FLAG) and IB were as shown. Total lysate was 0.005% of the IP. The black line indicates that intervening lanes were spliced out. (B and C) MCF-10A cells were silenced with siRNA specific for total Numb, Numb-1/2, or Numb-3/4 and then were analyzed by RT-qPCR (B) or IB (C). For RT-qPCR, each target was analyzed in triplicate, and the means ± SD are shown. (D) MCF-10A cells were silenced as indicated on the top and IB as shown on the right. Numb-1/2 is an antibody against amino acids 66–80 of human Numb isoform-1, which recognizes Numb-1 and Numb-2. Of note, Mdm2 levels are also reduced upon total Numb and Numb-1/2 silencing, making Mdm2 a transcriptional target of p53 in a negative feedback loop ( Barak et al., 1993 ; Haupt et al., 1997 ; Kubbutat et al., 1997 ; Wu and Levine, 1997 ). Molecular masses are given in kilodaltons.
Figure Legend Snippet: Role of Numb isoforms in the regulation of p53 levels. (A) HEK293 cells were transfected with the indicated FLAG-tagged Numb isoforms and Mdm2. IP (anti-FLAG) and IB were as shown. Total lysate was 0.005% of the IP. The black line indicates that intervening lanes were spliced out. (B and C) MCF-10A cells were silenced with siRNA specific for total Numb, Numb-1/2, or Numb-3/4 and then were analyzed by RT-qPCR (B) or IB (C). For RT-qPCR, each target was analyzed in triplicate, and the means ± SD are shown. (D) MCF-10A cells were silenced as indicated on the top and IB as shown on the right. Numb-1/2 is an antibody against amino acids 66–80 of human Numb isoform-1, which recognizes Numb-1 and Numb-2. Of note, Mdm2 levels are also reduced upon total Numb and Numb-1/2 silencing, making Mdm2 a transcriptional target of p53 in a negative feedback loop ( Barak et al., 1993 ; Haupt et al., 1997 ; Kubbutat et al., 1997 ; Wu and Levine, 1997 ). Molecular masses are given in kilodaltons.

Techniques Used: Transfection, Quantitative RT-PCR

The PTB domain of Numb interacts directly with the acidic domain of Mdm2. (A) HEK293 cells were transfected with siRNA oligonucleotides targeting p53 (+) or control oligonucleotides (−) and after 24 h were further transfected with the catalytically inactive Mdm2-C464A mutant (to avoid p53 degradation) and FLAG–Numb-1. Immunoprecipitates with anti-FLAG or irrelevant IgG were immunoblotted as shown. Extract was 0.01% of the IP. (B) Domain structure of human Numb isoform-1 (Numb-1) and Mdm2. The two regions (Ex3 and Ex9) deriving from differential splicing of exon 3 and 9 are both present in Numb-1. PRR, proline-rich region. (C) Pulldown of endogenous Numb (from 1 mg of MCF-10A lysate) with GST-Mdm2 fragments (0.5 µM). (Top) Numb IB; (bottom) Ponceau staining. Endogenous Numb is frequently resolved as a doublet (top band, Numb-1 and -3; bottom band, Numb-2 and -4). Extract was 0.025% of the IP. (D) The purified GST-Mdm2 fragments shown at the top (0.4 µM) were used to pull down the Numb fragments (from Numb-1; expressed as FLAG-tagged in HEK293 cells; 0.75 mg of lysate) shown at the bottom. Top, FLAG IB; bottom, Ponceau staining. Extract was 0.025% of the IP. (E) Comparative pulldown of Numb fragments as in D with purified GST-Mdm2 134–334 (numbering according to UniProtKB/Swiss-Prot: Q00987 ). FL, full length. (F) The GST-Mdm2 fragments (1 µM) were used to pull down the purified Numb fragment 20–337 and Numb PTBi (bacterially expressed as His fusions; 5 µM). Detection was done by Coomassie staining. Asterisks mark Numb 20–337 . Molecular masses are given in kilodaltons.
Figure Legend Snippet: The PTB domain of Numb interacts directly with the acidic domain of Mdm2. (A) HEK293 cells were transfected with siRNA oligonucleotides targeting p53 (+) or control oligonucleotides (−) and after 24 h were further transfected with the catalytically inactive Mdm2-C464A mutant (to avoid p53 degradation) and FLAG–Numb-1. Immunoprecipitates with anti-FLAG or irrelevant IgG were immunoblotted as shown. Extract was 0.01% of the IP. (B) Domain structure of human Numb isoform-1 (Numb-1) and Mdm2. The two regions (Ex3 and Ex9) deriving from differential splicing of exon 3 and 9 are both present in Numb-1. PRR, proline-rich region. (C) Pulldown of endogenous Numb (from 1 mg of MCF-10A lysate) with GST-Mdm2 fragments (0.5 µM). (Top) Numb IB; (bottom) Ponceau staining. Endogenous Numb is frequently resolved as a doublet (top band, Numb-1 and -3; bottom band, Numb-2 and -4). Extract was 0.025% of the IP. (D) The purified GST-Mdm2 fragments shown at the top (0.4 µM) were used to pull down the Numb fragments (from Numb-1; expressed as FLAG-tagged in HEK293 cells; 0.75 mg of lysate) shown at the bottom. Top, FLAG IB; bottom, Ponceau staining. Extract was 0.025% of the IP. (E) Comparative pulldown of Numb fragments as in D with purified GST-Mdm2 134–334 (numbering according to UniProtKB/Swiss-Prot: Q00987 ). FL, full length. (F) The GST-Mdm2 fragments (1 µM) were used to pull down the purified Numb fragment 20–337 and Numb PTBi (bacterially expressed as His fusions; 5 µM). Detection was done by Coomassie staining. Asterisks mark Numb 20–337 . Molecular masses are given in kilodaltons.

Techniques Used: Transfection, Mutagenesis, Staining, Purification

Biochemical validation of the Mdm2 determinants required for binding to Numb-PTBi. (A–E) Pulldown experiments with the GST proteins indicated on top (1 µM; Mdm2 mutants are graphically shown in Fig. 5 C ; in Mdm2 QEL/A, the stretch 272–274 is mutated into three Ala) incubated with increasing concentrations (indicated by triangles, 2 and 4 µM, except for E, in which 1 and 2 µM were used) of PTBi (bacterially expressed and cleaved from the His moiety). Detection was by Coomassie staining. The position of the PTBi is indicated by asterisks. The other bands correspond with the GST fusion proteins. In A–D, Mdm2 mutants were engineered in the Mdm2 250–290 background. In E, Mdm2 mutants were engineered in the Mdm2 216–302 background so that mutagenesis of Ser246–248 could also be included. In C, the different migration of the Mdm2 mutants was probably caused by the alterations of the charges introduced by mutagenesis. (F) HEK293 cells were transfected with the Mdm2 constructs and FLAG-tagged Numb isoform-1 or -3 as shown on top. Mdm2-mut represents the F255A-Y282A-Y287A mutant carrying mutations in the Tyr and Phe residues important for direct interaction between the acidic domain and the PTBi (see also Fig. S3 G). Lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted as indicated. As shown, the Mdm2 mutant could not be efficiently coimmunoprecipitated with the PTBi-containing Numb-1. Numb-3 was used as a negative control for the specificity of the observed interactions. Total extract was 0.002% of the IP. Molecular masses are given in kilodaltons. (G) Schematic representation indicating that the PTBi–Mdm2 interaction interface represents a fuzzy complex, where two intrinsically disordered regions in both binding partners interact and retain dynamic and intrinsic disorder in the complex. Multiple negatively charged and hydrophobic motifs of Mdm2 216–302 (green) engage the PTBi insert by charge complementarity with Lys residues and stacking interactions with phenylalanines (red).
Figure Legend Snippet: Biochemical validation of the Mdm2 determinants required for binding to Numb-PTBi. (A–E) Pulldown experiments with the GST proteins indicated on top (1 µM; Mdm2 mutants are graphically shown in Fig. 5 C ; in Mdm2 QEL/A, the stretch 272–274 is mutated into three Ala) incubated with increasing concentrations (indicated by triangles, 2 and 4 µM, except for E, in which 1 and 2 µM were used) of PTBi (bacterially expressed and cleaved from the His moiety). Detection was by Coomassie staining. The position of the PTBi is indicated by asterisks. The other bands correspond with the GST fusion proteins. In A–D, Mdm2 mutants were engineered in the Mdm2 250–290 background. In E, Mdm2 mutants were engineered in the Mdm2 216–302 background so that mutagenesis of Ser246–248 could also be included. In C, the different migration of the Mdm2 mutants was probably caused by the alterations of the charges introduced by mutagenesis. (F) HEK293 cells were transfected with the Mdm2 constructs and FLAG-tagged Numb isoform-1 or -3 as shown on top. Mdm2-mut represents the F255A-Y282A-Y287A mutant carrying mutations in the Tyr and Phe residues important for direct interaction between the acidic domain and the PTBi (see also Fig. S3 G). Lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted as indicated. As shown, the Mdm2 mutant could not be efficiently coimmunoprecipitated with the PTBi-containing Numb-1. Numb-3 was used as a negative control for the specificity of the observed interactions. Total extract was 0.002% of the IP. Molecular masses are given in kilodaltons. (G) Schematic representation indicating that the PTBi–Mdm2 interaction interface represents a fuzzy complex, where two intrinsically disordered regions in both binding partners interact and retain dynamic and intrinsic disorder in the complex. Multiple negatively charged and hydrophobic motifs of Mdm2 216–302 (green) engage the PTBi insert by charge complementarity with Lys residues and stacking interactions with phenylalanines (red).

Techniques Used: Binding Assay, Incubation, Staining, Mutagenesis, Migration, Transfection, Construct, Immunoprecipitation, Negative Control

53) Product Images from "Suppression of Osteoclastogenesis by Melatonin: A Melatonin Receptor-Independent Action"

Article Title: Suppression of Osteoclastogenesis by Melatonin: A Melatonin Receptor-Independent Action

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18061142

The expressions of MT1 and MT2 melatonin receptors decreased during osteoclast differentiation. ( A ) BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for indicated days. After the culture, total RNAs were isolated and the expressions of MT1/MT2 mRNAs were examined by RT-PCR (reverse transcription-polymerase chain reaction) analyses. TRAP served as a marker for osteoclast differentiation; ( B ) BMMs were cultured as in panel ( A ) and whole cell lysates were prepared and MT1/MT2 protein expressions were determined by immunoblotting with anti-MT1 or anti-MT1 antibodies; ( C , D ) The mRNA expressions of MT1 and MT2 were analyzed by quantitative real-time PCR after culturing BMMs in the 30 ng/mL M-CSF and 100 ng/mL RANKL for the indicated days. All quantitative data are presented mean ± SD (* p
Figure Legend Snippet: The expressions of MT1 and MT2 melatonin receptors decreased during osteoclast differentiation. ( A ) BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for indicated days. After the culture, total RNAs were isolated and the expressions of MT1/MT2 mRNAs were examined by RT-PCR (reverse transcription-polymerase chain reaction) analyses. TRAP served as a marker for osteoclast differentiation; ( B ) BMMs were cultured as in panel ( A ) and whole cell lysates were prepared and MT1/MT2 protein expressions were determined by immunoblotting with anti-MT1 or anti-MT1 antibodies; ( C , D ) The mRNA expressions of MT1 and MT2 were analyzed by quantitative real-time PCR after culturing BMMs in the 30 ng/mL M-CSF and 100 ng/mL RANKL for the indicated days. All quantitative data are presented mean ± SD (* p

Techniques Used: Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Real-time Polymerase Chain Reaction

54) Product Images from "Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf"

Article Title: Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf

Journal: Nature Communications

doi: 10.1038/s41467-018-07853-z

Aberrantly upregulated p19Arf antagonizes T FH differentiation and survival. a Diagram showing the structure of WT and mutant forms of p19Arf, with their capacity of interacting with Mdm2 and Bcl6 summarized. b p19Arf and Bcl6 interaction capacity. WT or mutant p19Arf was co-transfected with WT Bcl6 expression vector into 293T cells. Cell lysates were immunoprecipitated with anti-Bcl6 and then immunoblotted with anti-p19Arf. Data are representative of two experiments. c Impact of p19Arf on T FH differentiation and survival. In vivo primed WT Smarta CD4 + T cells were infected with EV -GFP retrovirus or that expressing WT or mutant p19Arf, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi (corresponding to day 5 after initial priming), CD45.2 + CD4 + T cells were detected for GFP expression (top panels), and GFP + cells were analyzed for AnnexinV positivity (middle) or CXCR5 + SLAM lo T FH cells (bottom panels). d Interplay of p19Arf and Bcl6 in T FH differentiation. WT Smarta CD4 + T cells were transduced with EV- GFP or ArfΔ14 retrovirus in combination with EV -mCherry or Bcl6 retrovirus, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi, GFP + mCherry + CD45.2 + CD4 + T cells were analyzed for frequency of CXCR5 + SLAM lo T FH cells. e – f Effect of genetically ablating p19Arf on T FH and B cell responses to protein immunization. WT, Ezh2 –/– , or Ezh2 –/– Arf –/– CD45.2 + Smarta CD4 + T cells were adoptively transferred into CD45.1 + Bcl6 –/– recipients followed by GP61-KLH immunization. On day 5 post-immunization, CXCR5 + T FH cells were detected in the draining LNs ( e ), and on day 8, KLH-specific IgG was detected in the sera by ELISA ( f ). Data are in ( c – f ) are means ± s.d. from ≥2 experiments. * p
Figure Legend Snippet: Aberrantly upregulated p19Arf antagonizes T FH differentiation and survival. a Diagram showing the structure of WT and mutant forms of p19Arf, with their capacity of interacting with Mdm2 and Bcl6 summarized. b p19Arf and Bcl6 interaction capacity. WT or mutant p19Arf was co-transfected with WT Bcl6 expression vector into 293T cells. Cell lysates were immunoprecipitated with anti-Bcl6 and then immunoblotted with anti-p19Arf. Data are representative of two experiments. c Impact of p19Arf on T FH differentiation and survival. In vivo primed WT Smarta CD4 + T cells were infected with EV -GFP retrovirus or that expressing WT or mutant p19Arf, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi (corresponding to day 5 after initial priming), CD45.2 + CD4 + T cells were detected for GFP expression (top panels), and GFP + cells were analyzed for AnnexinV positivity (middle) or CXCR5 + SLAM lo T FH cells (bottom panels). d Interplay of p19Arf and Bcl6 in T FH differentiation. WT Smarta CD4 + T cells were transduced with EV- GFP or ArfΔ14 retrovirus in combination with EV -mCherry or Bcl6 retrovirus, followed by adoptive-transfer and LCMV-Arm infection. On 6 dpi, GFP + mCherry + CD45.2 + CD4 + T cells were analyzed for frequency of CXCR5 + SLAM lo T FH cells. e – f Effect of genetically ablating p19Arf on T FH and B cell responses to protein immunization. WT, Ezh2 –/– , or Ezh2 –/– Arf –/– CD45.2 + Smarta CD4 + T cells were adoptively transferred into CD45.1 + Bcl6 –/– recipients followed by GP61-KLH immunization. On day 5 post-immunization, CXCR5 + T FH cells were detected in the draining LNs ( e ), and on day 8, KLH-specific IgG was detected in the sera by ELISA ( f ). Data are in ( c – f ) are means ± s.d. from ≥2 experiments. * p

