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    Advantage RT for PCR Kit
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    TaKaRa advantage rt for pcr kit
    Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the <t>qRT-PCR</t> data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p

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    1) Product Images from "HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway"

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1214-5

    Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p

    Techniques Used: Quantitative RT-PCR, Infection, Mouse Assay, Western Blot, Expressing, Formalin-fixed Paraffin-Embedded, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay

    HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p
    Figure Legend Snippet: HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p

    Techniques Used: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression

    Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p
    Figure Legend Snippet: Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p

    Techniques Used: Expressing, Quantitative RT-PCR

    HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p
    Figure Legend Snippet: HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, In Vivo, Over Expression, Real-time Polymerase Chain Reaction

    2) Product Images from "Network-based approach to prediction and population-based validation of in silico drug repurposing"

    Article Title: Network-based approach to prediction and population-based validation of in silico drug repurposing

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05116-5

    Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p
    Figure Legend Snippet: Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p

    Techniques Used: Expressing, RNA Sequencing Assay, In Vitro, In Vivo, Activation Assay, Inhibition, Incubation, Quantitative RT-PCR

    3) Product Images from "Expression of Gab1 Lacking the Pleckstrin Homology Domain Is Associated with Neoplastic Progression"

    Article Title: Expression of Gab1 Lacking the Pleckstrin Homology Domain Is Associated with Neoplastic Progression

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.20.6895-6905.2001

    Exclusive expression of Gab1 mRNA led by LSP sequence in 10W−1, especially in 10W−1L cells. One microgram of RNA from 10W+8, 10W−1E, and 10W−1L cells was subjected to reverse transcription reaction using oligo(dT) 18 primer. For some samples, reverse transcriptase (RT) was not included in the reaction (lanes 1, 3, 5, 7, 9, and 11). cDNA, in turn, was provided for PCR with either −14F or LSP as 5′ primer and 2104R as 3′ primer. PCR products were analyzed by 1.2% agarose gel and visualized with ethidium bromide staining and UV exposure.
    Figure Legend Snippet: Exclusive expression of Gab1 mRNA led by LSP sequence in 10W−1, especially in 10W−1L cells. One microgram of RNA from 10W+8, 10W−1E, and 10W−1L cells was subjected to reverse transcription reaction using oligo(dT) 18 primer. For some samples, reverse transcriptase (RT) was not included in the reaction (lanes 1, 3, 5, 7, 9, and 11). cDNA, in turn, was provided for PCR with either −14F or LSP as 5′ primer and 2104R as 3′ primer. PCR products were analyzed by 1.2% agarose gel and visualized with ethidium bromide staining and UV exposure.

    Techniques Used: Expressing, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    4) Product Images from "Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression"

    Article Title: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression

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

    doi:

    Proteasome inhibitor LLnV induces Bax accumulation, cytochrome c release, and PARP cleavage in Bcl-2-overexpressing Jurkat T cells. ( A – C ) Jurkat T cells overexpressing Bcl-2 (0 h) were treated with 50 μM LLnV for up to 8 h, followed by preparation of cytosol and mitochondrial fractions. Both fractions were immunoblotted first by an antibody to cytochrome c (Cyto C, 17 kDa; A and C ) and then reblotted by anticytochrome oxidase subunit II (COX, 26 kDa; B ). Note: 20 μg protein from the cytosol, and 40 μg protein from the mitochondrial, preparation was used in each lane. ( D–G ) Whole-cell extracts (70 μg per lane) of the above treated cells were immunoblotted with specific antibodies to PARP ( D ), Bax (clone N-20; E ), Bcl-2 ( F ), or actin ( G ). Molecular masses of PARP, the PARP cleavage fragment (p85), Bax, Bcl-2, and actin are 113, 85, 21, 26, and 40 kDa, respectively. Positions of protein markers are indicated at right. ( H ) Bcl-2-expressing Jurkat cells (Control) were treated with 50 μM LLnV for 8 h, followed by reverse transcription–PCR (RT-PCR). For the first-strand cDNA synthesis, 0.2 (lanes 1 and 4), 0.6 (lanes 2 and 5), and 1.8 μg (lanes 3 and 6) of the total RNA were used. The positions of Bax (538 bp) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA (983 bp) are indicated. Lane M is DNA molecular weight marker.
    Figure Legend Snippet: Proteasome inhibitor LLnV induces Bax accumulation, cytochrome c release, and PARP cleavage in Bcl-2-overexpressing Jurkat T cells. ( A – C ) Jurkat T cells overexpressing Bcl-2 (0 h) were treated with 50 μM LLnV for up to 8 h, followed by preparation of cytosol and mitochondrial fractions. Both fractions were immunoblotted first by an antibody to cytochrome c (Cyto C, 17 kDa; A and C ) and then reblotted by anticytochrome oxidase subunit II (COX, 26 kDa; B ). Note: 20 μg protein from the cytosol, and 40 μg protein from the mitochondrial, preparation was used in each lane. ( D–G ) Whole-cell extracts (70 μg per lane) of the above treated cells were immunoblotted with specific antibodies to PARP ( D ), Bax (clone N-20; E ), Bcl-2 ( F ), or actin ( G ). Molecular masses of PARP, the PARP cleavage fragment (p85), Bax, Bcl-2, and actin are 113, 85, 21, 26, and 40 kDa, respectively. Positions of protein markers are indicated at right. ( H ) Bcl-2-expressing Jurkat cells (Control) were treated with 50 μM LLnV for 8 h, followed by reverse transcription–PCR (RT-PCR). For the first-strand cDNA synthesis, 0.2 (lanes 1 and 4), 0.6 (lanes 2 and 5), and 1.8 μg (lanes 3 and 6) of the total RNA were used. The positions of Bax (538 bp) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA (983 bp) are indicated. Lane M is DNA molecular weight marker.

    Techniques Used: Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

    5) Product Images from "Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells"

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells

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

    doi:

    Expression of receptors for various growth factors in human embryonic cells. RNA samples from ES cells, 5-day-old EBs, and 10-day-old DE cells were analyzed by RT-PCR for expression of specific receptors. As a control, RNA of 5-day-old EBs was analyzed without the prior generation of cDNA (−RT). For abbreviations of receptors used, see Materials and Methods .
    Figure Legend Snippet: Expression of receptors for various growth factors in human embryonic cells. RNA samples from ES cells, 5-day-old EBs, and 10-day-old DE cells were analyzed by RT-PCR for expression of specific receptors. As a control, RNA of 5-day-old EBs was analyzed without the prior generation of cDNA (−RT). For abbreviations of receptors used, see Materials and Methods .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC"

    Article Title: Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC

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

    doi: 10.1073/pnas.0409213102

    Tissue distribution of CD1. RT-PCR from RNA from ( Top ) six immunoisolated spleen cell populations and ( Middle ) 10 tissues, one macrophage line (HD11 cells), and isolated embryonic enterocytes. First-strand cDNA was prepared by incubation of 2 h at 42°C
    Figure Legend Snippet: Tissue distribution of CD1. RT-PCR from RNA from ( Top ) six immunoisolated spleen cell populations and ( Middle ) 10 tissues, one macrophage line (HD11 cells), and isolated embryonic enterocytes. First-strand cDNA was prepared by incubation of 2 h at 42°C

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

    7) Product Images from "Hypoxia-induced Suppression of c-Myc by HIF-2α in Human Pulmonary Endothelial Cells Attenuates TFAM Expression"

    Article Title: Hypoxia-induced Suppression of c-Myc by HIF-2α in Human Pulmonary Endothelial Cells Attenuates TFAM Expression

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2017.07.008

    Hypoxia suppresses TFAM expression. (A) Quantitative real-time PCR (qRT-PCR) with 0.5 μg RNA, using actin as an endogenous control, was performed to monitor TFAM (n=5) and PGC1β (n=3) gene expression after 24 h hypoxia. Shown are mean ± SD. (B) Western blot for TFAM and actin following 42 h hypoxia or normoxia. Graphs show the relative densitometries corrected for actin; mean ± SD (n=4) are plotted. (C) qRT-PCR with 12 μg RNA was used to monitor PGC1α gene expression in normoxia and hypoxia and to compare the Δ CT of PGC1α and PGC1β transcripts in normoxic conditions (normalized to actin) (n=4). *, significantly different (p
    Figure Legend Snippet: Hypoxia suppresses TFAM expression. (A) Quantitative real-time PCR (qRT-PCR) with 0.5 μg RNA, using actin as an endogenous control, was performed to monitor TFAM (n=5) and PGC1β (n=3) gene expression after 24 h hypoxia. Shown are mean ± SD. (B) Western blot for TFAM and actin following 42 h hypoxia or normoxia. Graphs show the relative densitometries corrected for actin; mean ± SD (n=4) are plotted. (C) qRT-PCR with 12 μg RNA was used to monitor PGC1α gene expression in normoxia and hypoxia and to compare the Δ CT of PGC1α and PGC1β transcripts in normoxic conditions (normalized to actin) (n=4). *, significantly different (p

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

    8) Product Images from "Molecular Characterization of Iron Binding Proteins from Glossina morsitans morsitans (Diptera: Glossinidae)"

    Article Title: Molecular Characterization of Iron Binding Proteins from Glossina morsitans morsitans (Diptera: Glossinidae)

    Journal: Insect biochemistry and molecular biology

    doi: 10.1016/j.ibmb.2006.09.003

    Transcript abundance of transferrin and ferritin during life stages and in isolated tissues of G. m. morsitans . ( A ). Northern blot analysis of G. m. morsitans RNA from early larva (EL), late larva (LL), early pupa (EP), late pupa (LP), and adult female fat body (A F ). ( B ). Tissue-specific expression of GmmTsf1, GmmFer1HCH , and GmmFer2LCH in adult male and female tsetse identified by semi-quantitative RT-PCR analysis. Amplification products were generated using cDNAs from midgut (MG), fat body (FB) and reproductive tissues (RT) as templates. ( C ). Northern blot analysis of GmmTsf1 transcript abundance in whole flies analyzed at 0, 6, 12, 24 and 48 hours after blood feeding. Flies were collected from points after their first blood meal post eclosion and acquisition of their second blood meal. The tsetse tubulin gene GmmTub was used in hybridizations as a loading control. ( D ). Quantification of GmmTsf1 expression levels. Transcript abundance for the Northern data in ( C ) was quantified and then normalized against the GmmTub loading control.
    Figure Legend Snippet: Transcript abundance of transferrin and ferritin during life stages and in isolated tissues of G. m. morsitans . ( A ). Northern blot analysis of G. m. morsitans RNA from early larva (EL), late larva (LL), early pupa (EP), late pupa (LP), and adult female fat body (A F ). ( B ). Tissue-specific expression of GmmTsf1, GmmFer1HCH , and GmmFer2LCH in adult male and female tsetse identified by semi-quantitative RT-PCR analysis. Amplification products were generated using cDNAs from midgut (MG), fat body (FB) and reproductive tissues (RT) as templates. ( C ). Northern blot analysis of GmmTsf1 transcript abundance in whole flies analyzed at 0, 6, 12, 24 and 48 hours after blood feeding. Flies were collected from points after their first blood meal post eclosion and acquisition of their second blood meal. The tsetse tubulin gene GmmTub was used in hybridizations as a loading control. ( D ). Quantification of GmmTsf1 expression levels. Transcript abundance for the Northern data in ( C ) was quantified and then normalized against the GmmTub loading control.