Techniques Used: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, In Vivo, Infection, Adoptive Transfer Assay, Transduction, Enzyme-linked Immunosorbent Assay

Ezh2 acts upstream of Bcl6 induction to promote T FH differentiation. a – b Impact of Ezh2 deficiency on monoclonal CD4 + T cell responses. CD45.2 + Smarta CD4 + T cells from WT or Ezh2 –/– Smarta-Tg mice were adoptively transferred into congenic mice, followed by infection with LCMV-Arm. On 4 dpi, CXCR5 + SLAM lo ( a ) and CXCR5 + Bcl6 + T FH cells ( b ) were detected in recipient spleens. Contour plots are representative of ≥2 experiments, and cumulative data on frequency and numbers of each subset are means ± s.d. (each dot represents a mouse). c Detection of Bcl6 transcripts in T FH cells. CD45.2 + CXCR5 + SLAM lo T FH cells were sorted from the recipient spleens on 4 dpi as in ( a ), and Bcl6 transcript was detected by quantitative RT-PCR. d Detection of Ezh2 binding to the Bcl6 promoter. WT CD45.2 + T FH and T H 1 cells were sorted from recipient spleens on 5 dpi , and together with WT naïve CD4 + T cells, were analyzed by ChIP with anti-Ezh2 antibody or control IgG. Enriched Ezh2 binding at the TSSs of Bcl6 or Hprt1 genes was determined by ChIP-qPCR. Data in c and d are means ± s.d. from ≥2 experiments. e – f Impact of forced expression of Bcl6 on T FH and antibody responses. WT or Ezh2 –/– Smarta CD4 + T cells were primed in vivo for 24 h and infected with empty vector ( EV )-mCherry or Bcl6 -mCherry retrovirus. Transduced Smarta CD4 + T cells were adoptively transferred into congenic mice, followed by LCMV-Arm infection ( e ), or into Bcl6 –/– recipients, followed by KLH-GP61 immunization ( f ). In e , mCherry + CXCR5 + SLAM lo T FH cells were detected in the recipient spleens on 3 dpi (corresponding to day 5 after initial priming), with representative contour plots and cumulative data on frequency and numbers of mCherry + CXCR5 + SLAM lo T FH cells shown. In f , KLH-specific IgG was detected in the recipient sera on day 8 post-immunization. All cumulative data are means ± s.d. * p
Figure Legend Snippet: Ezh2 acts upstream of Bcl6 induction to promote T FH differentiation. a – b Impact of Ezh2 deficiency on monoclonal CD4 + T cell responses. CD45.2 + Smarta CD4 + T cells from WT or Ezh2 –/– Smarta-Tg mice were adoptively transferred into congenic mice, followed by infection with LCMV-Arm. On 4 dpi, CXCR5 + SLAM lo ( a ) and CXCR5 + Bcl6 + T FH cells ( b ) were detected in recipient spleens. Contour plots are representative of ≥2 experiments, and cumulative data on frequency and numbers of each subset are means ± s.d. (each dot represents a mouse). c Detection of Bcl6 transcripts in T FH cells. CD45.2 + CXCR5 + SLAM lo T FH cells were sorted from the recipient spleens on 4 dpi as in ( a ), and Bcl6 transcript was detected by quantitative RT-PCR. d Detection of Ezh2 binding to the Bcl6 promoter. WT CD45.2 + T FH and T H 1 cells were sorted from recipient spleens on 5 dpi , and together with WT naïve CD4 + T cells, were analyzed by ChIP with anti-Ezh2 antibody or control IgG. Enriched Ezh2 binding at the TSSs of Bcl6 or Hprt1 genes was determined by ChIP-qPCR. Data in c and d are means ± s.d. from ≥2 experiments. e – f Impact of forced expression of Bcl6 on T FH and antibody responses. WT or Ezh2 –/– Smarta CD4 + T cells were primed in vivo for 24 h and infected with empty vector ( EV )-mCherry or Bcl6 -mCherry retrovirus. Transduced Smarta CD4 + T cells were adoptively transferred into congenic mice, followed by LCMV-Arm infection ( e ), or into Bcl6 –/– recipients, followed by KLH-GP61 immunization ( f ). In e , mCherry + CXCR5 + SLAM lo T FH cells were detected in the recipient spleens on 3 dpi (corresponding to day 5 after initial priming), with representative contour plots and cumulative data on frequency and numbers of mCherry + CXCR5 + SLAM lo T FH cells shown. In f , KLH-specific IgG was detected in the recipient sera on day 8 post-immunization. All cumulative data are means ± s.d. * p

Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, In Vivo, Plasmid Preparation

55) Product Images from "Ordered progression of stage-specific miRNA profiles in the mouse B2 B-cell lineage"

Article Title: Ordered progression of stage-specific miRNA profiles in the mouse B2 B-cell lineage

Journal: Blood

doi: 10.1182/blood-2010-10-316034

Validation of sequencing results by TaqMan LDAs. Excellent correlations were found between the normalized sequence counts and the TaqMan real-time qPCR LDA results for ∼ 200 known miRNAs.
Figure Legend Snippet: Validation of sequencing results by TaqMan LDAs. Excellent correlations were found between the normalized sequence counts and the TaqMan real-time qPCR LDA results for ∼ 200 known miRNAs.

Techniques Used: Sequencing, Real-time Polymerase Chain Reaction

56) Product Images from "Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation"

Article Title: Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation

Journal: Journal of Tissue Engineering

doi: 10.1177/2041731418802438

PCR assay for chondrogenic gene expressions in various gels at day 14: (a) RT-PCR and (b) qPCR. Statistical analysis was performed using one-way ANOVA followed by a post hoc LSD test. * indicates statistically significant differences with different gel groups (*p
Figure Legend Snippet: PCR assay for chondrogenic gene expressions in various gels at day 14: (a) RT-PCR and (b) qPCR. Statistical analysis was performed using one-way ANOVA followed by a post hoc LSD test. * indicates statistically significant differences with different gel groups (*p

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

57) Product Images from "The immune characterization of interferon‐β responses in tuberculosis patients"

Article Title: The immune characterization of interferon‐β responses in tuberculosis patients

Journal: Microbiology and Immunology

doi: 10.1111/1348-0421.12583

BCG induced IFN‐β expression in PFMC . (a) IFN‐β expression in PFMC ( n = 10) from freshly isolated tuberculous pleural effusions and PBMC from healthy donors ( n = 10). RNA was extracted, and IFN‐β expression was detected by RT‐PCR. (b) The statistical results depict the ratios of IFN‐β/GAPDH. (c) PFMC ( n = 10) were incubated with BCG for varying amounts of time, and IFN‐β expression was measured using a DNA thermal cycler. (d) Graph showing the ratios of IFN‐β/GAPDH. Data represent the mean ± SD of five separate experiments. * P
Figure Legend Snippet: BCG induced IFN‐β expression in PFMC . (a) IFN‐β expression in PFMC ( n = 10) from freshly isolated tuberculous pleural effusions and PBMC from healthy donors ( n = 10). RNA was extracted, and IFN‐β expression was detected by RT‐PCR. (b) The statistical results depict the ratios of IFN‐β/GAPDH. (c) PFMC ( n = 10) were incubated with BCG for varying amounts of time, and IFN‐β expression was measured using a DNA thermal cycler. (d) Graph showing the ratios of IFN‐β/GAPDH. Data represent the mean ± SD of five separate experiments. * P

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Incubation

58) Product Images from "The Assembly of EDC4 and Dcp1a into Processing Bodies Is Critical for the Translational Regulation of IL-6"

Article Title: The Assembly of EDC4 and Dcp1a into Processing Bodies Is Critical for the Translational Regulation of IL-6

Journal: PLoS ONE

doi: 10.1371/journal.pone.0123223

P-body knockdown affects the expression of IL-6-regulating molecules. THP-1 cells were transfected with a siRNA targeting EDC4 or Dcp1a, or with a non-targeting siRNA (siNeg). Twenty hours after transfection, the cells were differentiated with PMA, followed by activation later. The expression levels of (A) miRNAs and (B) NF-IL6 were determined by quantitative PCR as described in Methods. *p
Figure Legend Snippet: P-body knockdown affects the expression of IL-6-regulating molecules. THP-1 cells were transfected with a siRNA targeting EDC4 or Dcp1a, or with a non-targeting siRNA (siNeg). Twenty hours after transfection, the cells were differentiated with PMA, followed by activation later. The expression levels of (A) miRNAs and (B) NF-IL6 were determined by quantitative PCR as described in Methods. *p

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

P-body knockdown reduces LPS-stimulated IL-6 release in M1-THPs. THP-1 cells were transfected with siRNA targeting EDC4 (EDC), Dcp1a (D1a), or with a non-targeting siRNA (siNeg). Twenty hours after transfection, the cells were PMA-differentiated, activated toward an M1 or M2 phenotype, and subjected to LPS stimulation as described in Methods. (A) Depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected cells were polarized toward an M1 or M2 phenotype, harvested, and subjected to immunoblot analysis with the indicated antibodies; β-actin served as a loading control. (B) Effects of EDC4 or Dcp1a knockdown on cytokine mRNA expression. The siRNA-transfected cells were polarized toward an M1 or M2 phenotype, treated with LPS, harvested at the time points indicated, and analyzed by semi-quantitative RT-PCR. (C) P-body knockdown did not affect the levels of LPS-induced IL-6 and TNF-α mRNA expression in M1-THPs. Total RNA was isolated 0, 4, 8, or 24 hours after LPS stimulation and subjected to quantitative real-time RT-PCR analysis to detect IL-6 and TNF-α gene transcripts, as described in Methods. RNAs were normalized to HPRT RNA. The level of the IL-6 and TNF-α transcripts in siNeg-transfected cells at 4 and at 0 hours after LPS stimulation, respectively, was set to 1 to calculate the relative values in each sample. The IL-6 mRNA level before LPS stimulation (0 hour) was below the limit of detection. Data are averages of three independent experiments; error bars indicate SD. (D) P-body knockdown reduced the level of IL-6 released from M1-THPs cells. Supernatants were harvested at 0, 8, or 24 hours after LPS stimulation, and protein concentrations of IL-6 and TNF-α were measured by ELISA. IL-6 protein levels before LPS stimulation (0 hour) were below the limit of detection. Data are averages of three independent experiments; error bars indicate SD. ***p
Figure Legend Snippet: P-body knockdown reduces LPS-stimulated IL-6 release in M1-THPs. THP-1 cells were transfected with siRNA targeting EDC4 (EDC), Dcp1a (D1a), or with a non-targeting siRNA (siNeg). Twenty hours after transfection, the cells were PMA-differentiated, activated toward an M1 or M2 phenotype, and subjected to LPS stimulation as described in Methods. (A) Depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected cells were polarized toward an M1 or M2 phenotype, harvested, and subjected to immunoblot analysis with the indicated antibodies; β-actin served as a loading control. (B) Effects of EDC4 or Dcp1a knockdown on cytokine mRNA expression. The siRNA-transfected cells were polarized toward an M1 or M2 phenotype, treated with LPS, harvested at the time points indicated, and analyzed by semi-quantitative RT-PCR. (C) P-body knockdown did not affect the levels of LPS-induced IL-6 and TNF-α mRNA expression in M1-THPs. Total RNA was isolated 0, 4, 8, or 24 hours after LPS stimulation and subjected to quantitative real-time RT-PCR analysis to detect IL-6 and TNF-α gene transcripts, as described in Methods. RNAs were normalized to HPRT RNA. The level of the IL-6 and TNF-α transcripts in siNeg-transfected cells at 4 and at 0 hours after LPS stimulation, respectively, was set to 1 to calculate the relative values in each sample. The IL-6 mRNA level before LPS stimulation (0 hour) was below the limit of detection. Data are averages of three independent experiments; error bars indicate SD. (D) P-body knockdown reduced the level of IL-6 released from M1-THPs cells. Supernatants were harvested at 0, 8, or 24 hours after LPS stimulation, and protein concentrations of IL-6 and TNF-α were measured by ELISA. IL-6 protein levels before LPS stimulation (0 hour) were below the limit of detection. Data are averages of three independent experiments; error bars indicate SD. ***p

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

EDC4 or Dcp1a knockdown affects P-body assembly. THP-1 cells were transfected with the indicated siRNAs; 20 hours after transfection, the cells were PMA-differentiated and then activated toward a M1 phenotype. (A) The depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected M1-THP cells were harvested and analyzed by immunoblotting with the indicated antibodies; β-actin was used as a loading control. (B) EDC4 depletion inhibited P-body formation. The siRNA-transfected cells were polarized toward an M1 phenotype, fixed, stained with goat polyclonal antibody specific for EDC4 and mouse monoclonal antibody specific for Dcp1a, and finally stained with Alexa 488-conjugated donkey anti-goat and Alexa 568-conjugated donkey anti-mouse secondary antibodies.
Figure Legend Snippet: EDC4 or Dcp1a knockdown affects P-body assembly. THP-1 cells were transfected with the indicated siRNAs; 20 hours after transfection, the cells were PMA-differentiated and then activated toward a M1 phenotype. (A) The depletion of EDC4 or Dcp1a by siRNA. The siRNA-transfected M1-THP cells were harvested and analyzed by immunoblotting with the indicated antibodies; β-actin was used as a loading control. (B) EDC4 depletion inhibited P-body formation. The siRNA-transfected cells were polarized toward an M1 phenotype, fixed, stained with goat polyclonal antibody specific for EDC4 and mouse monoclonal antibody specific for Dcp1a, and finally stained with Alexa 488-conjugated donkey anti-goat and Alexa 568-conjugated donkey anti-mouse secondary antibodies.