    Techniques Used: Isolation, Northern Blot, Expressing, Quantitative RT-PCR, Amplification, Generated

    9) Product Images from "Comprehensive target capture/next-generation sequencing as a second-tier diagnostic approach for congenital muscular dystrophy in Taiwan"

    Article Title: Comprehensive target capture/next-generation sequencing as a second-tier diagnostic approach for congenital muscular dystrophy in Taiwan

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170517

    cDNA analysis for patient 19 to elucidate the pathogenicity of c.4311 G > A in LAMA2 . (A) RT-PCR analysis of LAMA2 mRNA of patient 4 and control individual. (B) Aberrant splicing of the mutant LAMA2 mRNA in patient 4.
    Figure Legend Snippet: cDNA analysis for patient 19 to elucidate the pathogenicity of c.4311 G > A in LAMA2 . (A) RT-PCR analysis of LAMA2 mRNA of patient 4 and control individual. (B) Aberrant splicing of the mutant LAMA2 mRNA in patient 4.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mutagenesis

    10) Product Images from "HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway"

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1214-5

    Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p

    Techniques Used: Quantitative RT-PCR, Infection, Mouse Assay, Western Blot, Expressing, Formalin-fixed Paraffin-Embedded, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay

    HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p
    Figure Legend Snippet: HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p

    Techniques Used: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression

    Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p
    Figure Legend Snippet: Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p

    Techniques Used: Expressing, Quantitative RT-PCR

    HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p
    Figure Legend Snippet: HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, In Vivo, Over Expression, Real-time Polymerase Chain Reaction

    11) Product Images from "Osteopontin is a Novel Marker of Pancreatic Ductal Tissues and of Undifferentiated Pancreatic Precursors in Mice"

    Article Title: Osteopontin is a Novel Marker of Pancreatic Ductal Tissues and of Undifferentiated Pancreatic Precursors in Mice

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    doi: 10.1002/dvdy.20729

    In developing pancreata, Opn and Vn are similarly expressed. A , Results of RT-PCR analysis of RNA from E14.5 pancreatic tissues show expression of Osteopontin ( Opn ) and Vitronectin ( Vn ) transcripts, but not of those encoding the matricellular protein Tenascin C ( Ten-C ) or the two SIBLING family members Bsp or Dmp . B, In the pancreatic region of E13.5 embryos, expression of Vn proteins (red fluorescence) is detected in the pancreatic epithelium (labeled with green; arrows) and at lower level, in some mesenchymal cells (arrowheads). C , Vn (red fluorescence and arrows) is normally expressed in the pancreata of E13.5 Opn-deficient embryos. D , At E13.5, expression of Opn proteins (green fluorescence) is detected on the apical side (arrowheads) or in the cytoplasm (arrows) of pancreatic epithelial cells. E , In these tissues a large population of epithelial cells also expresses Vn in the cytoplasm (arrows and red fluorescence). F , In E13.5 pancreata many Opn + cells co-express Vn (arrows), although some Opn + cells do not (arrowhead). D–F , are confocal images of the same section ( D and E are separate channels and F shows the overlapped images). Cell nuclei were stained with DAPI ( B, C ) or with TO-PRO-3 ( D–F ). Asterisks in F indicate the lumen of the epithelium. Scale bars: 200 μM.
    Figure Legend Snippet: In developing pancreata, Opn and Vn are similarly expressed. A , Results of RT-PCR analysis of RNA from E14.5 pancreatic tissues show expression of Osteopontin ( Opn ) and Vitronectin ( Vn ) transcripts, but not of those encoding the matricellular protein Tenascin C ( Ten-C ) or the two SIBLING family members Bsp or Dmp . B, In the pancreatic region of E13.5 embryos, expression of Vn proteins (red fluorescence) is detected in the pancreatic epithelium (labeled with green; arrows) and at lower level, in some mesenchymal cells (arrowheads). C , Vn (red fluorescence and arrows) is normally expressed in the pancreata of E13.5 Opn-deficient embryos. D , At E13.5, expression of Opn proteins (green fluorescence) is detected on the apical side (arrowheads) or in the cytoplasm (arrows) of pancreatic epithelial cells. E , In these tissues a large population of epithelial cells also expresses Vn in the cytoplasm (arrows and red fluorescence). F , In E13.5 pancreata many Opn + cells co-express Vn (arrows), although some Opn + cells do not (arrowhead). D–F , are confocal images of the same section ( D and E are separate channels and F shows the overlapped images). Cell nuclei were stained with DAPI ( B, C ) or with TO-PRO-3 ( D–F ). Asterisks in F indicate the lumen of the epithelium. Scale bars: 200 μM.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Labeling, Staining

    12) Product Images from "Transcriptional oncogenomic hot spots in Barrett's adenocarcinomas: Serial analysis of gene expression a"

    Article Title: Transcriptional oncogenomic hot spots in Barrett's adenocarcinomas: Serial analysis of gene expression a

    Journal: Genes, Chromosomes & Cancer

    doi: 10.1002/gcc.20479

    Visualization of RT‐PCR products on gel electrophoresis. Five matched tumor and normal samples that were analyzed using qRT‐PCR were subjected to 1.2% agarose gel electrophoresis and ethidium bromide staining. The intensity of bands confirms the PCR results, indicating higher mRNA expression levels of ANPEP, PP1201, EIF5A1 , and ECGF , as well as lower expression of GKN1 in most of the tumor samples as compared with their matched normal control samples. HPRT1 was used as a control to show similar levels in each matched normal and tumor samples.
    Figure Legend Snippet: Visualization of RT‐PCR products on gel electrophoresis. Five matched tumor and normal samples that were analyzed using qRT‐PCR were subjected to 1.2% agarose gel electrophoresis and ethidium bromide staining. The intensity of bands confirms the PCR results, indicating higher mRNA expression levels of ANPEP, PP1201, EIF5A1 , and ECGF , as well as lower expression of GKN1 in most of the tumor samples as compared with their matched normal control samples. HPRT1 was used as a control to show similar levels in each matched normal and tumor samples.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Quantitative RT-PCR, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Expressing

    Quantitative real‐time reverse‐transcription PCR showing fold expression changes at the mRNA level of five representative genes. qRT‐PCR analysis was performed using iCycler on 31 lower esophageal and GEJ adenocarcinoma samples (Tu) and 6 Barrett's esophagus (BE) samples in comparison with 26 normal glandular mucosa samples (N). The horizontal axis shows sample numbers, whereas the fold expression in tumor samples compared with that in normal samples is shown on the vertical axis. The fold expression was calculated according to the formula: as detailed in the “ Materials and Methods ” section. Each bar represents one sample. The displayed mean fold expression for each sample is calculated in comparison with the expression average of the 26 normal samples. The expression of each gene was normalized to the expression of HPRT1 , which showed minimal variation in all normal and neoplastic samples tested. GKN1 shows downregulation (≤0.4‐fold expression) whereas ANPEP , PP1201 , EIF5A1 , and ECGF1 demonstrate overexpression (≥2.5 fold expression) in primary tumors as compared to normal tissue samples.
    Figure Legend Snippet: Quantitative real‐time reverse‐transcription PCR showing fold expression changes at the mRNA level of five representative genes. qRT‐PCR analysis was performed using iCycler on 31 lower esophageal and GEJ adenocarcinoma samples (Tu) and 6 Barrett's esophagus (BE) samples in comparison with 26 normal glandular mucosa samples (N). The horizontal axis shows sample numbers, whereas the fold expression in tumor samples compared with that in normal samples is shown on the vertical axis. The fold expression was calculated according to the formula: as detailed in the “ Materials and Methods ” section. Each bar represents one sample. The displayed mean fold expression for each sample is calculated in comparison with the expression average of the 26 normal samples. The expression of each gene was normalized to the expression of HPRT1 , which showed minimal variation in all normal and neoplastic samples tested. GKN1 shows downregulation (≤0.4‐fold expression) whereas ANPEP , PP1201 , EIF5A1 , and ECGF1 demonstrate overexpression (≥2.5 fold expression) in primary tumors as compared to normal tissue samples.

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

    13) Product Images from "Enhanced Upregulation of CRH mRNA Expression in the Nucleus Accumbens of Male Rats after a Second Injection of Methamphetamine Given Thirty Days Later"

    Article Title: Enhanced Upregulation of CRH mRNA Expression in the Nucleus Accumbens of Male Rats after a Second Injection of Methamphetamine Given Thirty Days Later

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084665

    qPCR validation of METH-induced changes identified by microarray analysis. There is a significant correlation between METH-induced changes in the expression of genes identified by microarray analysis and validated by qRT-PCR.
    Figure Legend Snippet: qPCR validation of METH-induced changes identified by microarray analysis. There is a significant correlation between METH-induced changes in the expression of genes identified by microarray analysis and validated by qRT-PCR.

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

    14) Product Images from "Role of CRD-BP in the growth of human Basal Cell Carcinoma Cells"

    Article Title: Role of CRD-BP in the growth of human Basal Cell Carcinoma Cells

    Journal: The Journal of investigative dermatology

    doi: 10.1038/jid.2014.17

    Analysis of the expression of CRD-BP and the activity of Wnt and Hh signaling pathways in BCC a. Relative expression of CRD-BP, GLI1, β-TrCP1, and c-myc in mRNA isolated from 13 superficial BCCs, determined by quantitative RT-PCR. Each value represents the ratio between the expression of each mRNA in BCC and its expression in matching normal skin. b. Relative expression of Tcf1, Axin, PTCH1, and PTCH2 in mRNA isolated from 12 superficial BCCs, determined by quantitative RT-PCR. Each value represents the ratio between the expression of each mRNA in BCC and its expression in matching normal skin.
    Figure Legend Snippet: Analysis of the expression of CRD-BP and the activity of Wnt and Hh signaling pathways in BCC a. Relative expression of CRD-BP, GLI1, β-TrCP1, and c-myc in mRNA isolated from 13 superficial BCCs, determined by quantitative RT-PCR. Each value represents the ratio between the expression of each mRNA in BCC and its expression in matching normal skin. b. Relative expression of Tcf1, Axin, PTCH1, and PTCH2 in mRNA isolated from 12 superficial BCCs, determined by quantitative RT-PCR. Each value represents the ratio between the expression of each mRNA in BCC and its expression in matching normal skin.

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

    Characterization of a human BCC cell line (UW-BCC1) a. UW-BCC1 in culture at passage 11 and at passage 73. A BCC sample was sequentially trypsinized for 6, 12 and 18 hrs in a humidified cell culture incubator with 5% CO 2 and grown in low calcium media as described in the supplemental information . Scale bar = 30 μm. b. Protein expression of CRD-BP and GLI1 in UW-BCC1 determined by immunoblot analyses. β-actin was used as internal control. This is a representative of three independent experiments. c. Protein expression of keratin 5 and keratin 14 in UW-BCC1 determined by immunoblot analyses. β-actin was used as internal control. This is a representative of three independent experiments. d. Relative expression of CRD-BP, GLI1, PTCH1, and PTCH2 in mRNA isolated from UW-BCC1 and other cell lines determined by quantitative RT-PCR. e. UW-BCC1 and other cells as indicated were grown in 6 well plates and co-transfected with 8×3′GLI BS-LucII reporter plasmid and pSV-40 -galactosidase plasmid. 48 hrs after transfection, the luciferase activity was estimated using luciferase reporter assay reagent (Promega). β-galactosidase was used for normalization and estimated using β-galactosidase assay reagent (Pierce). Each experiment was done at least twice and in triplicates. Each value represents Mean+/-SD. *P≤0.05 f. UW-BCC1 was grown in two 100 mm plates and transfected with pTK-puro plasmid. Equal number of cells from each plate was seeded in five 100 mm plates. One set of plates was treated with Vismodegib (100 nM) for 72 hrs and puromycin (5 μg) for 10 days. The other set of plates was treated with puromycin (5 μg) alone for 10 days. Colonies were counted under the microscope. Each experiment was done at least twice and in triplicates. Each value represents Mean+/-SD. *P≤0.05.
    Figure Legend Snippet: Characterization of a human BCC cell line (UW-BCC1) a. UW-BCC1 in culture at passage 11 and at passage 73. A BCC sample was sequentially trypsinized for 6, 12 and 18 hrs in a humidified cell culture incubator with 5% CO 2 and grown in low calcium media as described in the supplemental information . Scale bar = 30 μm. b. Protein expression of CRD-BP and GLI1 in UW-BCC1 determined by immunoblot analyses. β-actin was used as internal control. This is a representative of three independent experiments. c. Protein expression of keratin 5 and keratin 14 in UW-BCC1 determined by immunoblot analyses. β-actin was used as internal control. This is a representative of three independent experiments. d. Relative expression of CRD-BP, GLI1, PTCH1, and PTCH2 in mRNA isolated from UW-BCC1 and other cell lines determined by quantitative RT-PCR. e. UW-BCC1 and other cells as indicated were grown in 6 well plates and co-transfected with 8×3′GLI BS-LucII reporter plasmid and pSV-40 -galactosidase plasmid. 48 hrs after transfection, the luciferase activity was estimated using luciferase reporter assay reagent (Promega). β-galactosidase was used for normalization and estimated using β-galactosidase assay reagent (Pierce). Each experiment was done at least twice and in triplicates. Each value represents Mean+/-SD. *P≤0.05 f. UW-BCC1 was grown in two 100 mm plates and transfected with pTK-puro plasmid. Equal number of cells from each plate was seeded in five 100 mm plates. One set of plates was treated with Vismodegib (100 nM) for 72 hrs and puromycin (5 μg) for 10 days. The other set of plates was treated with puromycin (5 μg) alone for 10 days. Colonies were counted under the microscope. Each experiment was done at least twice and in triplicates. Each value represents Mean+/-SD. *P≤0.05.

    Techniques Used: Cell Culture, Expressing, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Microscopy

    15) Product Images from "Aberrant amino acid signaling promotes growth and metastasis of hepatocellular carcinomas through Rab1A-dependent activation of mTORC1 by Rab1A"

    Article Title: Aberrant amino acid signaling promotes growth and metastasis of hepatocellular carcinomas through Rab1A-dependent activation of mTORC1 by Rab1A

    Journal: Oncotarget

    doi:

    Rab1A is overexpressed in HCC due to copy number increase A. Correlation plot of the Rab1A copy number and mRNA level in 187 HCC samples in the TCGA cancer genome database. B. Rab1A mRNA expression in a panel of immortalized liver and HCC cell lines as determined by RT-qPCR. C. Rab1A protein expression in the same panel of immortalized liver and HCC cell lines as determined by immunoblot analysis. GAPDH served as a loading control. D. Correlation plot of Rab1A protein and mRNA expression in the above panel of cell lines. E. MSP results for HCC (MHCC97H and PLC/PRF/5) and immortalized liver (LO2 and QSG-7701) cell lines. M, methylated products of MSP; U, unmethylated products of MSP. F. Upper panel shows the CpG island used for designing primers to detect methylation status of Rab1A locus. Lower panel shows the atlas of PCR fragments for BGS. Arrows indicate potential methylated sites in the LO2 and MHCC97H cell lines. TSS, transcription start site.
    Figure Legend Snippet: Rab1A is overexpressed in HCC due to copy number increase A. Correlation plot of the Rab1A copy number and mRNA level in 187 HCC samples in the TCGA cancer genome database. B. Rab1A mRNA expression in a panel of immortalized liver and HCC cell lines as determined by RT-qPCR. C. Rab1A protein expression in the same panel of immortalized liver and HCC cell lines as determined by immunoblot analysis. GAPDH served as a loading control. D. Correlation plot of Rab1A protein and mRNA expression in the above panel of cell lines. E. MSP results for HCC (MHCC97H and PLC/PRF/5) and immortalized liver (LO2 and QSG-7701) cell lines. M, methylated products of MSP; U, unmethylated products of MSP. F. Upper panel shows the CpG island used for designing primers to detect methylation status of Rab1A locus. Lower panel shows the atlas of PCR fragments for BGS. Arrows indicate potential methylated sites in the LO2 and MHCC97H cell lines. TSS, transcription start site.

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

    16) Product Images from "Expression Profiling of Galectin-3-Depleted Melanoma Cells Reveals its Major Role in Melanoma Cell Plasticity and Vasculogenic Mimicry"

    Article Title: Expression Profiling of Galectin-3-Depleted Melanoma Cells Reveals its Major Role in Melanoma Cell Plasticity and Vasculogenic Mimicry

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2008.080380

    Effect of Gal-3 silencing on the activity of the promoter reporters of VE-cadherin ( A ) and IL-8 ( B ), and the in vivo binding of EGR-1 transcription factor to the promoters of these genes ( C ). NTshRNA- or Gal-3shRNA-expressing C8161-c9 cells were transfected with the pGL3-basic (empty vector), the pGL3-VE-cadherin promoter-driven vector and pRL-CMV control vector ( A ) or the pGL3-IL-8 promoter-driven vector and pRL-CMV control vector ( B ). Luciferase activity was assayed 72 hours later using a dual luciferase reporter assay system. Gal-3 silencing resulted in a decrease in the VE-cadherin promoter-driven and IL-8 promoter-driven luciferase activities. Results are means from triplicate samples ± SD C: ChIP analysis of the binding of EGR-1 transcription factor to the promoters of VE-cadherin and IL-8. PCR amplification of a 200-bp region of the VE-cadherin or IL-8 promoter from input DNA, DNA immunoprecipitated by anti-EGR-1 antibody, or DNA immunoprecipitated by control IgG antibody from Gal-3shRNA knockdown cell line compared to NTshRNA cells. The increase in EGR-1 recruitment to the promoters of VE-cadherin and IL-8 was observed after Gal-3 silencing. Results shown are representative gels of at least three independent experiments.
    Figure Legend Snippet: Effect of Gal-3 silencing on the activity of the promoter reporters of VE-cadherin ( A ) and IL-8 ( B ), and the in vivo binding of EGR-1 transcription factor to the promoters of these genes ( C ). NTshRNA- or Gal-3shRNA-expressing C8161-c9 cells were transfected with the pGL3-basic (empty vector), the pGL3-VE-cadherin promoter-driven vector and pRL-CMV control vector ( A ) or the pGL3-IL-8 promoter-driven vector and pRL-CMV control vector ( B ). Luciferase activity was assayed 72 hours later using a dual luciferase reporter assay system. Gal-3 silencing resulted in a decrease in the VE-cadherin promoter-driven and IL-8 promoter-driven luciferase activities. Results are means from triplicate samples ± SD C: ChIP analysis of the binding of EGR-1 transcription factor to the promoters of VE-cadherin and IL-8. PCR amplification of a 200-bp region of the VE-cadherin or IL-8 promoter from input DNA, DNA immunoprecipitated by anti-EGR-1 antibody, or DNA immunoprecipitated by control IgG antibody from Gal-3shRNA knockdown cell line compared to NTshRNA cells. The increase in EGR-1 recruitment to the promoters of VE-cadherin and IL-8 was observed after Gal-3 silencing. Results shown are representative gels of at least three independent experiments.

    Techniques Used: Activity Assay, In Vivo, Binding Assay, Expressing, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation

    17) Product Images from "Thyroglobulin Repression of Thyroid Transcription Factor 1 (TTF-1) Gene Expression Is Mediated by Decreased DNA Binding of Nuclear Factor I Proteins Which Control Constitutive TTF-1 Expression"

    Article Title: Thyroglobulin Repression of Thyroid Transcription Factor 1 (TTF-1) Gene Expression Is Mediated by Decreased DNA Binding of Nuclear Factor I Proteins Which Control Constitutive TTF-1 Expression

    Journal: Molecular and Cellular Biology

    doi:

    NFI proteins in FRTL-5 cells; sequence and expression by in vitro translation. NFI subtypes were cloned by PCR using FRTL-5 cell RNA (see Materials and Methods). Each was sequenced (A) and subjected to in vitro translation (B) as described in Materials and Methods. In vitro translation was performed using 1 μg of expression vector carrying full cDNA sequences of each NFI subtype and [ 35 S]methionine as described in Materials and Methods. All proteins had the molecular masses expected from the sizes of the open reading frames.
    Figure Legend Snippet: NFI proteins in FRTL-5 cells; sequence and expression by in vitro translation. NFI subtypes were cloned by PCR using FRTL-5 cell RNA (see Materials and Methods). Each was sequenced (A) and subjected to in vitro translation (B) as described in Materials and Methods. In vitro translation was performed using 1 μg of expression vector carrying full cDNA sequences of each NFI subtype and [ 35 S]methionine as described in Materials and Methods. All proteins had the molecular masses expected from the sizes of the open reading frames.

    Techniques Used: Sequencing, Expressing, In Vitro, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation

    18) Product Images from "Silencing cAMP-response Element-binding Protein (CREB) Identifies CYR61 as a Tumor Suppressor Gene in Melanoma *"

    Article Title: Silencing cAMP-response Element-binding Protein (CREB) Identifies CYR61 as a Tumor Suppressor Gene in Melanoma *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.019836

    Validation of CCN1 / CYR61 overexpression after CREB silencing in melanoma cells. A , quantitative real-time PCR validation for CCN1 / CYR61 gene expression. Expression values shown are -fold change in each CREB-shRNA-transduced cell line relative to the NT-shRNA cells after normalization with 18s RNA. *, p
    Figure Legend Snippet: Validation of CCN1 / CYR61 overexpression after CREB silencing in melanoma cells. A , quantitative real-time PCR validation for CCN1 / CYR61 gene expression. Expression values shown are -fold change in each CREB-shRNA-transduced cell line relative to the NT-shRNA cells after normalization with 18s RNA. *, p

    Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Expressing, shRNA

    19) Product Images from "Regulation of Plasticity and Fibrogenic Activity of Trabecular Meshwork Cells by Rho GTPase Signaling"

    Article Title: Regulation of Plasticity and Fibrogenic Activity of Trabecular Meshwork Cells by Rho GTPase Signaling

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.24524

    Effects of MRTF-A, a transcriptional co-activator of serum response factor (SRF) and inhibitor of MRTF-A (CCG1423) on HTM cell fibrogenic activity. A . Total RNA derived from serum starved HTM cells (derived from two different human donors; age 58 and 73 year-old) expressing recombinant MRTF-A was subjected to q-PCR to detect changes in gene expression levels for FSP-1, collagen-1A1 and αSMA. Expression of all three genes was significantly elevated in MRTF-A expressing cells compared to GFP expressing control cells. GAPDH expression was used to normalize the cDNA content of test samples. Data represent values from two independent experiments performed in triplicates (n=6, Mean± SD) * P
    Figure Legend Snippet: Effects of MRTF-A, a transcriptional co-activator of serum response factor (SRF) and inhibitor of MRTF-A (CCG1423) on HTM cell fibrogenic activity. A . Total RNA derived from serum starved HTM cells (derived from two different human donors; age 58 and 73 year-old) expressing recombinant MRTF-A was subjected to q-PCR to detect changes in gene expression levels for FSP-1, collagen-1A1 and αSMA. Expression of all three genes was significantly elevated in MRTF-A expressing cells compared to GFP expressing control cells. GAPDH expression was used to normalize the cDNA content of test samples. Data represent values from two independent experiments performed in triplicates (n=6, Mean± SD) * P

    Techniques Used: Activity Assay, Derivative Assay, Expressing, Recombinant, Polymerase Chain Reaction

    Regulation of expression of transcriptional suppressors-Slug, Snail and Twist-1 in HTM cells and effects of Slug on HTM cell plasticity and fibrogenic activity. A . Total RNA derived from serum starved HTM cells (derived from two different human donors: 58 and 73 year-old) expressing either RhoAV14 or recombinant MRTF-A were subjected to q-PCR analysis to detect relative changes in gene expression for Snail, Slug and Twist-1. Cells expressing either RhoAV14 or MRTF-A exhibit a significant increase in the expression of Slug and Twist-1 as compared to the respective controls. Interestingly, under similar conditions, the levels of Snail expression were reduced significantly. Data represent two independent experiments in triplicates (n=6, Mean± SD) * P
    Figure Legend Snippet: Regulation of expression of transcriptional suppressors-Slug, Snail and Twist-1 in HTM cells and effects of Slug on HTM cell plasticity and fibrogenic activity. A . Total RNA derived from serum starved HTM cells (derived from two different human donors: 58 and 73 year-old) expressing either RhoAV14 or recombinant MRTF-A were subjected to q-PCR analysis to detect relative changes in gene expression for Snail, Slug and Twist-1. Cells expressing either RhoAV14 or MRTF-A exhibit a significant increase in the expression of Slug and Twist-1 as compared to the respective controls. Interestingly, under similar conditions, the levels of Snail expression were reduced significantly. Data represent two independent experiments in triplicates (n=6, Mean± SD) * P

    Techniques Used: Expressing, Activity Assay, Derivative Assay, Recombinant, Polymerase Chain Reaction

    Influence of RhoA, MRTF-A and TGF-β2 on the expression profile of miRNA29a, b and miRNA21 in HTM cells. RNA derived from the serum starved HTM cells expressing either the recombinant RhoAV14, MRTF-A or treated with TGF-β2 (10ng/ml) for 24 h was subjected to q-PCR to detect the relative changes in expression of miRNA29a, miRNA29b (A) and miRNA21 (B). While the levels of miRNA29b and miRNA21 were unaltered, there was a significant increase in miRNA29a expression in the cells expressing RhoAV14 and MRTF-A, or treated with TGF-β2 as compared to respective controls. RNA samples were normalized to the expression of U26 small nucleolar RNA (RNU26). Data represent values from two independent experiments in triplicates (n=6, Mean± SD) * P
    Figure Legend Snippet: Influence of RhoA, MRTF-A and TGF-β2 on the expression profile of miRNA29a, b and miRNA21 in HTM cells. RNA derived from the serum starved HTM cells expressing either the recombinant RhoAV14, MRTF-A or treated with TGF-β2 (10ng/ml) for 24 h was subjected to q-PCR to detect the relative changes in expression of miRNA29a, miRNA29b (A) and miRNA21 (B). While the levels of miRNA29b and miRNA21 were unaltered, there was a significant increase in miRNA29a expression in the cells expressing RhoAV14 and MRTF-A, or treated with TGF-β2 as compared to respective controls. RNA samples were normalized to the expression of U26 small nucleolar RNA (RNU26). Data represent values from two independent experiments in triplicates (n=6, Mean± SD) * P

    Techniques Used: Expressing, Derivative Assay, Recombinant, Polymerase Chain Reaction