Techniques Used: Transfection, Staining

59) Product Images from "DIFFERENCES IN CYSTEINE PROTEASE ACTIVITY IN SCHISTOSOMA MANSONI-RESISTANT AND -SUSCEPTIBLE BIOMPHALARIA GLABRATA AND CHARACTERIZATION OF THE HEPATOPANCREAS CATHEPSIN B FULL-LENGTH cDNA"

Article Title: DIFFERENCES IN CYSTEINE PROTEASE ACTIVITY IN SCHISTOSOMA MANSONI-RESISTANT AND -SUSCEPTIBLE BIOMPHALARIA GLABRATA AND CHARACTERIZATION OF THE HEPATOPANCREAS CATHEPSIN B FULL-LENGTH cDNA

Journal: The Journal of parasitology

doi: 10.1645/GE-1410R.1

Real-time quantitative RT-PCR analysis of the snail hepatopancreas cathepsin B transcript in resistant (BS-90) and susceptible (NMRI and M-line) pre- and postexposure to miracidia. RT-PCR was performed with the cathepsin B gene-specific primers using RNA from individual snails after various times postexposure. Gene-specific primers for the housekeeping gene myoglobin were used to assess sample uniformity and to confirm that the template cDNA was used in equivalent amounts for each amplification reaction. RT-PCR reactions were performed using an Applied Biosystems 7300 Real-Time PCR System, and detection of the fluorescently labeled product was performed at the end of the amplification period. The -fold increase of gene expression was calculated by comparative Ct method as described in Materials and Methods. A value of > 1-fold increase was considered to be significant (1 = value for nonexposed snails).
Figure Legend Snippet: Real-time quantitative RT-PCR analysis of the snail hepatopancreas cathepsin B transcript in resistant (BS-90) and susceptible (NMRI and M-line) pre- and postexposure to miracidia. RT-PCR was performed with the cathepsin B gene-specific primers using RNA from individual snails after various times postexposure. Gene-specific primers for the housekeeping gene myoglobin were used to assess sample uniformity and to confirm that the template cDNA was used in equivalent amounts for each amplification reaction. RT-PCR reactions were performed using an Applied Biosystems 7300 Real-Time PCR System, and detection of the fluorescently labeled product was performed at the end of the amplification period. The -fold increase of gene expression was calculated by comparative Ct method as described in Materials and Methods. A value of > 1-fold increase was considered to be significant (1 = value for nonexposed snails).

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

60) Product Images from "Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum"

Article Title: Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum

Journal: BMC Genomics

doi: 10.1186/s12864-018-5316-3

Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P
Figure Legend Snippet: Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P

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

61) Product Images from "The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation"

Article Title: The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002479

Quantitative analysis of KSHV BAC36 wt, RTA 1st and BAC RTA all recombinant viruses infected PBMCs at 1dpi, 2dpi, 4dpi and 7dpi. (A) Immunofluorescence assay for PBMCs infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses at 2 dpi, 4 dpi and 7 dpi. Uninfected and infected PBMCs at 2 dpi, 4 dpi and 7 dpi were stained for LANA protein expression. PBMCs expressed GFP, indicating the presence of viral genome. (B, C). Quantitative analysis for determination of latent and lytic infection in PMBC cells infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses. Total RNAs were extracted, treated with DNase I, and reverse transcribed to cDNA after 1 dpi, 2 dpi, 4 dpi and 7dpi. Quantitative Real-time PCR analysis with the primers for LANA and RTA was performed using StepOnePlus Real-Time PCR System. Error bars indicate standard deviations from three separate experiments.
Figure Legend Snippet: Quantitative analysis of KSHV BAC36 wt, RTA 1st and BAC RTA all recombinant viruses infected PBMCs at 1dpi, 2dpi, 4dpi and 7dpi. (A) Immunofluorescence assay for PBMCs infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses at 2 dpi, 4 dpi and 7 dpi. Uninfected and infected PBMCs at 2 dpi, 4 dpi and 7 dpi were stained for LANA protein expression. PBMCs expressed GFP, indicating the presence of viral genome. (B, C). Quantitative analysis for determination of latent and lytic infection in PMBC cells infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses. Total RNAs were extracted, treated with DNase I, and reverse transcribed to cDNA after 1 dpi, 2 dpi, 4 dpi and 7dpi. Quantitative Real-time PCR analysis with the primers for LANA and RTA was performed using StepOnePlus Real-Time PCR System. Error bars indicate standard deviations from three separate experiments.

Techniques Used: BAC Assay, Recombinant, Infection, Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction

62) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

Journal: Cell

doi: 10.1016/j.cell.2017.11.008

Rescue of Enhancer-Promoter Interactions in Cells (A) Model depicting use of dCas9-YY1 to artificially tether YY1 to a site adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 in order to determine whether artificially tethered YY1 can rescue enhancer-promoter interactions. (B) Model depicting dCas9-YY1 rescue experiments. Etv4 promoter-proximal YY1 binding motif mutant cells were transduced with lentivirus to stably express either dCas9 or dCas9-YY1, and two sgRNAs to direct their localization to the sequences adjacent to the deleted YY1 binding motif in the Etv4 promoter-proximal region. The ability to rescue enhancer-promoter looping was assayed by 4C-seq. (C) Western blot results showing that Etv4 promoter-proximal YY1 binding motif mutant cells transduced with lentivirus to stably express either dCas9 or dCas9-YY1 successfully express dCas9 or dCas9-YY1. (D) Artificial tethering of YY1 using dCas9-YY1 was performed at sites adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 . The effects of tethering YY1 using dCas9-YY1 on enhancer-promoter looping and expression of the Etv4 gene were measured and compared to dCas9 alone. The genotype of the Etv4 promoter-proximal YY1 binding motif mutant cells and the 4C-seq viewpoint (VP) is shown. The 4C-seq signal is displayed as the smoothed average reads per million per base pair. The mean 4C-seq signal is represented as a line, and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and CAS9 ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. (E) Model depicting the loss of looping interactions after the inducible degradation of the structuring factors CTCF and YY1 followed by restoration of looping upon washout of degradation compounds. (F) Change in normalized interaction frequency (log2 fold change) after YY1 and CTCF degradation (treated) and recovery (washout) relative to untreated cells. For YY1 degradation, change in normalized interaction frequency is plotted for YY1-YY1 enhancer-promoter interactions. For CTCF degradation, change in normalized interaction frequency is plotted for CTCF-CTCF interactions. .
Figure Legend Snippet: Rescue of Enhancer-Promoter Interactions in Cells (A) Model depicting use of dCas9-YY1 to artificially tether YY1 to a site adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 in order to determine whether artificially tethered YY1 can rescue enhancer-promoter interactions. (B) Model depicting dCas9-YY1 rescue experiments. Etv4 promoter-proximal YY1 binding motif mutant cells were transduced with lentivirus to stably express either dCas9 or dCas9-YY1, and two sgRNAs to direct their localization to the sequences adjacent to the deleted YY1 binding motif in the Etv4 promoter-proximal region. The ability to rescue enhancer-promoter looping was assayed by 4C-seq. (C) Western blot results showing that Etv4 promoter-proximal YY1 binding motif mutant cells transduced with lentivirus to stably express either dCas9 or dCas9-YY1 successfully express dCas9 or dCas9-YY1. (D) Artificial tethering of YY1 using dCas9-YY1 was performed at sites adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 . The effects of tethering YY1 using dCas9-YY1 on enhancer-promoter looping and expression of the Etv4 gene were measured and compared to dCas9 alone. The genotype of the Etv4 promoter-proximal YY1 binding motif mutant cells and the 4C-seq viewpoint (VP) is shown. The 4C-seq signal is displayed as the smoothed average reads per million per base pair. The mean 4C-seq signal is represented as a line, and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and CAS9 ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. (E) Model depicting the loss of looping interactions after the inducible degradation of the structuring factors CTCF and YY1 followed by restoration of looping upon washout of degradation compounds. (F) Change in normalized interaction frequency (log2 fold change) after YY1 and CTCF degradation (treated) and recovery (washout) relative to untreated cells. For YY1 degradation, change in normalized interaction frequency is plotted for YY1-YY1 enhancer-promoter interactions. For CTCF degradation, change in normalized interaction frequency is plotted for CTCF-CTCF interactions. .

Techniques Used: Binding Assay, Mutagenesis, Transduction, Stable Transfection, Western Blot, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Deletion of YY1 Binding Sites Causes Loss of Enhancer-Promoter Interactions (A) Model depicting CRISPR/Cas9-mediated deletion of a YY1 binding motif in the regulatory region of a gene. (B and C) CRISPR/Cas9-mediated deletion of YY1 binding motifs in the regulatory regions of two genes, Raf1 (B) and Etv4 ), and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. .
Figure Legend Snippet: Deletion of YY1 Binding Sites Causes Loss of Enhancer-Promoter Interactions (A) Model depicting CRISPR/Cas9-mediated deletion of a YY1 binding motif in the regulatory region of a gene. (B and C) CRISPR/Cas9-mediated deletion of YY1 binding motifs in the regulatory regions of two genes, Raf1 (B) and Etv4 ), and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. .

Techniques Used: Binding Assay, CRISPR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

63) Product Images from "Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation"

Article Title: Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0672-5

Cell morphology and chondrogenic property of M-MSCs in vitro. a Cell morphology in two-dimensional culture and staining results of Safranin O or type II collagen (Col II) after chondrogenic differentiation for 21 days in three-dimensional cell pellets. b Size of pellets (diameter). c Expression levels of chondrogenic markers (Col2a1, Acan, and Sox9) and hypertrophic markers (Col10a1 and MMP13) determined by qRT-PCR assays. Values represent means ± SD ( n = 3 donors with three replicates per donor). * P
Figure Legend Snippet: Cell morphology and chondrogenic property of M-MSCs in vitro. a Cell morphology in two-dimensional culture and staining results of Safranin O or type II collagen (Col II) after chondrogenic differentiation for 21 days in three-dimensional cell pellets. b Size of pellets (diameter). c Expression levels of chondrogenic markers (Col2a1, Acan, and Sox9) and hypertrophic markers (Col10a1 and MMP13) determined by qRT-PCR assays. Values represent means ± SD ( n = 3 donors with three replicates per donor). * P

Techniques Used: In Vitro, Staining, Expressing, Quantitative RT-PCR

64) Product Images from "Uterine extracellular matrix components are altered during defective decidualization in interleukin-11 receptor ? deficient mice"

Article Title: Uterine extracellular matrix components are altered during defective decidualization in interleukin-11 receptor ? deficient mice

Journal: Reproductive biology and endocrinology : RB & E

doi: 10.1186/1477-7827-2-76

Quantitative real-time RT-PCR for extracellular matrix components. Quantitative real-time RT-PCR for (A) COL3A1, (B) BGN, (C) SPARC and (D) NID1. Circled data points indicate samples used in the cDNA microarray analysis, and horizontal lines the mean of each genotype. Absolute values for mRNA abundance were normalized to that of 18S rRNA.
Figure Legend Snippet: Quantitative real-time RT-PCR for extracellular matrix components. Quantitative real-time RT-PCR for (A) COL3A1, (B) BGN, (C) SPARC and (D) NID1. Circled data points indicate samples used in the cDNA microarray analysis, and horizontal lines the mean of each genotype. Absolute values for mRNA abundance were normalized to that of 18S rRNA.

Techniques Used: Quantitative RT-PCR, Microarray

65) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

Journal: Cell

doi: 10.1016/j.cell.2017.11.008

Rescue of Enhancer-Promoter Interactions in Cells (A) Model depicting use of dCas9-YY1 to artificially tether YY1 to a site adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 in order to determine whether artificially tethered YY1 can rescue enhancer-promoter interactions. (B) Model depicting dCas9-YY1 rescue experiments. Etv4 promoter-proximal YY1 binding motif mutant cells were transduced with lentivirus to stably express either dCas9 or dCas9-YY1, and two sgRNAs to direct their localization to the sequences adjacent to the deleted YY1 binding motif in the Etv4 promoter-proximal region. The ability to rescue enhancer-promoter looping was assayed by 4C-seq. (C) Western blot results showing that Etv4 promoter-proximal YY1 binding motif mutant cells transduced with lentivirus to stably express either dCas9 or dCas9-YY1 successfully express dCas9 or dCas9-YY1. (D) Artificial tethering of YY1 using dCas9-YY1 was performed at sites adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 . The effects of tethering YY1 using dCas9-YY1 on enhancer-promoter looping and expression of the Etv4 gene were measured and compared to dCas9 alone. The genotype of the Etv4 promoter-proximal YY1 binding motif mutant cells and the 4C-seq viewpoint (VP) is shown. The 4C-seq signal is displayed as the smoothed average reads per million per base pair. The mean 4C-seq signal is represented as a line, and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and CAS9 ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. (E) Model depicting the loss of looping interactions after the inducible degradation of the structuring factors CTCF and YY1 followed by restoration of looping upon washout of degradation compounds. (F) Change in normalized interaction frequency (log2 fold change) after YY1 and CTCF degradation (treated) and recovery (washout) relative to untreated cells. For YY1 degradation, change in normalized interaction frequency is plotted for YY1-YY1 enhancer-promoter interactions. For CTCF degradation, change in normalized interaction frequency is plotted for CTCF-CTCF interactions. .
Figure Legend Snippet: Rescue of Enhancer-Promoter Interactions in Cells (A) Model depicting use of dCas9-YY1 to artificially tether YY1 to a site adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 in order to determine whether artificially tethered YY1 can rescue enhancer-promoter interactions. (B) Model depicting dCas9-YY1 rescue experiments. Etv4 promoter-proximal YY1 binding motif mutant cells were transduced with lentivirus to stably express either dCas9 or dCas9-YY1, and two sgRNAs to direct their localization to the sequences adjacent to the deleted YY1 binding motif in the Etv4 promoter-proximal region. The ability to rescue enhancer-promoter looping was assayed by 4C-seq. (C) Western blot results showing that Etv4 promoter-proximal YY1 binding motif mutant cells transduced with lentivirus to stably express either dCas9 or dCas9-YY1 successfully express dCas9 or dCas9-YY1. (D) Artificial tethering of YY1 using dCas9-YY1 was performed at sites adjacent to the YY1 binding site mutation in the promoter-proximal region of Etv4 . The effects of tethering YY1 using dCas9-YY1 on enhancer-promoter looping and expression of the Etv4 gene were measured and compared to dCas9 alone. The genotype of the Etv4 promoter-proximal YY1 binding motif mutant cells and the 4C-seq viewpoint (VP) is shown. The 4C-seq signal is displayed as the smoothed average reads per million per base pair. The mean 4C-seq signal is represented as a line, and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and CAS9 ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. (E) Model depicting the loss of looping interactions after the inducible degradation of the structuring factors CTCF and YY1 followed by restoration of looping upon washout of degradation compounds. (F) Change in normalized interaction frequency (log2 fold change) after YY1 and CTCF degradation (treated) and recovery (washout) relative to untreated cells. For YY1 degradation, change in normalized interaction frequency is plotted for YY1-YY1 enhancer-promoter interactions. For CTCF degradation, change in normalized interaction frequency is plotted for CTCF-CTCF interactions. .