    Constitutively active RhoA (RhoAV14)-induced expression of fibrogenic and myofibroblast markers in HTM cells. A . Analysis of total RNA from serum starved HTM cells (derived from 58 and 73 years old donor eyes) expressing recombinant RhoAV14/GFP by q-PCR revealed a significant increase in expression of FSP-1, collagen-1A1 (Col1A1) and αSMA as compared to GFP expressing control cells. Values represent mean ± SD of two independent experiments performed in triplicate. * P
    Figure Legend Snippet: Constitutively active RhoA (RhoAV14)-induced expression of fibrogenic and myofibroblast markers in HTM cells. A . Analysis of total RNA from serum starved HTM cells (derived from 58 and 73 years old donor eyes) expressing recombinant RhoAV14/GFP by q-PCR revealed a significant increase in expression of FSP-1, collagen-1A1 (Col1A1) and αSMA as compared to GFP expressing control cells. Values represent mean ± SD of two independent experiments performed in triplicate. * P

    Techniques Used: Expressing, Derivative Assay, Recombinant, Polymerase Chain Reaction

    20) Product Images from "GABAA receptors are expressed and facilitate relaxation in airway smooth muscle"

    Article Title: GABAA receptors are expressed and facilitate relaxation in airway smooth muscle

    Journal:

    doi: 10.1152/ajplung.00287.2007

    RNA isolation and RT-PCR
    Figure Legend Snippet: RNA isolation and RT-PCR

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    Representative gel images of RT-PCR analysis of total RNA using primers specific for each known GABA A channel subunit. Total RNA extracted from freshly dissected human tracheal airway smooth muscle or primary cultures of human airway smooth muscle (huASM)
    Figure Legend Snippet: Representative gel images of RT-PCR analysis of total RNA using primers specific for each known GABA A channel subunit. Total RNA extracted from freshly dissected human tracheal airway smooth muscle or primary cultures of human airway smooth muscle (huASM)

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    RNA isolation and RT-PCR
    Figure Legend Snippet: RNA isolation and RT-PCR

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    Representative gel images of RT-PCR analysis of total RNA using primers specific for selected GABA A channel subunits using total RNA extracted from freshly dissected guinea pig tracheal airway smooth muscle. Lanes 1 : bp standards; lanes 2 : negative control
    Figure Legend Snippet: Representative gel images of RT-PCR analysis of total RNA using primers specific for selected GABA A channel subunits using total RNA extracted from freshly dissected guinea pig tracheal airway smooth muscle. Lanes 1 : bp standards; lanes 2 : negative control

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Negative Control

    21) Product Images from "Kindlin-1 and -2 Have Overlapping Functions in Epithelial Cells"

    Article Title: Kindlin-1 and -2 Have Overlapping Functions in Epithelial Cells

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2010.11.053

    In keratinocytes derived from normal and KS skin, kindlin-2 expression was down-regulated after treatment with UV-B. A: Representative agarose gel electrophoresis showing RT-PCR products on total RNA extracted from NHK-E6E7 (NHK) and KS-NM-E6E7 (KS-NM)
    Figure Legend Snippet: In keratinocytes derived from normal and KS skin, kindlin-2 expression was down-regulated after treatment with UV-B. A: Representative agarose gel electrophoresis showing RT-PCR products on total RNA extracted from NHK-E6E7 (NHK) and KS-NM-E6E7 (KS-NM)

    Techniques Used: Derivative Assay, Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    22) Product Images from "Formation of Monoterpenes in Antirrhinum majus and Clarkia breweri Flowers Involves Heterodimeric Geranyl Diphosphate Synthases"

    Article Title: Formation of Monoterpenes in Antirrhinum majus and Clarkia breweri Flowers Involves Heterodimeric Geranyl Diphosphate Synthases

    Journal: The Plant Cell

    doi: 10.1105/tpc.020156

    Tissue Specificity of A. majus GPPS.SSU and GPPS.LSU Gene Expression. (A) RNA gel blot of total RNA (5 μg per lane) isolated from leaves, upper and lower petal lobes, tubes, pistils, stamens, and ovaries of 3-d-old A. majus flowers. The top gel represents the results of hybridization with a coding region of the GPPS.SSU gene as a probe. Autoradiography was performed overnight. The blot was rehybridized with an 18S rDNA probe (bottom gel) to standardize samples. (B) RT-PCR with GPPS.LSU gene-specific primers was performed on RNA isolated from leaves, upper and lower petal lobes, tubes, pistils, stamens, and ovaries of 3-d-old A. majus flowers. The amplified products were run on 1.2% agarose gel, blotted onto nitrocellulose membrane, and hybridized with the GPPS.LSU gene as a probe. The RT-PCR products for rRNA are shown at the bottom.
    Figure Legend Snippet: Tissue Specificity of A. majus GPPS.SSU and GPPS.LSU Gene Expression. (A) RNA gel blot of total RNA (5 μg per lane) isolated from leaves, upper and lower petal lobes, tubes, pistils, stamens, and ovaries of 3-d-old A. majus flowers. The top gel represents the results of hybridization with a coding region of the GPPS.SSU gene as a probe. Autoradiography was performed overnight. The blot was rehybridized with an 18S rDNA probe (bottom gel) to standardize samples. (B) RT-PCR with GPPS.LSU gene-specific primers was performed on RNA isolated from leaves, upper and lower petal lobes, tubes, pistils, stamens, and ovaries of 3-d-old A. majus flowers. The amplified products were run on 1.2% agarose gel, blotted onto nitrocellulose membrane, and hybridized with the GPPS.LSU gene as a probe. The RT-PCR products for rRNA are shown at the bottom.

    Techniques Used: Expressing, Western Blot, Isolation, Hybridization, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    Developmental Changes in Steady State GPPS.SSU and GPPS.LSU mRNA Levels in Upper and Lower Lobes of A. majus Petals. (A) Representative RNA gel blot hybridization with mRNA isolated from upper and lower petal lobes at different stages of development, from mature flower buds to day 12 after anthesis. Each lane contained 5 μg of total RNA. A coding region of the GPPS.SSU gene was used as a probe. Autoradiography was performed overnight. The blots were rehybridized with an 18S rDNA probe (bottom gel) to standardize samples. RNA gel blots were scanned with a Storm 860 PhosphorImager, and the values were corrected by standardizing for the amounts of 18S rRNA measured in the same runs. The maximum transcript level was taken as 1. Each point is the average of six different experiments (including the one shown in [A] ). Standard error values are indicated by vertical bars. (B) Representative RT-PCR with GPPS.LSU gene-specific primers on RNA isolated from upper and lower petal lobes at different stages of development, from mature flower buds to day 12 after anthesis. The amplified products were run on 1.2% agarose gel, blotted onto nitrocellulose membrane, and hybridized with the GPPS.LSU gene as a probe. The RT-PCR products for rRNA are shown at the bottom. Hybridization signals were analyzed using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics). The maximum transcript level was taken as 1. Each point is the average of four different experiments (including the one shown in [B] ). Standard error values are indicated by vertical bars.
    Figure Legend Snippet: Developmental Changes in Steady State GPPS.SSU and GPPS.LSU mRNA Levels in Upper and Lower Lobes of A. majus Petals. (A) Representative RNA gel blot hybridization with mRNA isolated from upper and lower petal lobes at different stages of development, from mature flower buds to day 12 after anthesis. Each lane contained 5 μg of total RNA. A coding region of the GPPS.SSU gene was used as a probe. Autoradiography was performed overnight. The blots were rehybridized with an 18S rDNA probe (bottom gel) to standardize samples. RNA gel blots were scanned with a Storm 860 PhosphorImager, and the values were corrected by standardizing for the amounts of 18S rRNA measured in the same runs. The maximum transcript level was taken as 1. Each point is the average of six different experiments (including the one shown in [A] ). Standard error values are indicated by vertical bars. (B) Representative RT-PCR with GPPS.LSU gene-specific primers on RNA isolated from upper and lower petal lobes at different stages of development, from mature flower buds to day 12 after anthesis. The amplified products were run on 1.2% agarose gel, blotted onto nitrocellulose membrane, and hybridized with the GPPS.LSU gene as a probe. The RT-PCR products for rRNA are shown at the bottom. Hybridization signals were analyzed using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics). The maximum transcript level was taken as 1. Each point is the average of four different experiments (including the one shown in [B] ). Standard error values are indicated by vertical bars.

    Techniques Used: Western Blot, Hybridization, Isolation, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Software

    23) Product Images from "Rac1 mediates STAT3 activation by autocrine IL-6"

    Article Title: Rac1 mediates STAT3 activation by autocrine IL-6

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

    doi: 10.1073/pnas.161281298

    Effect of Rac V12 expression on STAT3 activation and specific gene induction. ( A ) HeLa cells were either untransfected or transfected with either pCDNA3 (as a transfection control) or Rac V12. Total RNA was collected and examined by reverse transcription–PCR for IL-6, IL-6 receptor, and GAPDH mRNA expression. The ethidium bromide-stained 2% agarose gel shows the IL-6 and GAPDH cDNA fragments ( Left ) and IL-6 receptor and GAPDH cDNA fragments ( Right ) amplified from the same amounts of cDNA. ( B ) HeLa cells were transfected with STAT3-GFP alone or with Rac V12 and were incubated for 18 h in serum-free DMEM in the presence of neutralizing IL-6 receptor antibody (1 μg/ml). Cells either were left untreated ( a , d , and e ) or were treated with soluble IL-6 receptor protein (100 ng/ml) (to enhance IL-6 responsiveness) and IL-6 (20 ng/ml) for 20 min ( b and c ). Cells were fixed and examined by fluorescent microscopy. ( C ) HeLa cells and HT1080 cells were cotransfected with (Gas) 3 -Luc, SV40-LacZ, and either pCDNA3 (control) or STAT3 alone or with Rac V12 and placed in serum-free DMEM in the absence (open bars) or presence (solid bars) of neutralizing IL-6 receptor antibodies (1 μg/ml). Cell lysates were prepared 48 h after transfection and were used to measure both luciferase and β-gal activity. The data shown are means ± SEM and are representative of similar experiments.
    Figure Legend Snippet: Effect of Rac V12 expression on STAT3 activation and specific gene induction. ( A ) HeLa cells were either untransfected or transfected with either pCDNA3 (as a transfection control) or Rac V12. Total RNA was collected and examined by reverse transcription–PCR for IL-6, IL-6 receptor, and GAPDH mRNA expression. The ethidium bromide-stained 2% agarose gel shows the IL-6 and GAPDH cDNA fragments ( Left ) and IL-6 receptor and GAPDH cDNA fragments ( Right ) amplified from the same amounts of cDNA. ( B ) HeLa cells were transfected with STAT3-GFP alone or with Rac V12 and were incubated for 18 h in serum-free DMEM in the presence of neutralizing IL-6 receptor antibody (1 μg/ml). Cells either were left untreated ( a , d , and e ) or were treated with soluble IL-6 receptor protein (100 ng/ml) (to enhance IL-6 responsiveness) and IL-6 (20 ng/ml) for 20 min ( b and c ). Cells were fixed and examined by fluorescent microscopy. ( C ) HeLa cells and HT1080 cells were cotransfected with (Gas) 3 -Luc, SV40-LacZ, and either pCDNA3 (control) or STAT3 alone or with Rac V12 and placed in serum-free DMEM in the absence (open bars) or presence (solid bars) of neutralizing IL-6 receptor antibodies (1 μg/ml). Cell lysates were prepared 48 h after transfection and were used to measure both luciferase and β-gal activity. The data shown are means ± SEM and are representative of similar experiments.

    Techniques Used: Expressing, Activation Assay, Transfection, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Amplification, Incubation, Microscopy, Luciferase, Activity Assay

    24) Product Images from "Up-regulation of the tumor promoter Glyoxalase-1 indicates poor prognosis in breast cancer"

    Article Title: Up-regulation of the tumor promoter Glyoxalase-1 indicates poor prognosis in breast cancer

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    Glo1 is frequently up-regulated in human breast cancer cells and tissues. A. Glo1 mRNA levels in breast cancer cell lines was observably higher than mammary epithelial cell (MCF-10A) analyzed by Real-time PCR. B. The tumor tissue to adjacent nontumorous tissue (ANT) ratio of Glo1 expression was quantified by real-time RT-PCR. C. Glo1 protein was detected by Western blot and also showed that Glo1 protein level in breast cancer cell lines was observably higher than that in mammary epithelial cell (MCF-10A). D. Western blot analysis of Glo1 expression in each of the primary IDC tissue (T) and paired ANT (N) from four patients. E. IHC confirmed that the Glo1 protein was significantly elevated in primary IDC tissues (T) compared to that of paired ANT (N) from eight patients. Magnifications: ×100.
    Figure Legend Snippet: Glo1 is frequently up-regulated in human breast cancer cells and tissues. A. Glo1 mRNA levels in breast cancer cell lines was observably higher than mammary epithelial cell (MCF-10A) analyzed by Real-time PCR. B. The tumor tissue to adjacent nontumorous tissue (ANT) ratio of Glo1 expression was quantified by real-time RT-PCR. C. Glo1 protein was detected by Western blot and also showed that Glo1 protein level in breast cancer cell lines was observably higher than that in mammary epithelial cell (MCF-10A). D. Western blot analysis of Glo1 expression in each of the primary IDC tissue (T) and paired ANT (N) from four patients. E. IHC confirmed that the Glo1 protein was significantly elevated in primary IDC tissues (T) compared to that of paired ANT (N) from eight patients. Magnifications: ×100.

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

    To confirm the silencing efficiency on Glo1. A. Silencing efficiency of Glo1 in mRNA level by siRNA in indicated cells were identified by qRT-PCR. ** P
    Figure Legend Snippet: To confirm the silencing efficiency on Glo1. A. Silencing efficiency of Glo1 in mRNA level by siRNA in indicated cells were identified by qRT-PCR. ** P

    Techniques Used: Quantitative RT-PCR

    25) Product Images from "Quantitative Proteomics Reveals Cellular Targets of Celastrol"

    Article Title: Quantitative Proteomics Reveals Cellular Targets of Celastrol

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026634

    Time- and dose-dependent effects of celastrol on transcriptional activation and cellular viability activity. (A) Transcript levels from genes involved in the heat shock response, ER unfolded protein response, anti-oxidant response, and mitochondrial unfolded protein response. The mRNA levels were measured by quantitative RT-PCR following incubation for 8 or 24 h in 1 µM celastrol. Relative mRNA levels were normalized using GAPDH mRNA as a reference and presented relative to levels in untreated control cells. Data are mean of two independent experiments (error bars show the range) and each cDNA was analyzed in triplicate PCR reactions. (B) Time- and dose-dependent inhibition of general gene transcription. The HSPA1B , HSPD1 , and ACTB mRNA levels in cells treated with 0.2, 0.5, or 1 µM celastrol for either 8 or 24 h were measured by quantitative RT-PCR. The mRNA levels were normalized to the amount of total RNA used for the RT-PCR reaction and presented relative to levels in untreated cells (arbitrary units, au). Data are mean of two independent experiments (error bars show the range) and each cDNA was analyzed in triplicate PCR reactions. (C) Dose-dependent inhibition of MTT metabolic activity. The metabolic activity/viability following 24 h treatment with varying celastrol concentrations was addressed by quantifying the cellular reduction of MTT into formazan. The amount of MTT reduced in a 4 h period was measured spectrophotometrically by the absorbance Abs [A 595 –A 655 ] of the formazan product. Each datapoint are the mean of three experiments. Insert: Metabolic activity of cells exposed to 0.8 µM celastrol for 0, 1, 4, and 24 h. Data are mean (+/−SD) of three experiments.
    Figure Legend Snippet: Time- and dose-dependent effects of celastrol on transcriptional activation and cellular viability activity. (A) Transcript levels from genes involved in the heat shock response, ER unfolded protein response, anti-oxidant response, and mitochondrial unfolded protein response. The mRNA levels were measured by quantitative RT-PCR following incubation for 8 or 24 h in 1 µM celastrol. Relative mRNA levels were normalized using GAPDH mRNA as a reference and presented relative to levels in untreated control cells. Data are mean of two independent experiments (error bars show the range) and each cDNA was analyzed in triplicate PCR reactions. (B) Time- and dose-dependent inhibition of general gene transcription. The HSPA1B , HSPD1 , and ACTB mRNA levels in cells treated with 0.2, 0.5, or 1 µM celastrol for either 8 or 24 h were measured by quantitative RT-PCR. The mRNA levels were normalized to the amount of total RNA used for the RT-PCR reaction and presented relative to levels in untreated cells (arbitrary units, au). Data are mean of two independent experiments (error bars show the range) and each cDNA was analyzed in triplicate PCR reactions. (C) Dose-dependent inhibition of MTT metabolic activity. The metabolic activity/viability following 24 h treatment with varying celastrol concentrations was addressed by quantifying the cellular reduction of MTT into formazan. The amount of MTT reduced in a 4 h period was measured spectrophotometrically by the absorbance Abs [A 595 –A 655 ] of the formazan product. Each datapoint are the mean of three experiments. Insert: Metabolic activity of cells exposed to 0.8 µM celastrol for 0, 1, 4, and 24 h. Data are mean (+/−SD) of three experiments.

    Techniques Used: Activation Assay, Activity Assay, Quantitative RT-PCR, Incubation, Polymerase Chain Reaction, Inhibition, Reverse Transcription Polymerase Chain Reaction, MTT Assay

    26) Product Images from "IL-10 and NOS2 Modulate Antigen-Specific Reactivity and Nerve Infiltration by T Cells in Experimental Leprosy"

    Article Title: IL-10 and NOS2 Modulate Antigen-Specific Reactivity and Nerve Infiltration by T Cells in Experimental Leprosy

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003149

    Induration, M. leprae viability, and IFN-γ expression in infected FP. (A) B6 (black circles), IL-10−/− (inverted triangles), NOS2−/− (diamonds), and 10NOS2−/− (triangles) FP were inoculated with 3×10 7 M. leprae and FP induration was measured using a Vernier caliper. (B) IFN-γ expression in FP at 1, 4 and 8 months post-infection in B6 (white bars), IL-10−/− (gray bars), NOS2−/− (striped bars) and 10NOS2−/− (black bars). RNA was purified and subjected to real-time RT-PCR for IFN-γ. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA expression and reported as fold increase over uninfected FP. At 1 day and four months post infection, M. leprae per FP were (C) enumerated via RLEP Taqman and (D) bacterial viability was assessed by esx A RT-PCR in B6 (white bars), IL-10−/− (gray bars), NOS2−/− (striped bars) and 10NOS2−/− (black bars). Data shown are means +/− SD and are representative of two independent experiments. * p ≤0.05; ** p ≤0.01; *** p ≤0.001.
    Figure Legend Snippet: Induration, M. leprae viability, and IFN-γ expression in infected FP. (A) B6 (black circles), IL-10−/− (inverted triangles), NOS2−/− (diamonds), and 10NOS2−/− (triangles) FP were inoculated with 3×10 7 M. leprae and FP induration was measured using a Vernier caliper. (B) IFN-γ expression in FP at 1, 4 and 8 months post-infection in B6 (white bars), IL-10−/− (gray bars), NOS2−/− (striped bars) and 10NOS2−/− (black bars). RNA was purified and subjected to real-time RT-PCR for IFN-γ. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA expression and reported as fold increase over uninfected FP. At 1 day and four months post infection, M. leprae per FP were (C) enumerated via RLEP Taqman and (D) bacterial viability was assessed by esx A RT-PCR in B6 (white bars), IL-10−/− (gray bars), NOS2−/− (striped bars) and 10NOS2−/− (black bars). Data shown are means +/− SD and are representative of two independent experiments. * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

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

    27) Product Images from "Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: Caveolin-2 is up-regulated in response to cell injury"

    Article Title: Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: Caveolin-2 is up-regulated in response to cell injury

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

    doi:

    Expression of caveolins 1 and 2 in DRG neurons. ( A ) Immunoblot analysis. Lysates were prepared from cultures of rat DRG neurons and divided into Triton-soluble (S) and Triton-insoluble (I) fractions. Caveolin-1 (pAb N-20) and caveolin-2 (mAb 65) specific antibody probes were then used to probe these cellular extracts by immunoblotting. Note that both caveolins 1 and 2 are expressed and behave as Triton-insoluble components. ( B ) RT-PCR analysis. Total RNA isolated from PC12 cells and DRG neurons was subjected to RT-PCR analysis by using primers directed against the known sequences of caveolins 1 and 2; NIH 3T3 cells served as a postitive control.
    Figure Legend Snippet: Expression of caveolins 1 and 2 in DRG neurons. ( A ) Immunoblot analysis. Lysates were prepared from cultures of rat DRG neurons and divided into Triton-soluble (S) and Triton-insoluble (I) fractions. Caveolin-1 (pAb N-20) and caveolin-2 (mAb 65) specific antibody probes were then used to probe these cellular extracts by immunoblotting. Note that both caveolins 1 and 2 are expressed and behave as Triton-insoluble components. ( B ) RT-PCR analysis. Total RNA isolated from PC12 cells and DRG neurons was subjected to RT-PCR analysis by using primers directed against the known sequences of caveolins 1 and 2; NIH 3T3 cells served as a postitive control.

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

    28) Product Images from "Gap Junctional Coupling and Patterns of Connexin Expression among Neonatal Rat Lumbar Spinal Motor Neurons"

    Article Title: Gap Junctional Coupling and Patterns of Connexin Expression among Neonatal Rat Lumbar Spinal Motor Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-24-10813.1999

    RT-PCR analysis of connexins expressed by embryonic motor neurons and in neonatal spinal cord. RT-PCR analysis was performed on motor neuron RNA. PCR products were amplified using primers for each of the 13 known rodent connexins. Bands were typically observed in the motor neuron cDNA lanes, but not RNA lanes, using primers specific for Cx36, Cx37, Cx40, Cx43, and Cx45. Primers specific for Cx36 amplified a 979 bp band in motor neuron and eye cDNA. Primers specific for Cx37, Cx40, Cx43, and Cx45 amplified 422, 308, 292, and 1217 bp bands, respectively, in motor neuron and heart cDNA. In contrast, primers against the other known rodent connexins amplified the predicted size band from tissues known to express that particular connexin (for example, skin, heart, liver, eye, and testis) but failed to amplify the same size band in motor neurons. In each case, PCR products were eluted from gels, cloned, and sequenced to verify their identity. Horizontal lines at left indicate markers (from top to bottom, 1.2, 0.6, and 0.3 kB).
    Figure Legend Snippet: RT-PCR analysis of connexins expressed by embryonic motor neurons and in neonatal spinal cord. RT-PCR analysis was performed on motor neuron RNA. PCR products were amplified using primers for each of the 13 known rodent connexins. Bands were typically observed in the motor neuron cDNA lanes, but not RNA lanes, using primers specific for Cx36, Cx37, Cx40, Cx43, and Cx45. Primers specific for Cx36 amplified a 979 bp band in motor neuron and eye cDNA. Primers specific for Cx37, Cx40, Cx43, and Cx45 amplified 422, 308, 292, and 1217 bp bands, respectively, in motor neuron and heart cDNA. In contrast, primers against the other known rodent connexins amplified the predicted size band from tissues known to express that particular connexin (for example, skin, heart, liver, eye, and testis) but failed to amplify the same size band in motor neurons. In each case, PCR products were eluted from gels, cloned, and sequenced to verify their identity. Horizontal lines at left indicate markers (from top to bottom, 1.2, 0.6, and 0.3 kB).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Clone Assay

    29) Product Images from "Identification of the adenylyl cyclase-activating 5-hydroxytryptamine receptor subtypes expressed in the rat submandibular gland"

    Article Title: Identification of the adenylyl cyclase-activating 5-hydroxytryptamine receptor subtypes expressed in the rat submandibular gland

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0703303

    RT–PCR analysis of 5-HT receptor subtype mRNA expression in the rat SMG. 5-HT receptor subtype-specific oligonucleotide primers were designed and used in the RT–PCR as described in Methods. Migration of 500, 400 and 300 base pair (bp) ladder bands (L) in 2% agarose gel electrophoresis are shown on the left and labels above each lane indicate the cDNA source. Primers used and size of product obtained are indicated below each band. Absence of genomic DNA contamination in the cDNA samples was verified by PCR without prior incubation with reverse transcriptase.
    Figure Legend Snippet: RT–PCR analysis of 5-HT receptor subtype mRNA expression in the rat SMG. 5-HT receptor subtype-specific oligonucleotide primers were designed and used in the RT–PCR as described in Methods. Migration of 500, 400 and 300 base pair (bp) ladder bands (L) in 2% agarose gel electrophoresis are shown on the left and labels above each lane indicate the cDNA source. Primers used and size of product obtained are indicated below each band. Absence of genomic DNA contamination in the cDNA samples was verified by PCR without prior incubation with reverse transcriptase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Migration, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Incubation

    30) Product Images from "(E)-?-Ocimene and Myrcene Synthase Genes of Floral Scent Biosynthesis in Snapdragon"

    Article Title: (E)-?-Ocimene and Myrcene Synthase Genes of Floral Scent Biosynthesis in Snapdragon

    Journal: The Plant Cell

    doi: 10.1105/tpc.011015

    Tissue Specificity of Snapdragon Monoterpene Synthase Gene Expression. (A) RNA gel blot of total RNA (5 μg per lane) isolated from young leaves, sepals, pistils, stamens, upper and lower petal lobes, and tubes of 3-day- old snapdragon flowers. The top gel represents the results of hybridization with a coding region of the monoterpene synthase genes derived from ama1e20 as a probe. The length of the monoterpene synthase mRNA was estimated as 2.8 kb using RNA molecular markers in an adjacent lane. Autoradiography was performed for 48 h. The blot was rehybridized with an 18S rRNA probe (bottom gel) to standardize samples. (B) Contribution of each monoterpene synthase to total monoterpene synthase gene expression. RT-PCR with gene-specific primers was performed on RNA isolated from upper and lower petal lobes of 6-day-old snapdragon flowers. Relative expression was estimated after hybridization of RT-PCR products with the same gene probe used in the RNA gel blot analysis (A) . Hybridization signals were analyzed using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics).
    Figure Legend Snippet: Tissue Specificity of Snapdragon Monoterpene Synthase Gene Expression. (A) RNA gel blot of total RNA (5 μg per lane) isolated from young leaves, sepals, pistils, stamens, upper and lower petal lobes, and tubes of 3-day- old snapdragon flowers. The top gel represents the results of hybridization with a coding region of the monoterpene synthase genes derived from ama1e20 as a probe. The length of the monoterpene synthase mRNA was estimated as 2.8 kb using RNA molecular markers in an adjacent lane. Autoradiography was performed for 48 h. The blot was rehybridized with an 18S rRNA probe (bottom gel) to standardize samples. (B) Contribution of each monoterpene synthase to total monoterpene synthase gene expression. RT-PCR with gene-specific primers was performed on RNA isolated from upper and lower petal lobes of 6-day-old snapdragon flowers. Relative expression was estimated after hybridization of RT-PCR products with the same gene probe used in the RNA gel blot analysis (A) . Hybridization signals were analyzed using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics).