Techniques Used: Binding Assay, Mutagenesis, Transduction, Stable Transfection, Western Blot, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Deletion of YY1 Binding Sites Causes Loss of Enhancer-Promoter Interactions (A) Model depicting CRISPR/Cas9-mediated deletion of a YY1 binding motif in the regulatory region of a gene. (B and C) CRISPR/Cas9-mediated deletion of YY1 binding motifs in the regulatory regions of two genes, Raf1 (B) and Etv4 ), and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. .
Figure Legend Snippet: Deletion of YY1 Binding Sites Causes Loss of Enhancer-Promoter Interactions (A) Model depicting CRISPR/Cas9-mediated deletion of a YY1 binding motif in the regulatory region of a gene. (B and C) CRISPR/Cas9-mediated deletion of YY1 binding motifs in the regulatory regions of two genes, Raf1 (B) and Etv4 ), and the shaded area represents the 95% confidence interval. Three biological replicates were assayed for 4C-seq and ChIP-qPCR experiments, and six biological replicates were assayed for RT-qPCR experiments. Error bars represent the SD. All p values were determined using the Student’s t test. .

Techniques Used: Binding Assay, CRISPR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

66) Product Images from "Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier"

Article Title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144273

AMPK activation contributes to the induction of UCP2 and the down-regulation of MPC1. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 1 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of UCP2 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) for 1 and 48 h or with Compound C (CC, 5 μM) for 1 h. (C) qRT-PCR analysis of MPC1 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) or with Compound C (CC, 5 μM), both for 1 h. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p
Figure Legend Snippet: AMPK activation contributes to the induction of UCP2 and the down-regulation of MPC1. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 1 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of UCP2 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) for 1 and 48 h or with Compound C (CC, 5 μM) for 1 h. (C) qRT-PCR analysis of MPC1 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) or with Compound C (CC, 5 μM), both for 1 h. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p

Techniques Used: Activation Assay, Western Blot, Negative Control, Quantitative RT-PCR, Expressing

shGTPBP3 cells show increased antioxidant capacity. (A) Determination of the mitochondrial membrane potential by flow cytometry in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells with the fluorescent probe MitoTracker Red. NC cells treated for 30 min with sodium azide (SA) at 25 mM were included in the analysis as a positive control for the membrane potential drop. (B) Determination of oxygen consumption rate with a Clark-type oxygen electrode in shGTPBP3-1, shGTPBP3-2 and NC cells. (C) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells with hydroethidine. (D) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) for 2 h with 0.3 mM H 2 O 2 with dihydrorhodamine 123. (E) Measurements of antioxidant enzyme activities: Catalase, SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase). Data in A, C, D and E are expressed as % of NC. (F) qRT-PCR analysis of Thioredoxin-1 , Thioredoxin-2 , Peroxiredoxin-3 , Peroxiredoxin-5 and Uncoupling protein-2 ( UCP2 ) mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells. (G) Western blot analysis of UCP2 in shGTPBP3-1, shGTPBP3-2 and NC cells. The filter was also probed with porin as a loading control. (H) Densitometric analysis of UCP2 normalized to loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p
Figure Legend Snippet: shGTPBP3 cells show increased antioxidant capacity. (A) Determination of the mitochondrial membrane potential by flow cytometry in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells with the fluorescent probe MitoTracker Red. NC cells treated for 30 min with sodium azide (SA) at 25 mM were included in the analysis as a positive control for the membrane potential drop. (B) Determination of oxygen consumption rate with a Clark-type oxygen electrode in shGTPBP3-1, shGTPBP3-2 and NC cells. (C) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells with hydroethidine. (D) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) for 2 h with 0.3 mM H 2 O 2 with dihydrorhodamine 123. (E) Measurements of antioxidant enzyme activities: Catalase, SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase). Data in A, C, D and E are expressed as % of NC. (F) qRT-PCR analysis of Thioredoxin-1 , Thioredoxin-2 , Peroxiredoxin-3 , Peroxiredoxin-5 and Uncoupling protein-2 ( UCP2 ) mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells. (G) Western blot analysis of UCP2 in shGTPBP3-1, shGTPBP3-2 and NC cells. The filter was also probed with porin as a loading control. (H) Densitometric analysis of UCP2 normalized to loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

Techniques Used: Flow Cytometry, Cytometry, Negative Control, Positive Control, Quantitative RT-PCR, Expressing, Western Blot

Stable knock-down of GTPBP3 disturbs Complex I assembly and reduces the expression of Complex I assembly factors NDUFAF3 and NDUFAF4 (A) Western blot analysis of OXPHOS subunits ND1, NDUFS3 and NDUFB8 (Complex I), SDHA (Complex II), COXII and COXIV (Complex IV), and β-subunit (Complex V) in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. The filter was also probed with porin as a loading control. (B) Densitometric analysis of OXPHOS subunits normalized to porin and represented as % of NC. (C) Representative Blue Native-PAGE of OXPHOS complexes in shGTPBP3-1, shGTPBP3-2 and NC cells. (D) Densitometric analysis of OXPHOS Complexes normalized to Complex-II (loading control) and represented as % of NC. (E) qRT-PCR analysis of C20ORF7 , NUBPL , NDUFAF3 and NDUFAF4 mRNA expression in shGTPBP3 and NC cells. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p
Figure Legend Snippet: Stable knock-down of GTPBP3 disturbs Complex I assembly and reduces the expression of Complex I assembly factors NDUFAF3 and NDUFAF4 (A) Western blot analysis of OXPHOS subunits ND1, NDUFS3 and NDUFB8 (Complex I), SDHA (Complex II), COXII and COXIV (Complex IV), and β-subunit (Complex V) in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. The filter was also probed with porin as a loading control. (B) Densitometric analysis of OXPHOS subunits normalized to porin and represented as % of NC. (C) Representative Blue Native-PAGE of OXPHOS complexes in shGTPBP3-1, shGTPBP3-2 and NC cells. (D) Densitometric analysis of OXPHOS Complexes normalized to Complex-II (loading control) and represented as % of NC. (E) qRT-PCR analysis of C20ORF7 , NUBPL , NDUFAF3 and NDUFAF4 mRNA expression in shGTPBP3 and NC cells. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

Techniques Used: Expressing, Western Blot, Negative Control, Blue Native PAGE, Quantitative RT-PCR

Increased mRNA expression in shGTPBP3 cells of genes involved in glycolysis and fatty acid oxidation. qRT-PCR analysis of mRNA expression of genes related to: 1) glycolysis ( GLUT1 : glucose transporter 1, PKF1 : phosphofructokinase, and LDHA and LDHB : lactate dehydrogenase A and B, respectively), 2) fatty acid oxidation ( CPT1 : carnitine palmitoyltransferase I, LCAD : long-chain acyl-CoA dehydrogenase, and MCAD : medium-chain acyl-CoA dehydrogenase), and 3) glutaminolysis ( ASCT2 : glutamine/amino acid transporter 2, SN2 : glutamine/amino acid transporter system N, and GLS : glutaminase) in shGTPBP3-1, shGTPBP3-2 and NC cells. Data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p
Figure Legend Snippet: Increased mRNA expression in shGTPBP3 cells of genes involved in glycolysis and fatty acid oxidation. qRT-PCR analysis of mRNA expression of genes related to: 1) glycolysis ( GLUT1 : glucose transporter 1, PKF1 : phosphofructokinase, and LDHA and LDHB : lactate dehydrogenase A and B, respectively), 2) fatty acid oxidation ( CPT1 : carnitine palmitoyltransferase I, LCAD : long-chain acyl-CoA dehydrogenase, and MCAD : medium-chain acyl-CoA dehydrogenase), and 3) glutaminolysis ( ASCT2 : glutamine/amino acid transporter 2, SN2 : glutamine/amino acid transporter system N, and GLS : glutaminase) in shGTPBP3-1, shGTPBP3-2 and NC cells. Data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

Techniques Used: Expressing, Quantitative RT-PCR

Expression of GTPBP3 is down regulated in shGTPBP3 cells. (A) qRT-PCR analysis of GTPBP3 mRNA expression in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. (B) Western blot analysis of GTPBP3 protein in shGTPBP3-1, shGTPBP3-2 and NC cells, using porin as a loading control. Positions of molecular-mass markers (in kDa) are indicated on the left. The arrow denotes a non-specific band. (C) Densitometric analysis of GTPBP3 protein normalized to loading control and represented as % of NC. In A and C, mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at ***p
Figure Legend Snippet: Expression of GTPBP3 is down regulated in shGTPBP3 cells. (A) qRT-PCR analysis of GTPBP3 mRNA expression in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. (B) Western blot analysis of GTPBP3 protein in shGTPBP3-1, shGTPBP3-2 and NC cells, using porin as a loading control. Positions of molecular-mass markers (in kDa) are indicated on the left. The arrow denotes a non-specific band. (C) Densitometric analysis of GTPBP3 protein normalized to loading control and represented as % of NC. In A and C, mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at ***p

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

AMPK contributes to the down regulation of NDUFAF4. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 48 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of NDUFAF4 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR or with Compound C (CC) as in (A). (C and D) Blue Native-PAGE analysis of Complex I in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) with AICAR (C) or with Compound C (D) as in (A). (E) Densitometric analysis of Complex I normalized to the loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p
Figure Legend Snippet: AMPK contributes to the down regulation of NDUFAF4. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 48 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of NDUFAF4 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR or with Compound C (CC) as in (A). (C and D) Blue Native-PAGE analysis of Complex I in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) with AICAR (C) or with Compound C (D) as in (A). (E) Densitometric analysis of Complex I normalized to the loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p

Techniques Used: Western Blot, Negative Control, Quantitative RT-PCR, Expressing, Blue Native PAGE

67) Product Images from "miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus"

Article Title: miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052555

TBC1D2 and LDLR are directly targeted by miR-17. ( A ) miR-17 targets 3′UTRs of TBC1D2 and LDLR as determined by a luciferase reporter assay. HeLa cells were co-transfected with the reporters containing wild-type 3′UTRs of LDLR and TBC1D2 and mutated 3′UTR of TBC1D2, Pre-miR-17 and Anti-miR-17. Luciferase activity was measured 24 h following co-transfection. The activity of luciferase for each experiment was normalized to the activity of the control samples, co-transfected with the respective reporter vector and control Anti-miR or Pre-miR (see Methods ). The bars show mean fold changes of luciferase activity and the error bars show s.e.m. derived from 3 independent experiments. Over-expression of miR-17 decreased the expression level of LDLR ( B ) and TBC1D2 ( C ) mRNAs as determined by qRT-PCR. The relative expression levels of TBC1D2 and LDLR after treatment with siRNAs and Pre-miR-17 were normalized to the expression level when control siRNAs and control Pre-miR were transfected (see Methods ). Bars show mean of the relative mRNA expression levels and error bars show s.e.m. derived from 2 independent experiments. *, p≤0,05; **, p≤0,01; ***, p≤0,001.
Figure Legend Snippet: TBC1D2 and LDLR are directly targeted by miR-17. ( A ) miR-17 targets 3′UTRs of TBC1D2 and LDLR as determined by a luciferase reporter assay. HeLa cells were co-transfected with the reporters containing wild-type 3′UTRs of LDLR and TBC1D2 and mutated 3′UTR of TBC1D2, Pre-miR-17 and Anti-miR-17. Luciferase activity was measured 24 h following co-transfection. The activity of luciferase for each experiment was normalized to the activity of the control samples, co-transfected with the respective reporter vector and control Anti-miR or Pre-miR (see Methods ). The bars show mean fold changes of luciferase activity and the error bars show s.e.m. derived from 3 independent experiments. Over-expression of miR-17 decreased the expression level of LDLR ( B ) and TBC1D2 ( C ) mRNAs as determined by qRT-PCR. The relative expression levels of TBC1D2 and LDLR after treatment with siRNAs and Pre-miR-17 were normalized to the expression level when control siRNAs and control Pre-miR were transfected (see Methods ). Bars show mean of the relative mRNA expression levels and error bars show s.e.m. derived from 2 independent experiments. *, p≤0,05; **, p≤0,01; ***, p≤0,001.

Techniques Used: Luciferase, Reporter Assay, Transfection, Activity Assay, Cotransfection, Plasmid Preparation, Derivative Assay, Over Expression, Expressing, Quantitative RT-PCR

68) Product Images from "Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris"

Article Title: Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

Journal: BioMed Research International

doi: 10.1155/2014/487050

CvHSP90 mRNA expression levels in different heat shock times (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, and 12 h) at 35°C were analyzed by real-time quantitative RT-PCR. CvHSP90 mRNA expression was normalized to the control group, and β -actin gene was used as internal control to calibrate the cDNA template for all the samples. Each bar represents the mean value from five determinations with standard error. Significant differences across control were indicated with an asterisk at P
Figure Legend Snippet: CvHSP90 mRNA expression levels in different heat shock times (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, and 12 h) at 35°C were analyzed by real-time quantitative RT-PCR. CvHSP90 mRNA expression was normalized to the control group, and β -actin gene was used as internal control to calibrate the cDNA template for all the samples. Each bar represents the mean value from five determinations with standard error. Significant differences across control were indicated with an asterisk at P

Techniques Used: Expressing, Quantitative RT-PCR

CvHSP90 mRNA expression levels relative to β -actin mRNA levels under stress of different salt concentrations analyzed by real-time quantitative RT-PCR. The β -actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean ± SE ( N = 5). Significant differences across control were indicated with an asterisk at P
Figure Legend Snippet: CvHSP90 mRNA expression levels relative to β -actin mRNA levels under stress of different salt concentrations analyzed by real-time quantitative RT-PCR. The β -actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean ± SE ( N = 5). Significant differences across control were indicated with an asterisk at P

Techniques Used: Expressing, Quantitative RT-PCR

69) Product Images from "Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1"

Article Title: Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1

Journal: Toxins

doi: 10.3390/toxins7082723

( A ) The changes in the amount of OTA product at different time points during the growth of A. ochraceus fc-1. OTA concentrations were determined by HPLC-FLD using an OTA standard; ( B ) Relative expression of the AoOTApks-1 and -2 genes was assayed at different time points using a 7500 real-time PCR system. The glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was used as a control.
Figure Legend Snippet: ( A ) The changes in the amount of OTA product at different time points during the growth of A. ochraceus fc-1. OTA concentrations were determined by HPLC-FLD using an OTA standard; ( B ) Relative expression of the AoOTApks-1 and -2 genes was assayed at different time points using a 7500 real-time PCR system. The glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was used as a control.

Techniques Used: High Performance Liquid Chromatography, Expressing, Real-time Polymerase Chain Reaction

70) Product Images from "piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts"

Article Title: piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1464

Human full-length dystrophin expression in PB -transposed C2C12 myoblast-derived differentiated myotubes. ( A ) The transcript levels of the full-length human dystrophin (black bars) and the GFP (white bars) were detected by qRT-PCR in C2C12 cells transposed with hyPB + PB-SPc-DYS-Pgk-GFP , while in proliferation or in differentiated myotubes (D: differentiated sample). Results were presented as mean±SEM of triplicate qRT-PCR analyses performed for three independent biological replicates; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ns: not significant). ( B ) RT-PCR showing expression of full-length human dystrophin transcript in myotubes derived from C2C12 cells that had undergone transposition after co-transfection with hyPB and PB-SPc-DYS-Pgk-GFP . Three different primer pairs were used that recognize respectively (1) the N-terminal, (2) a central region and (3) the C-terminal sequences, yielding amplicons of 221 bp, 222 bp and 228 bp, respectively. A schematic representation of the PCR primers relative to the different regions of the dystrophin transcript is depicted. Human skeletal muscle cells (SKM) differentiated in myotubes were used as a positive control. Negative controls included untreated differentiated C2C12 cells (designated as C2C12) or differentiated C2C12 cells co-transfected with the PB-SPc-DYS-Pgk-GFP transposon and an empty expression plasmid without transposase (designated as Empty). L: 50 bp ladder. ( C ) Western blot analysis demonstrating expression of the full-length human dystrophin protein (427 kDa) in myotubes derived from C2C12 cells that had undergone transposition after co-transfection with hyPB and PB-SPc-DYS-Pgk-GFP . The antibody NCL-DYS3 was used to detect the human dystrophin. Non-transfected differentiated C2C12 cells or differentiated C2C12 cells co-transfected with the PB-SPc-DYS-Pgk-GFP transposon and an empty expression plasmid without transposase were included as negative controls. Normal human skeletal muscle cells were used as positive control. In vitro skeletal muscle differentiation was confirmed for all the samples by the expression of the MyHC protein (223 kDa). β-Tubulin (50 kDa) was used to normalize the amount of loaded proteins. ( D, E ) Immunofluorescence staining on C2C12 myotubes upon 4 days in conditioning medium (24 days post-EP) C2C12 cells transposed with hyPB + PB-SPc-DYS-Pgk-GFP showed a co-localized expression of GFP (D, in green) and full-length human dystrophin (D, E in red) when differentiated in myotubes, as confirmed by the myosin expression (E, in white). The nuclei were stained with Hoechst (Scale bar 100 μm). The Ab mouse anti-human dystrophin NCL-DYS3 was used.
Figure Legend Snippet: Human full-length dystrophin expression in PB -transposed C2C12 myoblast-derived differentiated myotubes. ( A ) The transcript levels of the full-length human dystrophin (black bars) and the GFP (white bars) were detected by qRT-PCR in C2C12 cells transposed with hyPB + PB-SPc-DYS-Pgk-GFP , while in proliferation or in differentiated myotubes (D: differentiated sample). Results were presented as mean±SEM of triplicate qRT-PCR analyses performed for three independent biological replicates; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ns: not significant). ( B ) RT-PCR showing expression of full-length human dystrophin transcript in myotubes derived from C2C12 cells that had undergone transposition after co-transfection with hyPB and PB-SPc-DYS-Pgk-GFP . Three different primer pairs were used that recognize respectively (1) the N-terminal, (2) a central region and (3) the C-terminal sequences, yielding amplicons of 221 bp, 222 bp and 228 bp, respectively. A schematic representation of the PCR primers relative to the different regions of the dystrophin transcript is depicted. Human skeletal muscle cells (SKM) differentiated in myotubes were used as a positive control. Negative controls included untreated differentiated C2C12 cells (designated as C2C12) or differentiated C2C12 cells co-transfected with the PB-SPc-DYS-Pgk-GFP transposon and an empty expression plasmid without transposase (designated as Empty). L: 50 bp ladder. ( C ) Western blot analysis demonstrating expression of the full-length human dystrophin protein (427 kDa) in myotubes derived from C2C12 cells that had undergone transposition after co-transfection with hyPB and PB-SPc-DYS-Pgk-GFP . The antibody NCL-DYS3 was used to detect the human dystrophin. Non-transfected differentiated C2C12 cells or differentiated C2C12 cells co-transfected with the PB-SPc-DYS-Pgk-GFP transposon and an empty expression plasmid without transposase were included as negative controls. Normal human skeletal muscle cells were used as positive control. In vitro skeletal muscle differentiation was confirmed for all the samples by the expression of the MyHC protein (223 kDa). β-Tubulin (50 kDa) was used to normalize the amount of loaded proteins. ( D, E ) Immunofluorescence staining on C2C12 myotubes upon 4 days in conditioning medium (24 days post-EP) C2C12 cells transposed with hyPB + PB-SPc-DYS-Pgk-GFP showed a co-localized expression of GFP (D, in green) and full-length human dystrophin (D, E in red) when differentiated in myotubes, as confirmed by the myosin expression (E, in white). The nuclei were stained with Hoechst (Scale bar 100 μm). The Ab mouse anti-human dystrophin NCL-DYS3 was used.

Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Cotransfection, Polymerase Chain Reaction, Positive Control, Transfection, Plasmid Preparation, Western Blot, In Vitro, Immunofluorescence, Staining

PB -mediated transposition of the full-length human dystrophin CDS in GRMD MABs. ( A ) The bar graph shows the percentage of GFP+ GRMD MABs at the day of the sorting performed at 7 days post-EP. Optimized and equimolar doses have been used in the different conditions. The statistically significant difference in transfection efficiency detected when PB-SPc-DYS-Pgk-GFP or PB-Pgk-GFP transposon are deployed reflects the different sizes of the plasmids. Results were presented as mean±SEM of three independent biological replicates; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ns: not significant). ( B ) The GFP+ populations were monitored at different time points for 28 days post sorting. GRMD MABs co-transfected with the empty plasmid and the same transposons, and untransfected cells were used as controls. Shown are mean±SD on a biological replicate; two-way ANOVA (Bonferroni's multiple comparison test); *** P ≤ 0.001. ( C ) Live cell imaging showing the GFP expression of the different conditions at day 21 post sorting (Scale bar 100 μm). ( D ) Bar graph representing the transposon copies per diploid genome detected by qPCR in transposed GRMD MABs ( PB-SPc-DYS-Pgk-GFP 0.5 ± 0.03 copies/cell; PB-Pgk-GFP 1.7 ± 0.04 copies/cell). Shown are mean±SEM of triplicate qRT-PCR analyses performed for a biological replicate; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ** P ≤ 0.01).
Figure Legend Snippet: PB -mediated transposition of the full-length human dystrophin CDS in GRMD MABs. ( A ) The bar graph shows the percentage of GFP+ GRMD MABs at the day of the sorting performed at 7 days post-EP. Optimized and equimolar doses have been used in the different conditions. The statistically significant difference in transfection efficiency detected when PB-SPc-DYS-Pgk-GFP or PB-Pgk-GFP transposon are deployed reflects the different sizes of the plasmids. Results were presented as mean±SEM of three independent biological replicates; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ns: not significant). ( B ) The GFP+ populations were monitored at different time points for 28 days post sorting. GRMD MABs co-transfected with the empty plasmid and the same transposons, and untransfected cells were used as controls. Shown are mean±SD on a biological replicate; two-way ANOVA (Bonferroni's multiple comparison test); *** P ≤ 0.001. ( C ) Live cell imaging showing the GFP expression of the different conditions at day 21 post sorting (Scale bar 100 μm). ( D ) Bar graph representing the transposon copies per diploid genome detected by qPCR in transposed GRMD MABs ( PB-SPc-DYS-Pgk-GFP 0.5 ± 0.03 copies/cell; PB-Pgk-GFP 1.7 ± 0.04 copies/cell). Shown are mean±SEM of triplicate qRT-PCR analyses performed for a biological replicate; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ** P ≤ 0.01).

Techniques Used: Transfection, Two Tailed Test, Plasmid Preparation, Live Cell Imaging, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

PB- mediated expression of MD1 and MD2 in C2C12 myoblasts. ( A ) The bar graphs show respectively the percentage of GFP+ (left) and the mean fluorescence intensity (MFI; right) of C2C12 cells at 48 and 72 h post-electroporation (EP) with the PB-SPc-GFP transposon when co-transfected with the native mPB transposase or the empty plasmid (ratio of 1.26 pmol transposase DNA: 3.48 pmol transposon DNA). Untransfected cells are also shown. Shown are mean±SEM of three independent biological replicates; two-tailed unpaired Student's t-test (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant). ( B ) Live cell imaging showing the GFP expression of the conditions described in (A) at 24, 48 and 72 h post-EP (UNTR: untransfected; scale bar 100 μm). ( C,D ) The bar graphs depict the transcript levels of GFP (C) or MD1 and MD2 (D) detected by qRT-PCR in PB -transposed C2C12 myoblasts in proliferation (white bars) and myotubes in differentiation (black bars). The transcript levels of the human dystrophin from the human skeletal muscle (SKM) cells and myotubes were used as positive control. Values were normalized for the myosin heavy chain (MyHC) and shown as relative expression. Shown are mean±SEM of triplicate qRT-PCR analyses performed for three independent biological replicates; two-tailed unpaired Student's t-test (*** P ≤ 0.001; ** P ≤ 0.01; ns: not significant). ( E,F ) Immunofluorescence staining on transposed C2C12 cells upon skeletal muscle differentiation in vitro has been used to detect the GFP expression (E, in green) in MyHC positive myotubes (E, in red), or the human microdystrophins MD1 and MD2 expression (F, in green) in myosin positive myotubes (F in red). The nuclei were stained with Hoechst (Scale bar 100 μm). The mouse anti-human dystrophin NCL-DYS3 antibody was used.
Figure Legend Snippet: PB- mediated expression of MD1 and MD2 in C2C12 myoblasts. ( A ) The bar graphs show respectively the percentage of GFP+ (left) and the mean fluorescence intensity (MFI; right) of C2C12 cells at 48 and 72 h post-electroporation (EP) with the PB-SPc-GFP transposon when co-transfected with the native mPB transposase or the empty plasmid (ratio of 1.26 pmol transposase DNA: 3.48 pmol transposon DNA). Untransfected cells are also shown. Shown are mean±SEM of three independent biological replicates; two-tailed unpaired Student's t-test (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant). ( B ) Live cell imaging showing the GFP expression of the conditions described in (A) at 24, 48 and 72 h post-EP (UNTR: untransfected; scale bar 100 μm). ( C,D ) The bar graphs depict the transcript levels of GFP (C) or MD1 and MD2 (D) detected by qRT-PCR in PB -transposed C2C12 myoblasts in proliferation (white bars) and myotubes in differentiation (black bars). The transcript levels of the human dystrophin from the human skeletal muscle (SKM) cells and myotubes were used as positive control. Values were normalized for the myosin heavy chain (MyHC) and shown as relative expression. Shown are mean±SEM of triplicate qRT-PCR analyses performed for three independent biological replicates; two-tailed unpaired Student's t-test (*** P ≤ 0.001; ** P ≤ 0.01; ns: not significant). ( E,F ) Immunofluorescence staining on transposed C2C12 cells upon skeletal muscle differentiation in vitro has been used to detect the GFP expression (E, in green) in MyHC positive myotubes (E, in red), or the human microdystrophins MD1 and MD2 expression (F, in green) in myosin positive myotubes (F in red). The nuclei were stained with Hoechst (Scale bar 100 μm). The mouse anti-human dystrophin NCL-DYS3 antibody was used.

Techniques Used: Expressing, Fluorescence, Electroporation, Transfection, Plasmid Preparation, Two Tailed Test, Live Cell Imaging, Quantitative RT-PCR, Positive Control, Immunofluorescence, Staining, In Vitro

Correction of GRMD MABs by the PB -mediated expression of the full-length human dystrophin. ( A ) The transcript levels of the full-length human dystrophin (black bars) and GFP (white bars) were detected by qRT-PCR analysis in GRMD MABs transposed with hyPB + PB-SPc-DYS-Pgk-GFP or PB-Pgk-GFP . SKM cells have been used as positive control. Samples are indicated with (D) when differentiated in myocytes/myotubes. Results were presented as mean±SEM of triplicate qRT-PCR analyses performed for a biological replicate; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ** P ≤ 0.01; ns: not significant; D: differentiated sample). ( B ) RT-PCR demonstrating expression of full-length human dystrophin transcript in differentiated GRMD MABs that had undergone transposition after co-transfection with hyPB and PB-SPc-DYS-Pgk-GFP . Three different primer pairs were used that recognize respectively (1) the N-terminal, (2) a central region and (3) the C-terminal sequences, yielding amplicons of 221 bp, 222 bp and 228 bp, respectively. A schematic representation of the PCR primers relative to the different regions of the dystrophin transcript is depicted. Controls included differentiated human SKM, untreated differentiated MABs (designated as GRMD MABs) and differentiated GRMD MABs co-transfected with the PB-SPc-DYS-Pgk-GFP transposon and an empty expression plasmid without transposase (designated as Empty). L: 50 bp ladder. ( C,D ) At 21 days post-EP, GRMD MABs corrected with hyPB + PB-SPc-DYS-Pgk-GFP were induced to differentiate in skeletal muscle in vitro upon MyoD-ER overexpression and 6 days in conditioning medium. Immunofluorescence analysis confirms the expression and co-localization of the full-length human dystrophin (C, D in red) with the GFP (C, in green), or with the myosin (D, in white). The conditions empty plasmid + PB-SPc-DYS-Pgk-GFP as well as the untransfected cells are also shown as negative controls. The nuclei were stained with Hoechst (Scale bar 100 μm). The mouse anti-dystrophin NCL-DYS1 and NCL-DYS2 antibodies were used.
Figure Legend Snippet: Correction of GRMD MABs by the PB -mediated expression of the full-length human dystrophin. ( A ) The transcript levels of the full-length human dystrophin (black bars) and GFP (white bars) were detected by qRT-PCR analysis in GRMD MABs transposed with hyPB + PB-SPc-DYS-Pgk-GFP or PB-Pgk-GFP . SKM cells have been used as positive control. Samples are indicated with (D) when differentiated in myocytes/myotubes. Results were presented as mean±SEM of triplicate qRT-PCR analyses performed for a biological replicate; two-tailed unpaired Student's t -test (*** P ≤ 0.001; ** P ≤ 0.01; ns: not significant; D: differentiated sample). ( B ) RT-PCR demonstrating expression of full-length human dystrophin transcript in differentiated GRMD MABs that had undergone transposition after co-transfection with hyPB and PB-SPc-DYS-Pgk-GFP . Three different primer pairs were used that recognize respectively (1) the N-terminal, (2) a central region and (3) the C-terminal sequences, yielding amplicons of 221 bp, 222 bp and 228 bp, respectively. A schematic representation of the PCR primers relative to the different regions of the dystrophin transcript is depicted. Controls included differentiated human SKM, untreated differentiated MABs (designated as GRMD MABs) and differentiated GRMD MABs co-transfected with the PB-SPc-DYS-Pgk-GFP transposon and an empty expression plasmid without transposase (designated as Empty). L: 50 bp ladder. ( C,D ) At 21 days post-EP, GRMD MABs corrected with hyPB + PB-SPc-DYS-Pgk-GFP were induced to differentiate in skeletal muscle in vitro upon MyoD-ER overexpression and 6 days in conditioning medium. Immunofluorescence analysis confirms the expression and co-localization of the full-length human dystrophin (C, D in red) with the GFP (C, in green), or with the myosin (D, in white). The conditions empty plasmid + PB-SPc-DYS-Pgk-GFP as well as the untransfected cells are also shown as negative controls. The nuclei were stained with Hoechst (Scale bar 100 μm). The mouse anti-dystrophin NCL-DYS1 and NCL-DYS2 antibodies were used.