    Techniques Used: Expressing, Western Blot, Isolation, Hybridization, Derivative Assay, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Software

    31) Product Images from "Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice"

    Article Title: Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002404

    Enumeration and quantification of M. leprae in mouse FPs by molecular assays. BALB/c (gray bars) and athymic nu/nu (black bars) mice were infected in with 3×10 7 M. leprae . FP tissues were fixed in 70% ethanol on day 1 and at 4, 8, 12, and 17 weeks post infection. DNA and RNA were purified using a FastPrep protocol. M. leprae were enumerated by RLEP PCR on the DNA fraction (A). cDNA was prepared from an RNA equivalent of 3×10 3 M. leprae for determination of viability by hsp18 (B) and esxA (C) qRT-PCR. Bars represent mean and standard deviation for each group. The value at each time point was compared to its respective value at 1 day. * = probability of statistical significance (p)
    Figure Legend Snippet: Enumeration and quantification of M. leprae in mouse FPs by molecular assays. BALB/c (gray bars) and athymic nu/nu (black bars) mice were infected in with 3×10 7 M. leprae . FP tissues were fixed in 70% ethanol on day 1 and at 4, 8, 12, and 17 weeks post infection. DNA and RNA were purified using a FastPrep protocol. M. leprae were enumerated by RLEP PCR on the DNA fraction (A). cDNA was prepared from an RNA equivalent of 3×10 3 M. leprae for determination of viability by hsp18 (B) and esxA (C) qRT-PCR. Bars represent mean and standard deviation for each group. The value at each time point was compared to its respective value at 1 day. * = probability of statistical significance (p)

    Techniques Used: Mouse Assay, Infection, Purification, Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    Enumeration and quantification via molecular assays of M. leprae from RMP and RPT treated mice. Athymic nu/nu (black bars) mice were infected in the FPs with 3×10 7 M. leprae . At 18 weeks post infection, groups of mice were treated with 1 dose (1×), 5 daily doses (5×), or 20 daily doses (20×) of RMP (striped bars) or RPT (crosshatched bars), each at 10 mg/kg. Control mice received vehicle alone (gray bars). One month after the last dose of each regimen, FP tissues were fixed in 70% ethanol. DNA and RNA were purified using a FastPrep protocol. M. leprae were enumerated by RLEP PCR on the DNA fraction, and cDNA was prepared from an RNA equivalent of 3×10 3 M. leprae for determination of viability by hsp18 (A) and esxA (B) qRT-PCR. Bars represent mean and standard deviation for each group. * = probability of statistical significance (p)
    Figure Legend Snippet: Enumeration and quantification via molecular assays of M. leprae from RMP and RPT treated mice. Athymic nu/nu (black bars) mice were infected in the FPs with 3×10 7 M. leprae . At 18 weeks post infection, groups of mice were treated with 1 dose (1×), 5 daily doses (5×), or 20 daily doses (20×) of RMP (striped bars) or RPT (crosshatched bars), each at 10 mg/kg. Control mice received vehicle alone (gray bars). One month after the last dose of each regimen, FP tissues were fixed in 70% ethanol. DNA and RNA were purified using a FastPrep protocol. M. leprae were enumerated by RLEP PCR on the DNA fraction, and cDNA was prepared from an RNA equivalent of 3×10 3 M. leprae for determination of viability by hsp18 (A) and esxA (B) qRT-PCR. Bars represent mean and standard deviation for each group. * = probability of statistical significance (p)

    Techniques Used: Mouse Assay, Infection, Purification, Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    32) Product Images from "Palmitate induces cisternal ER expansion via the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells"

    Article Title: Palmitate induces cisternal ER expansion via the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells

    Journal: Lipids in Health and Disease

    doi: 10.1186/s12944-015-0077-3

    Tunicamycin does not induces cisternal ER expansion in RAW 264.7 cells. Cells were treated with 10 μg/ml tunicamycin for 24 h. The phosphorylation of PERK, and the expression of BiP and CHOP were determined by western blot analysis ( a ). Total RNA was extracted and reverse transcribed to cDNA. Full-length and spliced XBP-1 cDNAs were amplified by PCR with mouse XBP-1 primers. Gapdh cDNA was included as an internal loading control ( b ). Changes in the ER structure were examined by transmission electron microscopy ( c )
    Figure Legend Snippet: Tunicamycin does not induces cisternal ER expansion in RAW 264.7 cells. Cells were treated with 10 μg/ml tunicamycin for 24 h. The phosphorylation of PERK, and the expression of BiP and CHOP were determined by western blot analysis ( a ). Total RNA was extracted and reverse transcribed to cDNA. Full-length and spliced XBP-1 cDNAs were amplified by PCR with mouse XBP-1 primers. Gapdh cDNA was included as an internal loading control ( b ). Changes in the ER structure were examined by transmission electron microscopy ( c )

    Techniques Used: Expressing, Western Blot, Amplification, Polymerase Chain Reaction, Transmission Assay, Electron Microscopy

    Palmitate-induced cisternal ER expansion is dependent on ER stress in RAW 264.7 cells. Cells were pre-treated with or without 4 mM 4-PBA or 40 μM TUDCA for 1 h and followed by treatment with 400 μM palmitate for 24 h. The phosphorylation of PERK, and the expression of BiP and CHOP were determined by western blot analysis ( a ). Total RNA was extracted and reverse transcribed to cDNA. Full-length and spliced XBP-1 cDNAs were amplified by PCR with mouse XBP-1 primers. Gapdh cDNA was included as an internal loading control ( b ). Changes in ER structure were examined by transmission electron microscopy ( c )
    Figure Legend Snippet: Palmitate-induced cisternal ER expansion is dependent on ER stress in RAW 264.7 cells. Cells were pre-treated with or without 4 mM 4-PBA or 40 μM TUDCA for 1 h and followed by treatment with 400 μM palmitate for 24 h. The phosphorylation of PERK, and the expression of BiP and CHOP were determined by western blot analysis ( a ). Total RNA was extracted and reverse transcribed to cDNA. Full-length and spliced XBP-1 cDNAs were amplified by PCR with mouse XBP-1 primers. Gapdh cDNA was included as an internal loading control ( b ). Changes in ER structure were examined by transmission electron microscopy ( c )

    Techniques Used: Expressing, Western Blot, Amplification, Polymerase Chain Reaction, Transmission Assay, Electron Microscopy

    Palmitate induces ER stress in RAW 264.7 cells. Cells were treated with palmitate at the indicated concentration for 24 h ( a and b ) and with 400 μM palmitate for the indicated times ( c and d ). The phosphorylation of PERK, and the expression of BiP and CHOP were determined by western blot analysis ( a and c ). Total RNA was extracted and reverse transcribed to cDNA. Full-length and spliced XBP-1 cDNAs were amplified by PCR with mouse XBP-1 primers. Gapdh cDNA was included as an internal loading control ( b and d )
    Figure Legend Snippet: Palmitate induces ER stress in RAW 264.7 cells. Cells were treated with palmitate at the indicated concentration for 24 h ( a and b ) and with 400 μM palmitate for the indicated times ( c and d ). The phosphorylation of PERK, and the expression of BiP and CHOP were determined by western blot analysis ( a and c ). Total RNA was extracted and reverse transcribed to cDNA. Full-length and spliced XBP-1 cDNAs were amplified by PCR with mouse XBP-1 primers. Gapdh cDNA was included as an internal loading control ( b and d )

    Techniques Used: Concentration Assay, Expressing, Western Blot, Amplification, Polymerase Chain Reaction

    33) Product Images from "Cis- and trans-regulations of pre-mRNA splicing by RNA editing enzymes influence cancer development"

    Article Title: Cis- and trans-regulations of pre-mRNA splicing by RNA editing enzymes influence cancer development

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14621-5

    ADAR2 binding to dsRNA that involves the Py-tract blocks U2AF65. a Schematic diagram of RELL2 minigene and serial deletions in exon 3 and intron 3. Editing site is highlighted in red. b RT-PCR analysis of exon 3 inclusion of exogenous RELL2 transcripts in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct ( n = 2 biological replicates for each). c In silico prediction of RNA secondary structure by RNAfold. Base-pair probabilities are shown by a color spectrum. Asterisk indicates the editing site. d RT-PCR analysis of exon 3 inclusion of exogenous RELL2 transcripts in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct ( n = 2 biological replicates for each). GA-rich sequences #1 and #2 are highlighted in red and blue. e REMSA analysis of the binding of ADAR2 protein to RELL2 transcripts in vitro, using 32 P-labeled wild-type or mutant RELL2 Py-ex3 dsRNA probe with the increasing amount of recombinant ADAR2 protein. %Shift is calculated as shift band intensity over the sum of free probe and shift band intensities. f RIP-qPCR analysis of the binding of ADAR2 protein to exogenous RELL2 transcripts in vivo. WB analysis of FLAG-RIP immunoprecipitates is shown in the top panel. Input indicates 1% of the total cell lysate. Data are presented as mean ± S.D. of %input derived from qPCR technical triplicates from a representative experiment. Each dot represents a technical replicate. Statistical significance is determined by unpaired, two-tailed Student’s t -test (**** P
    Figure Legend Snippet: ADAR2 binding to dsRNA that involves the Py-tract blocks U2AF65. a Schematic diagram of RELL2 minigene and serial deletions in exon 3 and intron 3. Editing site is highlighted in red. b RT-PCR analysis of exon 3 inclusion of exogenous RELL2 transcripts in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct ( n = 2 biological replicates for each). c In silico prediction of RNA secondary structure by RNAfold. Base-pair probabilities are shown by a color spectrum. Asterisk indicates the editing site. d RT-PCR analysis of exon 3 inclusion of exogenous RELL2 transcripts in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct ( n = 2 biological replicates for each). GA-rich sequences #1 and #2 are highlighted in red and blue. e REMSA analysis of the binding of ADAR2 protein to RELL2 transcripts in vitro, using 32 P-labeled wild-type or mutant RELL2 Py-ex3 dsRNA probe with the increasing amount of recombinant ADAR2 protein. %Shift is calculated as shift band intensity over the sum of free probe and shift band intensities. f RIP-qPCR analysis of the binding of ADAR2 protein to exogenous RELL2 transcripts in vivo. WB analysis of FLAG-RIP immunoprecipitates is shown in the top panel. Input indicates 1% of the total cell lysate. Data are presented as mean ± S.D. of %input derived from qPCR technical triplicates from a representative experiment. Each dot represents a technical replicate. Statistical significance is determined by unpaired, two-tailed Student’s t -test (**** P

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Over Expression, Construct, In Silico, In Vitro, Labeling, Mutagenesis, Recombinant, Real-time Polymerase Chain Reaction, In Vivo, Western Blot, Derivative Assay, Two Tailed Test