Techniques Used: Expressing, Quantitative RT-PCR, Positive Control, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Cotransfection, Polymerase Chain Reaction, Transfection, Plasmid Preparation, In Vitro, Over Expression, Immunofluorescence, Staining

71) Product Images from "Tri6 Is a Global Transcription Regulator in the Phytopathogen Fusarium graminearum"

Article Title: Tri6 Is a Global Transcription Regulator in the Phytopathogen Fusarium graminearum

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002266

Tri6 auto-regulates its own expression in nutrient-rich conditions. A ) Arrangement of the Tri6 gene and location of Tri6 primers used in the RT-qPCR analysis of wildtype ( Wt ) and transgenic strains. The solid vertical box indicates the location of Tri6-ORF primers in the coding region of Tri6 (Filled horizontal box) of the wildtype and the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The dotted vertical box indicates the location of Tri6 5′ UTR primers in the wildtype ( Wt ), the Tri6 mutant ( tri6 Δand the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The location of the Hygromycin gene within the Tri6 coding region of the tri6 Δstrain ( tri6 Δis indicated by the striped horizontal box. Gpd indicates the promoter used to over-express Tri6 [41] . The solid horizontal lines indicate 5′ and 3′ flanking regions of the Tri6 gene. B ) The quantitative real-time PCR (RT-qPCR) analysis of Tri genes in wildtype, tri6 Δand the tri6 Δ Tri6 strains grown in nutrient-rich conditions. RT-qPCR reactions were performed in triplicates using Applied Biosystems Power SYBR Green kit and the Applied Biosystems Step One Plus Real-Time PCR System. A list of qPCR primers for all the Tri genes is listed in the Table S2 . The β-tubulin gene ( FGSG_09530 ) was used as the internal control and the data was imported and Relative quantity (RQ) was derived by the Relative standard method included in the StepOne 2.1 software. C ) Identical to B ), except the internal control used was Gapdh ( FGSG_06257 ). The figures are representative of two independent biological replicates.
Figure Legend Snippet: Tri6 auto-regulates its own expression in nutrient-rich conditions. A ) Arrangement of the Tri6 gene and location of Tri6 primers used in the RT-qPCR analysis of wildtype ( Wt ) and transgenic strains. The solid vertical box indicates the location of Tri6-ORF primers in the coding region of Tri6 (Filled horizontal box) of the wildtype and the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The dotted vertical box indicates the location of Tri6 5′ UTR primers in the wildtype ( Wt ), the Tri6 mutant ( tri6 Δand the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The location of the Hygromycin gene within the Tri6 coding region of the tri6 Δstrain ( tri6 Δis indicated by the striped horizontal box. Gpd indicates the promoter used to over-express Tri6 [41] . The solid horizontal lines indicate 5′ and 3′ flanking regions of the Tri6 gene. B ) The quantitative real-time PCR (RT-qPCR) analysis of Tri genes in wildtype, tri6 Δand the tri6 Δ Tri6 strains grown in nutrient-rich conditions. RT-qPCR reactions were performed in triplicates using Applied Biosystems Power SYBR Green kit and the Applied Biosystems Step One Plus Real-Time PCR System. A list of qPCR primers for all the Tri genes is listed in the Table S2 . The β-tubulin gene ( FGSG_09530 ) was used as the internal control and the data was imported and Relative quantity (RQ) was derived by the Relative standard method included in the StepOne 2.1 software. C ) Identical to B ), except the internal control used was Gapdh ( FGSG_06257 ). The figures are representative of two independent biological replicates.

Techniques Used: Expressing, Quantitative RT-PCR, Transgenic Assay, Mutagenesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Derivative Assay, Software

72) Product Images from "MqsR/MqsA Toxin/Antitoxin System Regulates Persistence and Biofilm Formation in Pseudomonas putida KT2440"

Article Title: MqsR/MqsA Toxin/Antitoxin System Regulates Persistence and Biofilm Formation in Pseudomonas putida KT2440

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00840

MqsA negatively regulates algU, PP_3288 and nadB . (A) Fold changes of expression of three genes in ΔmqsA vs. KT2440, and ΔmqsA/ pHGE- mqsA vs. ΔmqsA/ pHGE were quantified by qRT-PCR. (B) Mid-log phase cells of the reporter strains harboring pMQ70, pMQ70- mqsRA , and pMQ70- mqsA (with 10 mM L -arabinose) were tested for β-galactosidase activity, respectively. Three independent cultures for each strain were used and the data are shown as means ± standard deviations. Asterisks represent a statistically significant difference. ( P
Figure Legend Snippet: MqsA negatively regulates algU, PP_3288 and nadB . (A) Fold changes of expression of three genes in ΔmqsA vs. KT2440, and ΔmqsA/ pHGE- mqsA vs. ΔmqsA/ pHGE were quantified by qRT-PCR. (B) Mid-log phase cells of the reporter strains harboring pMQ70, pMQ70- mqsRA , and pMQ70- mqsA (with 10 mM L -arabinose) were tested for β-galactosidase activity, respectively. Three independent cultures for each strain were used and the data are shown as means ± standard deviations. Asterisks represent a statistically significant difference. ( P

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

73) Product Images from "Osteopontin Expression in the Brain Triggers Localized Inflammation and Cell Death When Immune Cells Are Activated by Pertussis Toxin"

Article Title: Osteopontin Expression in the Brain Triggers Localized Inflammation and Cell Death When Immune Cells Are Activated by Pertussis Toxin

Journal: Mediators of Inflammation

doi: 10.1155/2014/358218

Proinflammatory molecules modified by OPN. QRT-PCR was used to evaluate levels of chemokine receptors and proinflammatory molecules using SABiosciences PCR array, and SyBr Green/ROX detectors in an ABI HT7900 Fast apparatus. We measured 84 genes involved in inflammatory responses, including chemokines and receptors (PAMM-022Z, Qiagen). Results show the genes that were significantly changed. (a) Genes upregulated in OPN-injected brain lobes compared to β -gal controls. (b) Genes downregulated in OPN compared to β -gal controls. Results represent average ± SD of 6 experimental lobes injected with either β -gal or OPN-encoding vector. Experiments were performed in triplicate. * P
Figure Legend Snippet: Proinflammatory molecules modified by OPN. QRT-PCR was used to evaluate levels of chemokine receptors and proinflammatory molecules using SABiosciences PCR array, and SyBr Green/ROX detectors in an ABI HT7900 Fast apparatus. We measured 84 genes involved in inflammatory responses, including chemokines and receptors (PAMM-022Z, Qiagen). Results show the genes that were significantly changed. (a) Genes upregulated in OPN-injected brain lobes compared to β -gal controls. (b) Genes downregulated in OPN compared to β -gal controls. Results represent average ± SD of 6 experimental lobes injected with either β -gal or OPN-encoding vector. Experiments were performed in triplicate. * P

Techniques Used: Modification, Quantitative RT-PCR, Polymerase Chain Reaction, SYBR Green Assay, Injection, Plasmid Preparation

74) Product Images from "Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells"

Article Title: Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku346

Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. ( A ) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. ( B, C ) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. ( D ) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. ( E ) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( F ) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = C t sample - C t 18S ) values were plotted. ( G ) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D . Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). ( H ) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. ( I ) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( J ) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or > 100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. ( K ) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. ( L ) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. ( M ) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments ( n = 3) when ns: non-significant, * P
Figure Legend Snippet: Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. ( A ) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. ( B, C ) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. ( D ) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. ( E ) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( F ) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = C t sample - C t 18S ) values were plotted. ( G ) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D . Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). ( H ) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. ( I ) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( J ) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or > 100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. ( K ) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. ( L ) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. ( M ) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments ( n = 3) when ns: non-significant, * P

Techniques Used: Co-Culture Assay, Activity Assay, Transfection, Cell Culture, Expressing, FACS, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction

IGF1 secreted by HepG2 reduces activity and expression of miR-122 in Huh7 cells. ( A ) Effect of GW4869 on transfer of anti-miR-122 signal from HepG2 to Huh7 cells. HepG2 cells and Huh7 cells were either co-cultured together or mixed after being cultured separately for 48 h in presence and in absence of GW4869. Real-time quantification of miR-122 was then done to detect the level of miR-122 in both control and co-cultured samples in presence or absence of GW4869. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( B ) Effect of different growth factors on miR-122 activity in Huh7 cells. Huh7 cells transfected with RL reporters were incubated for indicated concentrations (ng/ml) of Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor β (TGF-β), Insulin-like Growth Facto1 (IGF1) and Insulin-like Growth Factor 2 (IGF2) overnight in DMEM and luciferase activities were measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. ( C, D ) Effect of IGF1 on miR-122 activity (C) and Level (D) in Huh7 cells. Cells were incubated with exosome depleted Huh7 CM alone or supplemented with 100 ng/ml IGFI for 72 h with fresh changes after every 36 h. HepG2 CM was used as a positive control. ( E ) Dose response curve to determine the effect of various concentrations of recombinant IGF1 (ng/ml) on the miR-122 level of Huh7 cells. Huh7 cells were incubated for 24 h with DMEM containing IGF1 (0–50 ng/ml). Total RNA was extracted from the cells and qPCR was done to determine the miR-122 level. We found that the decrease in miR-122 level starts from 5 ng/ml of IGF1. For panel C experiments were performed in triplicate and P value was calculated by using unpaired t test. For panels D and E data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-24 and let-7a level change detected by real-time quantification in Huh7 cells treated with IGF1. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( G ) Effect of IGF1 on miR-122 level in primary mouse hepatocytes treated with IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Quantification of pre-miR-122, and other hepatic nuclear factor expression in Huh7 cells incubated for 72 h either with HepG2 CM or exosome depleted Huh7 CM containing 0 and 50 ng/ml of IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) Effect of IGF1 depletion in HepG2 or IGF1R depletion in Huh7 on miR-122 activity in Huh7 cells in presence of HepG2 CM. Huh7 cells (control or IGF1R depleted), expressing miR-122 RL reporter, were incubated with CM from normal or IGF1 depleted HepG2 for 72 h to determine the specificity of IGF1 to decrease miR-122 activity in Huh7 cells. For control experiments, non-target siRNA was used. Incubation of HepG2 CM with αIGF1 antibody removed the anti-miR-122 activity. nIgG was used as a control. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by taking the control as 1 and expressing repression values relative to 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( J ) Effect of IGF1 depletion on miR-122 level in Huh7 cells incubated with CM from HepG2 cells transfected with a non-target or IGF1 specific siRNAs. The miR-122 level of the cells was detected by RT-PCR. Data represents five independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( K ) miR-122 level in Huh7 cells depleted for IGF1R (siIGF1R transfected) against control siRNA transfected cells in presence of HepG2 CM. Cellular miR-122 levels were quantified by RT-PCR. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( L ) Effect of HepG2 CM on IGFR1R expression in Huh7 cells. Huh7 cells incubated for 72 h with HepG2 CM were analysed for IGF1R mRNA levels by qRT-PCR. Normalization was done by 18S rRNA. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P
Figure Legend Snippet: IGF1 secreted by HepG2 reduces activity and expression of miR-122 in Huh7 cells. ( A ) Effect of GW4869 on transfer of anti-miR-122 signal from HepG2 to Huh7 cells. HepG2 cells and Huh7 cells were either co-cultured together or mixed after being cultured separately for 48 h in presence and in absence of GW4869. Real-time quantification of miR-122 was then done to detect the level of miR-122 in both control and co-cultured samples in presence or absence of GW4869. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( B ) Effect of different growth factors on miR-122 activity in Huh7 cells. Huh7 cells transfected with RL reporters were incubated for indicated concentrations (ng/ml) of Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor β (TGF-β), Insulin-like Growth Facto1 (IGF1) and Insulin-like Growth Factor 2 (IGF2) overnight in DMEM and luciferase activities were measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. ( C, D ) Effect of IGF1 on miR-122 activity (C) and Level (D) in Huh7 cells. Cells were incubated with exosome depleted Huh7 CM alone or supplemented with 100 ng/ml IGFI for 72 h with fresh changes after every 36 h. HepG2 CM was used as a positive control. ( E ) Dose response curve to determine the effect of various concentrations of recombinant IGF1 (ng/ml) on the miR-122 level of Huh7 cells. Huh7 cells were incubated for 24 h with DMEM containing IGF1 (0–50 ng/ml). Total RNA was extracted from the cells and qPCR was done to determine the miR-122 level. We found that the decrease in miR-122 level starts from 5 ng/ml of IGF1. For panel C experiments were performed in triplicate and P value was calculated by using unpaired t test. For panels D and E data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-24 and let-7a level change detected by real-time quantification in Huh7 cells treated with IGF1. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( G ) Effect of IGF1 on miR-122 level in primary mouse hepatocytes treated with IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Quantification of pre-miR-122, and other hepatic nuclear factor expression in Huh7 cells incubated for 72 h either with HepG2 CM or exosome depleted Huh7 CM containing 0 and 50 ng/ml of IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) Effect of IGF1 depletion in HepG2 or IGF1R depletion in Huh7 on miR-122 activity in Huh7 cells in presence of HepG2 CM. Huh7 cells (control or IGF1R depleted), expressing miR-122 RL reporter, were incubated with CM from normal or IGF1 depleted HepG2 for 72 h to determine the specificity of IGF1 to decrease miR-122 activity in Huh7 cells. For control experiments, non-target siRNA was used. Incubation of HepG2 CM with αIGF1 antibody removed the anti-miR-122 activity. nIgG was used as a control. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by taking the control as 1 and expressing repression values relative to 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( J ) Effect of IGF1 depletion on miR-122 level in Huh7 cells incubated with CM from HepG2 cells transfected with a non-target or IGF1 specific siRNAs. The miR-122 level of the cells was detected by RT-PCR. Data represents five independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( K ) miR-122 level in Huh7 cells depleted for IGF1R (siIGF1R transfected) against control siRNA transfected cells in presence of HepG2 CM. Cellular miR-122 levels were quantified by RT-PCR. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( L ) Effect of HepG2 CM on IGFR1R expression in Huh7 cells. Huh7 cells incubated for 72 h with HepG2 CM were analysed for IGF1R mRNA levels by qRT-PCR. Normalization was done by 18S rRNA. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P