    Skipping of RELL2 exon 3 results in AS-NMD. a Schematic diagram showing skipping of RELL2 exon 3 may trigger NMD. With exon 3 included, RELL2 pre-mRNA has a stop codon at the end of exon 6, which is then translated to a functional protein. In contrast, skipping of exon 3 results in a frameshift and creates a pre-mature termination codon at the beginning of exon 5, which locates 368 nt upstream of the last exon–exon junction. b RT-PCR analysis of different isoforms of the indicated transcripts in HEK293T cells upon inhibition of NMD. Cells were transfected with scramble shRNA (scr) or shRNA against UPF1 (sh UPF1 ) for UPF1 knockdown, or treated with DMSO or CHX. SRSF1 or ZDHHC16 serves as a positive or negative control, respectively, to ensure successful inhibition of NMD. Number sign indicates the NMD-sensitive isoform. c qPCR analysis of total RELL2 transcript level in HEK293T cells with the indicated treatment. Data are presented as the mean ± S.D. of relative expression derived from qPCR technical triplicates or duplicates from a representative experiment. Each dot represents a technical replicate. Statistical significance is determined by unpaired, two-tailed Student’s t -test (* P
    Figure Legend Snippet: Skipping of RELL2 exon 3 results in AS-NMD. a Schematic diagram showing skipping of RELL2 exon 3 may trigger NMD. With exon 3 included, RELL2 pre-mRNA has a stop codon at the end of exon 6, which is then translated to a functional protein. In contrast, skipping of exon 3 results in a frameshift and creates a pre-mature termination codon at the beginning of exon 5, which locates 368 nt upstream of the last exon–exon junction. b RT-PCR analysis of different isoforms of the indicated transcripts in HEK293T cells upon inhibition of NMD. Cells were transfected with scramble shRNA (scr) or shRNA against UPF1 (sh UPF1 ) for UPF1 knockdown, or treated with DMSO or CHX. SRSF1 or ZDHHC16 serves as a positive or negative control, respectively, to ensure successful inhibition of NMD. Number sign indicates the NMD-sensitive isoform. c qPCR analysis of total RELL2 transcript level in HEK293T cells with the indicated treatment. Data are presented as the mean ± S.D. of relative expression derived from qPCR technical triplicates or duplicates from a representative experiment. Each dot represents a technical replicate. Statistical significance is determined by unpaired, two-tailed Student’s t -test (* P

    Techniques Used: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Inhibition, Transfection, shRNA, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Two Tailed Test

    ADAR proteins regulate a subset of alternative splicing events. a WB analyses of ADAR1 and ADAR2 proteins in EC109 cells that were stably knocked down (sh ADAR1 #3 and #9; sh ADAR2 #939 and #942; and scramble shRNA (scr)) or overexpressed (pLenti- ADAR1 ; pLenti- ADAR2 ; and empty vector control) for ADAR1 or 2, using lentiviral system. β-actin (actin) was used as a loading control. b Pie charts representing the number (and percentage) of each type of alternative splicing events affected by ADAR1 (left) and ADAR2 (right). c Heat maps showing the differentially spliced cassette exon events, upon knockdown and overexpression of ADAR1 (left) and ADAR2 (right). Splicing index (SI) is calculated by the ratio of inclusion junction reads to the sum of inclusion and skipping junction reads, and ΔSI indicates the difference in SI between ADARs knockdown/overexpression and their corresponding control samples. d , e RT-PCR analyses of representative ADAR1- d or ADAR2- e affected cassette exons in original RNA-Seq EC109 cells, as well as HEK293T cells. PSI, percent spliced in. Source data are provided as a Source Data file.
    Figure Legend Snippet: ADAR proteins regulate a subset of alternative splicing events. a WB analyses of ADAR1 and ADAR2 proteins in EC109 cells that were stably knocked down (sh ADAR1 #3 and #9; sh ADAR2 #939 and #942; and scramble shRNA (scr)) or overexpressed (pLenti- ADAR1 ; pLenti- ADAR2 ; and empty vector control) for ADAR1 or 2, using lentiviral system. β-actin (actin) was used as a loading control. b Pie charts representing the number (and percentage) of each type of alternative splicing events affected by ADAR1 (left) and ADAR2 (right). c Heat maps showing the differentially spliced cassette exon events, upon knockdown and overexpression of ADAR1 (left) and ADAR2 (right). Splicing index (SI) is calculated by the ratio of inclusion junction reads to the sum of inclusion and skipping junction reads, and ΔSI indicates the difference in SI between ADARs knockdown/overexpression and their corresponding control samples. d , e RT-PCR analyses of representative ADAR1- d or ADAR2- e affected cassette exons in original RNA-Seq EC109 cells, as well as HEK293T cells. PSI, percent spliced in. Source data are provided as a Source Data file.

    Techniques Used: Western Blot, Stable Transfection, shRNA, Plasmid Preparation, Over Expression, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay

    ADAR1-mediated editing of an ISS enhances SRSF7 binding for exon skipping. a Sequence chromatograms illustrate the editing level of the indicated sites (1–4) at intron 8 of CCDC15 pre-mRNA in HEK293T cells that were transfected with empty vector control (EV), ADAR1 (0.25, 1.0, or 2.0 µg), or ADAR2 (2.0 µg) expression construct. Black arrowhead indicates editing position. Red arrows show the location of primers used for PCR amplification. b Upper panel: schematic diagram of wild-type (WT) CCDC15 exon 8–9–10 minigene. The positions where an A-to-G mutation was introduced are highlighted in red (sites 1, 2, and 4) and purple (site 3). The 13-bp region deleted in the Del minigene is shaded in orange. Lower panel: RT-PCR analysis of exon 9 inclusion of exogenous CCDC15 transcripts in HEK293T cells that were transfected with the indicated WT or mutant minigenes ( n = 3 or 2 biological replicates for each). 1 + 2 denotes both sites 1 and 2 were mutated from A to G. c In silico prediction of SRSF7 binding sites on the edited CCDC15 pre-mRNA by Human Splicing Finder (orange line) and RBPmap (blue line). The edited nucleotide at site 2 is highlighted in red. d RT-PCR analysis of exon 9 inclusion of exogenous CCDC15 transcripts in HEK293T cells that were co-transfected with WT or site 2-mutated (Mut 2) minigene together with EV or SRSF7 expression construct ( n = 3 biological replicates for each). e WB analysis of RNA pulldown products (eluate) shows binding of SRSF7 and hnRNPK protein to the WT or Mut 2 RNA probes. Sequence of the Mut 2 probe is shown in c and the WT probe is the same except site 2 that remains as an unedited adenosine. FT, flow-through. b , d Data are presented as the mean ± S.D. of percent spliced in (PSI) values from biological replicates. Each dot represents a biological replicate. Statistical significance is determined by paired t -test (* P
    Figure Legend Snippet: ADAR1-mediated editing of an ISS enhances SRSF7 binding for exon skipping. a Sequence chromatograms illustrate the editing level of the indicated sites (1–4) at intron 8 of CCDC15 pre-mRNA in HEK293T cells that were transfected with empty vector control (EV), ADAR1 (0.25, 1.0, or 2.0 µg), or ADAR2 (2.0 µg) expression construct. Black arrowhead indicates editing position. Red arrows show the location of primers used for PCR amplification. b Upper panel: schematic diagram of wild-type (WT) CCDC15 exon 8–9–10 minigene. The positions where an A-to-G mutation was introduced are highlighted in red (sites 1, 2, and 4) and purple (site 3). The 13-bp region deleted in the Del minigene is shaded in orange. Lower panel: RT-PCR analysis of exon 9 inclusion of exogenous CCDC15 transcripts in HEK293T cells that were transfected with the indicated WT or mutant minigenes ( n = 3 or 2 biological replicates for each). 1 + 2 denotes both sites 1 and 2 were mutated from A to G. c In silico prediction of SRSF7 binding sites on the edited CCDC15 pre-mRNA by Human Splicing Finder (orange line) and RBPmap (blue line). The edited nucleotide at site 2 is highlighted in red. d RT-PCR analysis of exon 9 inclusion of exogenous CCDC15 transcripts in HEK293T cells that were co-transfected with WT or site 2-mutated (Mut 2) minigene together with EV or SRSF7 expression construct ( n = 3 biological replicates for each). e WB analysis of RNA pulldown products (eluate) shows binding of SRSF7 and hnRNPK protein to the WT or Mut 2 RNA probes. Sequence of the Mut 2 probe is shown in c and the WT probe is the same except site 2 that remains as an unedited adenosine. FT, flow-through. b , d Data are presented as the mean ± S.D. of percent spliced in (PSI) values from biological replicates. Each dot represents a biological replicate. Statistical significance is determined by paired t -test (* P

    Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, Expressing, Construct, Polymerase Chain Reaction, Amplification, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, In Silico, Western Blot

    34) Product Images from "The Cryptococcus neoformans Rim101 Transcription Factor Directly Regulates Genes Required for Adaptation to the Host"

    Article Title: The Cryptococcus neoformans Rim101 Transcription Factor Directly Regulates Genes Required for Adaptation to the Host

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01359-13

    RNA extraction, cDNA preparation, and RT-PCR.
    Figure Legend Snippet: RNA extraction, cDNA preparation, and RT-PCR.

    Techniques Used: RNA Extraction, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "M-Like K+ Currents in Type I Hair Cells and Calyx Afferent Endings of the Developing Rat Utricle"

    Article Title: M-Like K+ Currents in Type I Hair Cells and Calyx Afferent Endings of the Developing Rat Utricle

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2596-06.2006

    RT-PCR on single cells revealed widespread expression of KCNQ and erg subunits. A , Agarose gels showing PCR products from P8 rat utricular hair cells for KCNQ3, KCNQ4, and KCNQ5 (top) and erg1, erg2, and erg3 (bottom). Top, Nine type I hair cells from one macula were probed for KCNQ3–KCNQ5. Six cells (3, 4, 5, 6, 8, 9) were positive for KCNQ3 (160 bp). Seven cells (1, 2, 3, 4, 5, 7, 8) were positive for KCNQ5 (106 bp). The band for cell 1 was faint. The identities of products at other molecular weights are not known. None were positive for KCNQ4 (380 bp). Bottom, Six type I hair cells from another macula were probed for erg1, erg2, and erg3. Cell 2 was positive for all three subunits, cell 4 was positive for erg2 and erg3 (the band corresponding to erg2 is faint), and five of six cells (cells 1–5) were positive for erg3. B , Percentages of positive PCR products for the following: left, single type I hair cells tested with erg1, erg2, erg3, KCNQ3, KCNQ4, and KCNQ5 probes at P1 and P8, and with erg1, erg2, erg 3, and KCNQ4 probes at P14; right, single type II cells tested with KCNQ3 and KCNQ5 probes at P8. We did not test type II cells at other ages, nor did we test them for KCNQ4 at any age. The number of cells tested in each condition is given at the top of the histogram bars.
    Figure Legend Snippet: RT-PCR on single cells revealed widespread expression of KCNQ and erg subunits. A , Agarose gels showing PCR products from P8 rat utricular hair cells for KCNQ3, KCNQ4, and KCNQ5 (top) and erg1, erg2, and erg3 (bottom). Top, Nine type I hair cells from one macula were probed for KCNQ3–KCNQ5. Six cells (3, 4, 5, 6, 8, 9) were positive for KCNQ3 (160 bp). Seven cells (1, 2, 3, 4, 5, 7, 8) were positive for KCNQ5 (106 bp). The band for cell 1 was faint. The identities of products at other molecular weights are not known. None were positive for KCNQ4 (380 bp). Bottom, Six type I hair cells from another macula were probed for erg1, erg2, and erg3. Cell 2 was positive for all three subunits, cell 4 was positive for erg2 and erg3 (the band corresponding to erg2 is faint), and five of six cells (cells 1–5) were positive for erg3. B , Percentages of positive PCR products for the following: left, single type I hair cells tested with erg1, erg2, erg3, KCNQ3, KCNQ4, and KCNQ5 probes at P1 and P8, and with erg1, erg2, erg 3, and KCNQ4 probes at P14; right, single type II cells tested with KCNQ3 and KCNQ5 probes at P8. We did not test type II cells at other ages, nor did we test them for KCNQ4 at any age. The number of cells tested in each condition is given at the top of the histogram bars.