Techniques Used: Activity Assay, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Incubation, Luciferase, Positive Control, Recombinant, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. ( A ) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. ( B ) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( C ) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( D ) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( E ) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. ( G ) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( J ) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2 −ΔCt where ΔC t was calculated by subtracting the C t for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P
Figure Legend Snippet: HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. ( A ) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. ( B ) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( C ) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( D ) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( E ) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. ( G ) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( J ) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2 −ΔCt where ΔC t was calculated by subtracting the C t for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P

Techniques Used: Expressing, Co-Culture Assay, Activity Assay, Cell Culture, Transfection, Luciferase, Lysis, Isolation, Real-time Polymerase Chain Reaction, Plasmid Preparation, Incubation, Quantitative RT-PCR, Chromatin Immunoprecipitation, In Vivo, Immunoprecipitation, Binding Assay

75) Product Images from "Replication Study: BET bromodomain inhibition as a therapeutic strategy to target c-Myc"

Article Title: Replication Study: BET bromodomain inhibition as a therapeutic strategy to target c-Myc

Journal: eLife

doi: 10.7554/eLife.21253

MYC expression in JQ1-treated MM.1S-luc cells. MM.1S-luc cells were treated with 500 nM (+)-JQ1, 500 nM (−)-JQ1, or an equivalent volume of DMSO. Total RNA was isolated at 0 hr, 1 hr, and 8 hr after treatment and qRT-PCR analysis was performed to detect MYC and GAPDH levels. Relative expression ( MYC/GAPDH ) is presented for each time point and condition normalized to (+)-JQ1 treated cells at 0 hr. Means reported and error bars represent s.d. from five independent biological repeats. Mixed-design analysis of variance (ANOVA) with time (0 hr, 1 hr, and 8 hr) as the within-subjects factor and treatment ((+)-JQ1, (−)-JQ1, or vehicle) as the between-subjects factor; interaction effect: F (4,24) = 268.9, p =1.49x10 −19 , treatment main effect: F (2,12) = 393.5, p =1.15x10 −11 , time main effect: F (2, 24) = 368.0, p =9.84x10 −19 . Planned paired t -test of MM.1S-luc cells harvested 8 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 38.92, uncorrected p =2.60x10 −6 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =5.21x10 −6 ). Planned paired t -test of MM.1S-luc cells harvested 1 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 25.10, uncorrected p =1.50x10 −5 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =2.99x10 −5 ). Additional details for this experiment can be found at https://osf.io/9swnx/ . DOI: http://dx.doi.org/10.7554/eLife.21253.002
Figure Legend Snippet: MYC expression in JQ1-treated MM.1S-luc cells. MM.1S-luc cells were treated with 500 nM (+)-JQ1, 500 nM (−)-JQ1, or an equivalent volume of DMSO. Total RNA was isolated at 0 hr, 1 hr, and 8 hr after treatment and qRT-PCR analysis was performed to detect MYC and GAPDH levels. Relative expression ( MYC/GAPDH ) is presented for each time point and condition normalized to (+)-JQ1 treated cells at 0 hr. Means reported and error bars represent s.d. from five independent biological repeats. Mixed-design analysis of variance (ANOVA) with time (0 hr, 1 hr, and 8 hr) as the within-subjects factor and treatment ((+)-JQ1, (−)-JQ1, or vehicle) as the between-subjects factor; interaction effect: F (4,24) = 268.9, p =1.49x10 −19 , treatment main effect: F (2,12) = 393.5, p =1.15x10 −11 , time main effect: F (2, 24) = 368.0, p =9.84x10 −19 . Planned paired t -test of MM.1S-luc cells harvested 8 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 38.92, uncorrected p =2.60x10 −6 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =5.21x10 −6 ). Planned paired t -test of MM.1S-luc cells harvested 1 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 25.10, uncorrected p =1.50x10 −5 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =2.99x10 −5 ). Additional details for this experiment can be found at https://osf.io/9swnx/ . DOI: http://dx.doi.org/10.7554/eLife.21253.002

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

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Synthesized:

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Construct:

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Real-time Polymerase Chain Reaction:

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Expressing:

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Western Blot:

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SYBR Green Assay:

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Northern Blot:

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Reverse Transcription Polymerase Chain Reaction:

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Polymerase Chain Reaction:

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Article Snippet: .. The PCR programme was 95 °C for 15 min, 40 cycles of 94 °C for 15 s, 57 °C for 30 s and 72 °C for 30 s as described by Xue et al. [ ] and was run on the Applied Biosystems QuantStudio 7 Flex Real Time PCR system (Thermo Fisher Scientific, Paisley, UK). ..

Article Title: Immune-enhancing effects of anionic macromolecules extracted from Codium fragile on cyclophosphamide-treated mice
Article Snippet: .. Analysis of immune gene expression using real-time PCR Quantification of immune gene expression in peritoneal macrophages and splenocytes was performed using the QuantStudio™ 7 FlexReal-Time PCR System (ThermoFisher scientific, USA) and SYBR® Premix Ex Taq™ II (Takara Bio Inc., Japan). .. Relative gene expression was calculated using the 2-ΔΔC T method [ ] and β-Actin as the reference gene ( ).

Article Title: Immune-enhancing effects of anionic macromolecules extracted from Codium fragile on cyclophosphamide-treated mice
Article Snippet: .. After extraction of total RNA, TNF-α expression was analyzed using the QuantStudio™ 7 FlexReal-Time PCR System, as described above. .. Statistical analysis Statistical analysis was performed using the Statistix 8.1 software.

Article Title: Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus
Article Snippet: .. For RT-qPCR, cDNA template was mixed with 2X SYBR Green PCR master mix (Applied Biosystems) with the Applied Biosystems QuantStudio 12 K Flex Real-Time PCR system (Life Technologies). ..

RNA Sequencing Assay:

Article Title: Computational identification and validation of alternative splicing in ZSF1 rat RNA-seq data, a preclinical model for type 2 diabetic nephropathy
Article Snippet: The same total RNA samples that were subjected to RNA-seq were used for the qRT-PCR analysis. .. All qRT-PCR reactions were performed using a Taqman RNA-to-Ct 1-Step Kit (Thermo Fisher; catalogue number 4392656) following the manufacturer’s protocol, on an Applied Biosystems Quantstudio 12 K Flex Real-Time PCR System (Thermo Fisher).

Isolation:

Article Title: CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation
Article Snippet: RNA was isolated by phenol-chloroform extraction using Trizol reagent (Invitrogen, Carlsbad, CA). .. Following quantification, 1 ug of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). qRT-PCR was performed using a QuantStudio 12 K Flex Real-Time PCR System, reagents and software (Applied Biosystems, Carlsbad, CA).

Negative Control:

Article Title: Improving engraftment of hepatocyte transplantation using alpha-1 antitrypsin as an immune modulator
Article Snippet: Both female rat hepatocytes and sham liver samples were used as a negative control. .. The PCR programme was 95 °C for 15 min, 40 cycles of 94 °C for 15 s, 57 °C for 30 s and 72 °C for 30 s as described by Xue et al. [ ] and was run on the Applied Biosystems QuantStudio 7 Flex Real Time PCR system (Thermo Fisher Scientific, Paisley, UK).

Purification:

Article Title: Precocious obesity predisposes the development of more severe cisplatin-induced acute kidney injury in young adult mice
Article Snippet: Total RNA was purified from the renal tissue using the phenol and guanidine isothiocyanate-cesium chloride method (TRIzol, Life Technologies, Carlsbad, CA, USA), and total amount was quantified in a spectrophotometer (NanoVue, GE Healthcare Life Science, UK). cDNA was synthetized from 2 μg of total RNA using a mixture containing 0.5 mg/ml oligo (dT), 10 mM dithiothreitol (DTT), 0.5 mM deoxynucleoside triphosphates (Amersham Pharmacia Biotech, Uppsala, Sweden), and 200 U of reverse transcriptase enzyme (SuperScript RT, ThermoScientific, USA) in the presence of 1 U DNase (Promega, Madison, USA) to eliminate genomic DNA contamination. .. The mRNA expression levels were estimated using quantitative real-time PCR (QuantStudio Flex 7 Real-Time PCR System, Applied Biosystems, Carlsbad, CA, USA).

Transgenic Assay:

Article Title: Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus
Article Snippet: To detect the accumulation of CymMV and ORSV in WT and transgenic line after infection or grafting, RNA was extracted from various tissues and reverse transcribed into cDNA as described above. .. For RT-qPCR, cDNA template was mixed with 2X SYBR Green PCR master mix (Applied Biosystems) with the Applied Biosystems QuantStudio 12 K Flex Real-Time PCR system (Life Technologies).

Quantitative RT-PCR:

Article Title: Precocious obesity predisposes the development of more severe cisplatin-induced acute kidney injury in young adult mice
Article Snippet: Gene expression of cytokines and oxidative stress markers Quantification of the mRNA levels of inflammatory and oxidative stress markers was performed by using real-time RT-PCR. .. The mRNA expression levels were estimated using quantitative real-time PCR (QuantStudio Flex 7 Real-Time PCR System, Applied Biosystems, Carlsbad, CA, USA).

Article Title: CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation
Article Snippet: .. Following quantification, 1 ug of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). qRT-PCR was performed using a QuantStudio 12 K Flex Real-Time PCR System, reagents and software (Applied Biosystems, Carlsbad, CA). .. Reactions were performed in triplicate, including no template controls and amplification of an endogenous control transcript, Larger Ribosomal Protein (RPLPO), to assess template concentration and loading precision.

Article Title: Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus
Article Snippet: .. For RT-qPCR, cDNA template was mixed with 2X SYBR Green PCR master mix (Applied Biosystems) with the Applied Biosystems QuantStudio 12 K Flex Real-Time PCR system (Life Technologies). ..

Article Title: Computational identification and validation of alternative splicing in ZSF1 rat RNA-seq data, a preclinical model for type 2 diabetic nephropathy
Article Snippet: .. All qRT-PCR reactions were performed using a Taqman RNA-to-Ct 1-Step Kit (Thermo Fisher; catalogue number 4392656) following the manufacturer’s protocol, on an Applied Biosystems Quantstudio 12 K Flex Real-Time PCR System (Thermo Fisher). ..

IA:

Article Title: Computational identification and validation of alternative splicing in ZSF1 rat RNA-seq data, a preclinical model for type 2 diabetic nephropathy
Article Snippet: qRT-PCR validation Custom primer probe sequences labelled with 6-carboxyfluorescein (FAM) were ordered from Integrated DNA Technologies (IDT, Coralville, IA). .. All qRT-PCR reactions were performed using a Taqman RNA-to-Ct 1-Step Kit (Thermo Fisher; catalogue number 4392656) following the manufacturer’s protocol, on an Applied Biosystems Quantstudio 12 K Flex Real-Time PCR System (Thermo Fisher).

Software:

Article Title: Inter-population Differences in Retrogene Loss and Expression in Humans
Article Snippet: Real-time PCR Real-time polymerase chain reaction was performed in Applied Biosystems QuantStudio 6 & 7 Flex Real-Time PCR System using Power SYBR Green PCR Master Mix (Applied Biosystems) in 40 cycles and with Tm = 60°C. .. Results were analyzed using QuantStudio Real-Time PCR Software v1.0 (Applied Biosystems).

Article Title: CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation
Article Snippet: .. Following quantification, 1 ug of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). qRT-PCR was performed using a QuantStudio 12 K Flex Real-Time PCR System, reagents and software (Applied Biosystems, Carlsbad, CA). .. Reactions were performed in triplicate, including no template controls and amplification of an endogenous control transcript, Larger Ribosomal Protein (RPLPO), to assess template concentration and loading precision.

Electrophoresis:

Article Title: Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus
Article Snippet: The RT-PCR products were monitored by electrophoresis on 1.5% agarose gel in TBE buffer (Tris-borate-EDTA) and visualized with SYBR Green I core reagent (Life Technologies). .. For RT-qPCR, cDNA template was mixed with 2X SYBR Green PCR master mix (Applied Biosystems) with the Applied Biosystems QuantStudio 12 K Flex Real-Time PCR system (Life Technologies).

RNA Extraction:

Article Title: CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation
Article Snippet: Paragraph title: RNA Extraction and Gene Expression Analysis ... Following quantification, 1 ug of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). qRT-PCR was performed using a QuantStudio 12 K Flex Real-Time PCR System, reagents and software (Applied Biosystems, Carlsbad, CA).

Agarose Gel Electrophoresis:

Article Title: Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus
Article Snippet: The RT-PCR products were monitored by electrophoresis on 1.5% agarose gel in TBE buffer (Tris-borate-EDTA) and visualized with SYBR Green I core reagent (Life Technologies). .. For RT-qPCR, cDNA template was mixed with 2X SYBR Green PCR master mix (Applied Biosystems) with the Applied Biosystems QuantStudio 12 K Flex Real-Time PCR system (Life Technologies).

Spectrophotometry:

Article Title: Precocious obesity predisposes the development of more severe cisplatin-induced acute kidney injury in young adult mice
Article Snippet: Total RNA was purified from the renal tissue using the phenol and guanidine isothiocyanate-cesium chloride method (TRIzol, Life Technologies, Carlsbad, CA, USA), and total amount was quantified in a spectrophotometer (NanoVue, GE Healthcare Life Science, UK). cDNA was synthetized from 2 μg of total RNA using a mixture containing 0.5 mg/ml oligo (dT), 10 mM dithiothreitol (DTT), 0.5 mM deoxynucleoside triphosphates (Amersham Pharmacia Biotech, Uppsala, Sweden), and 200 U of reverse transcriptase enzyme (SuperScript RT, ThermoScientific, USA) in the presence of 1 U DNase (Promega, Madison, USA) to eliminate genomic DNA contamination. .. The mRNA expression levels were estimated using quantitative real-time PCR (QuantStudio Flex 7 Real-Time PCR System, Applied Biosystems, Carlsbad, CA, USA).

Article Title: CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation
Article Snippet: RNA was assessed for purity by A260/A280 ratio and quantified using a Nanodrop spectrophotometer. .. Following quantification, 1 ug of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). qRT-PCR was performed using a QuantStudio 12 K Flex Real-Time PCR System, reagents and software (Applied Biosystems, Carlsbad, CA).

Concentration Assay:

Article Title: CXCL12/CXCR4-Mediated Procollagen Secretion Is Coupled To Cullin-RING Ubiquitin Ligase Activation
Article Snippet: Following quantification, 1 ug of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). qRT-PCR was performed using a QuantStudio 12 K Flex Real-Time PCR System, reagents and software (Applied Biosystems, Carlsbad, CA). .. Reactions were performed in triplicate, including no template controls and amplification of an endogenous control transcript, Larger Ribosomal Protein (RPLPO), to assess template concentration and loading precision.

Variant Assay:

Article Title: Homozygous truncating mutation in NRAP gene identified by whole exome sequencing in a patient with dilated cardiomyopathy
Article Snippet: .. Genotyping SNP genotyping for NRAP variant was performed using TaqMan probes (Life Technologies) according to manufacturer protocol on QuantStudio 12 K Flex Real-Time PCR System (Applied Biosystems) in 231 unrelated DCM patients. .. Western blots Heart tissue were sampled in ice-cold PBS, snap-frozen in liquid nitrogen and stored at −80 °C.

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  • 99
    Thermo Fisher neon transfection system
    GRA18 activity is dependent on its interactions with GSK3 and PP2A-B56. ( A–B ) RAW264.7 cells were transfected with mCherry vector control or the FH-GRA18 expression vectors pcDNA-FH-GRA18 FL (GRA18 FL ), pcDNA-FH-GRA18 Nt (GRA18 Nt ), and pcDNA-FH-GRA18 Ct (GRA18 Ct ). At 18 hr after <t>transfection,</t> cells were harvested and ( A ) whole cell extracts were analyzed by immunoblot using the indicated antibodies, or ( B ) quantitative chemokine expression was determined by qRT-PCR as in Figure 6 . Asterisks indicate P -values (p
    Neon Transfection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1934 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/neon transfection system/product/Thermo Fisher
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    90
    Thermo Fisher quantstudio dx
    Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in serum and urine. a Normal human serum or b urine pooled from healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of each dilution were extracted using the small volume protocol (0.2 mL) and were tested with the Trioplex assay on the ABI7500 Fast Dx instrument. The same serum ( c ) and urine ( d ) dilutions were tested on the <t>QuantStudio</t> Dx instrument. Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)
    Quantstudio Dx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantstudio dx/product/Thermo Fisher
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    quantstudio dx - by Bioz Stars, 2020-04
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    99
    Thermo Fisher biosystems 7500 qrt pcr system
    Tissue-specific expression of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . The expression of AchnCYP86A1 (A) , AchnMYC2 (B) , AchnMYB107 (C) and AchnMYB41 (D) in different kiwifruit organs and fruit. RNA was isolated from roots, shoots, leaves, and fruit at different stages of development, and reverse transcribed. Gene transcript levels were analyzed by <t>qRT-PCR.</t> The expression level of the genes is relative to actin . Error bar represents the standard deviation of three biological replicates.
    Biosystems 7500 Qrt Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    biosystems 7500 qrt pcr system - by Bioz Stars, 2020-04
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    GRA18 activity is dependent on its interactions with GSK3 and PP2A-B56. ( A–B ) RAW264.7 cells were transfected with mCherry vector control or the FH-GRA18 expression vectors pcDNA-FH-GRA18 FL (GRA18 FL ), pcDNA-FH-GRA18 Nt (GRA18 Nt ), and pcDNA-FH-GRA18 Ct (GRA18 Ct ). At 18 hr after transfection, cells were harvested and ( A ) whole cell extracts were analyzed by immunoblot using the indicated antibodies, or ( B ) quantitative chemokine expression was determined by qRT-PCR as in Figure 6 . Asterisks indicate P -values (p

    Journal: eLife

    Article Title: Characterization of a Toxoplasma effector uncovers an alternative GSK3/β-catenin-regulatory pathway of inflammation

    doi: 10.7554/eLife.39887

    Figure Lengend Snippet: GRA18 activity is dependent on its interactions with GSK3 and PP2A-B56. ( A–B ) RAW264.7 cells were transfected with mCherry vector control or the FH-GRA18 expression vectors pcDNA-FH-GRA18 FL (GRA18 FL ), pcDNA-FH-GRA18 Nt (GRA18 Nt ), and pcDNA-FH-GRA18 Ct (GRA18 Ct ). At 18 hr after transfection, cells were harvested and ( A ) whole cell extracts were analyzed by immunoblot using the indicated antibodies, or ( B ) quantitative chemokine expression was determined by qRT-PCR as in Figure 6 . Asterisks indicate P -values (p

    Article Snippet: Transfection of RAW264.7 cells Cells were transfected by electroporation using the Neon Transfection System (Thermo Fisher Scientif).

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

    GRA18 promotes Ccl17, Ccl22 and Ccl24 chemokines expression in a β-catenin-dependent fashion. ( A ) Schematic diagram of gRNAs targeting the Ctnnb1 locus. The Protospacer Adjacent Motif (PAM) sequence is lined and highlighted in red; the targeting sequences is shown in green. Bi-directional arrow indicates the Cas9 cleavage site. DNA sequences from the wild-type and mutated RAW264.7-derived cell lines were analyzed by DNA sequencing; the identified deletions are indicated with (-). No mutations in control samples were observed. ( B ) Immunoblot analysis of β-catenin in parental RAW264.7 and the Ctnnb1 -/- mutant confirmed the absence of β-catenin expression. As positive controls, cells were treated with 3 mM of BIO or 2 mM of CHIR GSK3 inhibitors for 12 hr. TBP was used as loading control. ( C–E ) RAW264.7 (WT) and β-catenin-deficient ( Ctnnb1 -/- ) RAW264.7-derived cell lines were transfected with mCherry vector control (pcDNA-mCherry-HF) or the FH-GRA18 II expression vector (pcDNA-FH-GRA18 FL ). At 18 hr after transfection, cells were either ( C ) fixed for IFA using anti-HA (red) and anti-β-catenin (green) antibodies or ( D ) cells were harvested and analyzed by immunoblot using the indicated antibodies. Anti-HA was used to detect FH-GRA18 and mCherry-HF. In ( E ) and ( F ) transcripts for Ccl17, Ccl22, Ccl24 , and Ifnb1 were quantified by qPCR and normalized using Tbp . Data are mean value ± s.d. of three replicates. The P -values were calculated using two-tailed unpaired Student’s t -test or one-way ANOVA with Bonferroni posttests analysis of variance; *p

    Journal: eLife

    Article Title: Characterization of a Toxoplasma effector uncovers an alternative GSK3/β-catenin-regulatory pathway of inflammation

    doi: 10.7554/eLife.39887

    Figure Lengend Snippet: GRA18 promotes Ccl17, Ccl22 and Ccl24 chemokines expression in a β-catenin-dependent fashion. ( A ) Schematic diagram of gRNAs targeting the Ctnnb1 locus. The Protospacer Adjacent Motif (PAM) sequence is lined and highlighted in red; the targeting sequences is shown in green. Bi-directional arrow indicates the Cas9 cleavage site. DNA sequences from the wild-type and mutated RAW264.7-derived cell lines were analyzed by DNA sequencing; the identified deletions are indicated with (-). No mutations in control samples were observed. ( B ) Immunoblot analysis of β-catenin in parental RAW264.7 and the Ctnnb1 -/- mutant confirmed the absence of β-catenin expression. As positive controls, cells were treated with 3 mM of BIO or 2 mM of CHIR GSK3 inhibitors for 12 hr. TBP was used as loading control. ( C–E ) RAW264.7 (WT) and β-catenin-deficient ( Ctnnb1 -/- ) RAW264.7-derived cell lines were transfected with mCherry vector control (pcDNA-mCherry-HF) or the FH-GRA18 II expression vector (pcDNA-FH-GRA18 FL ). At 18 hr after transfection, cells were either ( C ) fixed for IFA using anti-HA (red) and anti-β-catenin (green) antibodies or ( D ) cells were harvested and analyzed by immunoblot using the indicated antibodies. Anti-HA was used to detect FH-GRA18 and mCherry-HF. In ( E ) and ( F ) transcripts for Ccl17, Ccl22, Ccl24 , and Ifnb1 were quantified by qPCR and normalized using Tbp . Data are mean value ± s.d. of three replicates. The P -values were calculated using two-tailed unpaired Student’s t -test or one-way ANOVA with Bonferroni posttests analysis of variance; *p

    Article Snippet: Transfection of RAW264.7 cells Cells were transfected by electroporation using the Neon Transfection System (Thermo Fisher Scientif).

    Techniques: Expressing, Sequencing, Derivative Assay, DNA Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Immunofluorescence, Real-time Polymerase Chain Reaction, Two Tailed Test

    Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in serum and urine. a Normal human serum or b urine pooled from healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of each dilution were extracted using the small volume protocol (0.2 mL) and were tested with the Trioplex assay on the ABI7500 Fast Dx instrument. The same serum ( c ) and urine ( d ) dilutions were tested on the QuantStudio Dx instrument. Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)

    Journal: Nature Communications

    Article Title: Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

    doi: 10.1038/s41467-018-03772-1

    Figure Lengend Snippet: Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in serum and urine. a Normal human serum or b urine pooled from healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of each dilution were extracted using the small volume protocol (0.2 mL) and were tested with the Trioplex assay on the ABI7500 Fast Dx instrument. The same serum ( c ) and urine ( d ) dilutions were tested on the QuantStudio Dx instrument. Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)

    Article Snippet: Standard curves were generated and tested with the Trioplex real-time RT-PCR assay on each qPCR instrument, ABI 7500 Fast Dx (ThermoFisher), and QuantStudio Dx (ThermoFisher).

    Techniques: RNA Extraction, Standard Deviation

    Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in whole blood (EDTA). A pool of whole blood donated by healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD) and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicas of every dilution were extracted using small volume protocol (0.2 mL) and tested with Trioplex assay on the ABI7500 Fast Dx instrument ( a ) or in the QuantStudio Dx instrument ( b ). Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)

    Journal: Nature Communications

    Article Title: Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

    doi: 10.1038/s41467-018-03772-1

    Figure Lengend Snippet: Analytical performance comparison between MP96 and LC 2.0 RNA extraction platforms in whole blood (EDTA). A pool of whole blood donated by healthy donors was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD) and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicas of every dilution were extracted using small volume protocol (0.2 mL) and tested with Trioplex assay on the ABI7500 Fast Dx instrument ( a ) or in the QuantStudio Dx instrument ( b ). Mean genome copy equivalents per milliliter (GCE/mL) of viral RNA detected are displayed at each dilution. Error bars represent GCE/mL standard deviation. Straight line represents linear regression of MP96 platform (Roche). Dashed line represents linear regression of LC 2.0 platform (Roche)

    Article Snippet: Standard curves were generated and tested with the Trioplex real-time RT-PCR assay on each qPCR instrument, ABI 7500 Fast Dx (ThermoFisher), and QuantStudio Dx (ThermoFisher).

    Techniques: RNA Extraction, Standard Deviation

    Tissue-specific expression of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . The expression of AchnCYP86A1 (A) , AchnMYC2 (B) , AchnMYB107 (C) and AchnMYB41 (D) in different kiwifruit organs and fruit. RNA was isolated from roots, shoots, leaves, and fruit at different stages of development, and reverse transcribed. Gene transcript levels were analyzed by qRT-PCR. The expression level of the genes is relative to actin . Error bar represents the standard deviation of three biological replicates.

    Journal: Frontiers in Plant Science

    Article Title: Three Transcription Activators of ABA Signaling Positively Regulate Suberin Monomer Synthesis by Activating Cytochrome P450 CYP86A1 in Kiwifruit

    doi: 10.3389/fpls.2019.01650

    Figure Lengend Snippet: Tissue-specific expression of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . The expression of AchnCYP86A1 (A) , AchnMYC2 (B) , AchnMYB107 (C) and AchnMYB41 (D) in different kiwifruit organs and fruit. RNA was isolated from roots, shoots, leaves, and fruit at different stages of development, and reverse transcribed. Gene transcript levels were analyzed by qRT-PCR. The expression level of the genes is relative to actin . Error bar represents the standard deviation of three biological replicates.

    Article Snippet: qRT-PCR Analysis The cDNA of kiwifruit and N. benthamiana genes for qRT-PCR were obtained by TBLASTX analysis against the kiwifruit genome database and the SOL Genomics Network database, respectively, using Arabidopsis genes as query ( ). qRT-PCR was performed in 96 well plates using Biosystems 7500 qRT-PCR system (Thermo Fisher Scientific Inc., USA).

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Standard Deviation

    Genes expression and the content of ω-hydroxyacids and α, ω-diacids in kiwifruit wound tissues treated with water, ABA and FLD. (A) Expression levels of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . Relative mRNA abundance was evaluated by qRT-PCR and presented as fold change relative to the initial value upon wounding. (B) Accumulation of ω-hydroxyacids and α, ω-diacids. Inset graph shows total content of ω-hydroxyacids and α, ω-diacids. ABA, abscisic acid; FLD, fluridone. Error bar represents the standard deviation of three biological replicates. Asterisk indicates significant difference ( t test, *p value

    Journal: Frontiers in Plant Science

    Article Title: Three Transcription Activators of ABA Signaling Positively Regulate Suberin Monomer Synthesis by Activating Cytochrome P450 CYP86A1 in Kiwifruit

    doi: 10.3389/fpls.2019.01650

    Figure Lengend Snippet: Genes expression and the content of ω-hydroxyacids and α, ω-diacids in kiwifruit wound tissues treated with water, ABA and FLD. (A) Expression levels of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . Relative mRNA abundance was evaluated by qRT-PCR and presented as fold change relative to the initial value upon wounding. (B) Accumulation of ω-hydroxyacids and α, ω-diacids. Inset graph shows total content of ω-hydroxyacids and α, ω-diacids. ABA, abscisic acid; FLD, fluridone. Error bar represents the standard deviation of three biological replicates. Asterisk indicates significant difference ( t test, *p value

    Article Snippet: qRT-PCR Analysis The cDNA of kiwifruit and N. benthamiana genes for qRT-PCR were obtained by TBLASTX analysis against the kiwifruit genome database and the SOL Genomics Network database, respectively, using Arabidopsis genes as query ( ). qRT-PCR was performed in 96 well plates using Biosystems 7500 qRT-PCR system (Thermo Fisher Scientific Inc., USA).

    Techniques: Expressing, Quantitative RT-PCR, Standard Deviation