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

    36) Product Images from "The dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle"

    Article Title: The dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00130.2011

    Representative gel images of RT-PCR analysis of total RNA using primers specific for each of the known human dopamine D 2 -like receptor subtypes (D 2 , D 3 , and D 4 ). Total RNA extracted from primary cultures of human airway smooth muscle (HASM) cells or freshly dissected human tracheal airway smooth muscle was analyzed. Lane 1 : base pair standards; lane 2 : negative control water blanks; lane 3 : total RNA from primary cultured HASM cells; lane 4 : total RNA from freshly dissected native human airway smooth muscle; lane 5 : total RNA from whole human brain.
    Figure Legend Snippet: Representative gel images of RT-PCR analysis of total RNA using primers specific for each of the known human dopamine D 2 -like receptor subtypes (D 2 , D 3 , and D 4 ). Total RNA extracted from primary cultures of human airway smooth muscle (HASM) cells or freshly dissected human tracheal airway smooth muscle was analyzed. Lane 1 : base pair standards; lane 2 : negative control water blanks; lane 3 : total RNA from primary cultured HASM cells; lane 4 : total RNA from freshly dissected native human airway smooth muscle; lane 5 : total RNA from whole human brain.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Negative Control, Cell Culture

    37) Product Images from "Network-based approach to prediction and population-based validation of in silico drug repurposing"

    Article Title: Network-based approach to prediction and population-based validation of in silico drug repurposing

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05116-5

    Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p
    Figure Legend Snippet: Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p

    Techniques Used: Expressing, RNA Sequencing Assay, In Vitro, In Vivo, Activation Assay, Inhibition, Incubation, Quantitative RT-PCR

    38) Product Images from "Expression of Id-1 is regulated by MCAM/MUC18: A missing link in melanoma progression"

    Article Title: Expression of Id-1 is regulated by MCAM/MUC18: A missing link in melanoma progression

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-3555

    Effect of MUC18 silencing on melanoma cell invasion and MMP-2 expression (A) Zymography shows decreased MMP-2 activity after MUC18 silencing in both A375SM and C8161 compared to NT transduced cells. Control of zymography gel demonstrates equal loading of samples as indicated by silver staining. Images are representative of three experiments performed. (B) Quantitative real-time PCR validates dimished MMP-2 expression following MUC18 silencing in both A375SM and C8161, compared to NT transduced cells(* p
    Figure Legend Snippet: Effect of MUC18 silencing on melanoma cell invasion and MMP-2 expression (A) Zymography shows decreased MMP-2 activity after MUC18 silencing in both A375SM and C8161 compared to NT transduced cells. Control of zymography gel demonstrates equal loading of samples as indicated by silver staining. Images are representative of three experiments performed. (B) Quantitative real-time PCR validates dimished MMP-2 expression following MUC18 silencing in both A375SM and C8161, compared to NT transduced cells(* p

    Techniques Used: Expressing, Zymography, Activity Assay, Silver Staining, Real-time Polymerase Chain Reaction

    39) Product Images from "Samarangenin B from Limonium sinense Suppresses Herpes Simplex Virus Type 1 Replication in Vero Cells by Regulation of Viral Macromolecular Synthesis"

    Article Title: Samarangenin B from Limonium sinense Suppresses Herpes Simplex Virus Type 1 Replication in Vero Cells by Regulation of Viral Macromolecular Synthesis

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.46.9.2854-2864.2002

    Effects of Sam B on HSV-1 γ gene expression in Vero cells detected by Western blot analysis and RT-PCR. Vero cells (5 × 10 6 ) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). (A) Lysates (20 μg of protein) were collected at 16 h p.i. and run on an SDS-10% PAGE gel and analyzed by immunoblotting with an anti-gB, -gC, -gD, -gG, or -ICP5 antibody. (B) The total cellular RNA was isolated from Vero cells at 16 h p.i. RT-PCR was done as described in Materials and Methods. Following the reaction, the amplified product was run on a 2% agarose gel. Lane 1, uninfected Vero cells; lane 2, HSV-1-infected cells; lane 3, cells treated with HSV-1 and Sam B at the same time; lane 4, infected cells treated with Sam B at 12 h to 16 h p.i. The graph indicates the ratio of γ protein or gB mRNA to β-actin mRNA. Each bar represents the mean of three independent experiments.
    Figure Legend Snippet: Effects of Sam B on HSV-1 γ gene expression in Vero cells detected by Western blot analysis and RT-PCR. Vero cells (5 × 10 6 ) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). (A) Lysates (20 μg of protein) were collected at 16 h p.i. and run on an SDS-10% PAGE gel and analyzed by immunoblotting with an anti-gB, -gC, -gD, -gG, or -ICP5 antibody. (B) The total cellular RNA was isolated from Vero cells at 16 h p.i. RT-PCR was done as described in Materials and Methods. Following the reaction, the amplified product was run on a 2% agarose gel. Lane 1, uninfected Vero cells; lane 2, HSV-1-infected cells; lane 3, cells treated with HSV-1 and Sam B at the same time; lane 4, infected cells treated with Sam B at 12 h to 16 h p.i. The graph indicates the ratio of γ protein or gB mRNA to β-actin mRNA. Each bar represents the mean of three independent experiments.

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Infection, Polyacrylamide Gel Electrophoresis, Isolation, Amplification, Agarose Gel Electrophoresis

    Effects of Sam B on HSV-1 ICP0 and ICP4 gene expression and formation of IEC in Vero cells detected by Western blotting, RT-PCR, and EMSA, respectively. Vero cells (5 × 10 6 ) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). (A) Lysates (20 μg of protein) were collected at 4 h p.i. and run on an SDS-10% PAGE gel and analyzed by immunoblotting with an anti-ICP0 or -ICP4 antibody. (C) Total cellular RNA was isolated from Vero cells at 4 h p.i. and analyzed by RT-PCR. Lane 1, uninfected Vero cells; lanes 2 and 4, HSV-1-infected cells treated with or without DMSO; lane 3, infected cells treated with Sam B. (B and D) Bar graphs indicating the ratio of ICP0 or ICP4 to β-actin proteins or mRNAs. Each bar represents the mean of three independent experiments. (E) EMSA was performed as described in Materials and Methods. Nuclear extracts from HSV-1-infected (lanes 1 and 4) or uninfected Vero cells (lane 3) were incubated with a 32 P-end-labeled GARAT probe. The effects of Sam B on the formation of IEC in virus-infected nuclear extracts pretreated with 25 μM Sam B for 5 min and then mixed with the probes (lane 2) were detected. Lane 5, results of adding a 50-fold excess of unlabeled probe to the reaction mixture.
    Figure Legend Snippet: Effects of Sam B on HSV-1 ICP0 and ICP4 gene expression and formation of IEC in Vero cells detected by Western blotting, RT-PCR, and EMSA, respectively. Vero cells (5 × 10 6 ) were infected with HSV-1 (MOI, 3) or were not infected in the presence or absence of Sam B (25 μM). (A) Lysates (20 μg of protein) were collected at 4 h p.i. and run on an SDS-10% PAGE gel and analyzed by immunoblotting with an anti-ICP0 or -ICP4 antibody. (C) Total cellular RNA was isolated from Vero cells at 4 h p.i. and analyzed by RT-PCR. Lane 1, uninfected Vero cells; lanes 2 and 4, HSV-1-infected cells treated with or without DMSO; lane 3, infected cells treated with Sam B. (B and D) Bar graphs indicating the ratio of ICP0 or ICP4 to β-actin proteins or mRNAs. Each bar represents the mean of three independent experiments. (E) EMSA was performed as described in Materials and Methods. Nuclear extracts from HSV-1-infected (lanes 1 and 4) or uninfected Vero cells (lane 3) were incubated with a 32 P-end-labeled GARAT probe. The effects of Sam B on the formation of IEC in virus-infected nuclear extracts pretreated with 25 μM Sam B for 5 min and then mixed with the probes (lane 2) were detected. Lane 5, results of adding a 50-fold excess of unlabeled probe to the reaction mixture.

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Infection, Polyacrylamide Gel Electrophoresis, Isolation, Incubation, Labeling

    40) Product Images from "Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse"

    Article Title: Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20061893

    CIP4 expression in NK cells. (A) RT-PCR for CIP4 message in NK cell lines and ex vivo NK cells. (B) Western blot (10 μg of protein per lane) for CIP4 in NK92, YTS, and ex vivo NK cells, as well as WASp and α-tubulin after stripping and reprobing membranes. (C) Intracellular CIP4 FACS using CIP4 mAb or IgG clone MOPC21 (in YTS cells as a specificity control, which was comparable with IgG control for the other cell types). Ex vivo NK cells were identified by FACS in total PBMCs by costaining for CD3 and CD56 and gating on CD3 − , CD56 + lymphocytes (NK). (D) The increase in CIP4 mean fluorescence intensity (MFI) over control IgG detected by FACS for YTS, NK92, ex vivo NK, and CIP4 YTS cells in three experiments and with six different donors of ex vivo cells is shown. IgG MFI was determined in parallel with each repeated assessment of CIP4. Error bars represent the SD.
    Figure Legend Snippet: CIP4 expression in NK cells. (A) RT-PCR for CIP4 message in NK cell lines and ex vivo NK cells. (B) Western blot (10 μg of protein per lane) for CIP4 in NK92, YTS, and ex vivo NK cells, as well as WASp and α-tubulin after stripping and reprobing membranes. (C) Intracellular CIP4 FACS using CIP4 mAb or IgG clone MOPC21 (in YTS cells as a specificity control, which was comparable with IgG control for the other cell types). Ex vivo NK cells were identified by FACS in total PBMCs by costaining for CD3 and CD56 and gating on CD3 − , CD56 + lymphocytes (NK). (D) The increase in CIP4 mean fluorescence intensity (MFI) over control IgG detected by FACS for YTS, NK92, ex vivo NK, and CIP4 YTS cells in three experiments and with six different donors of ex vivo cells is shown. IgG MFI was determined in parallel with each repeated assessment of CIP4. Error bars represent the SD.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Ex Vivo, Western Blot, Stripping Membranes, FACS, Fluorescence

    Related Articles

    Amplification:

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Article Title: Isolation and characterization of multipotent stem cells from human cruciate ligaments
    Article Snippet: .. Complementary DNA (cDNA) was obtained by RT of 1 µg total RNA using Advantage RT‐for‐PCR (Clontech, Palo Alto, CA, USA) per manufacturer's instructions. cDNA was amplified using ABI GeneAmp PCR system 2400 (PerkinElmer Applied Biosystems, Boston, MA, USA) at 94 °C for 40 s, 56 °C for 50 s, and 72 °C for 60 s for 30 cycles, after initial denaturation at 94 °C for 5 min. Primers used for amplification are listed in . .. Briefly, cells were treated with 0.06 µg/ml colcemid (Invitrogen) for 2–4 h, trypsinized, incubated in 0.075 m of KCl for 10 min, and fixed in Carnoy's fixative.

    Synthesized:

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway
    Article Snippet: .. The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes. ..

    Article Title: Network-based approach to prediction and population-based validation of in silico drug repurposing
    Article Snippet: .. RNA was collected from cells with the RNeasy kit (Qiagen) using the optional DNase I digestion. cDNA was synthesized from 0.5 μg of RNA using oligo dT primers and the Advantage RT-for-PCR kit (Clontech). .. Relative RNA levels were measured by quantitative RT-PCR method using the ΔΔCt method of analysis. β-Actin was used as the endogenous control.

    Isolation:

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway
    Article Snippet: .. The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes. ..

    Article Title: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression
    Article Snippet: .. To perform RT-PCR, total RNA was isolated from Jurkat T cells by an Advantage RT-for-PCR kit (CLONTECH). .. The primer pairs used for amplification of Bax mRNA (538 bp) were: forward, 5′-CAGCTCTGAGCAGATCATGAAGACA-3′ and reverse, 5′-GCCCATCTTCTTCCAGATGGTGAGC-3′ ( ).

    Incubation:

    Article Title: Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC
    Article Snippet: .. Total cDNA was produced by using the Advantage RT-for-PCR kit (Clontech), following the manufacturer's instructions, with oligo(dT) primer (GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTV), and incubated for 2 min at 70°C, then 1 h at 42°C, and then 5 min at 94°C. .. The coding region sequence of CD1.2 was from a clone with no 5′UTR and 51 bp of 3′UTR, derived using 0.5 μl (25 ng) of cDNA from various CB tissues and 20 pmol each primer [primer nos.

    Labeling:

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Produced:

    Article Title: Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC
    Article Snippet: .. Total cDNA was produced by using the Advantage RT-for-PCR kit (Clontech), following the manufacturer's instructions, with oligo(dT) primer (GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTV), and incubated for 2 min at 70°C, then 1 h at 42°C, and then 5 min at 94°C. .. The coding region sequence of CD1.2 was from a clone with no 5′UTR and 51 bp of 3′UTR, derived using 0.5 μl (25 ng) of cDNA from various CB tissues and 20 pmol each primer [primer nos.

    Polymerase Chain Reaction:

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Article Title: Isolation and characterization of multipotent stem cells from human cruciate ligaments
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    Article Snippet: .. First-strand cDNA was generated with 1 μg of total RNA and oligo(dT)18 primer using Advantage RT-for PCR kit (Clontech). ..

    Generated:

    Article Title: Expression of Gab1 Lacking the Pleckstrin Homology Domain Is Associated with Neoplastic Progression
    Article Snippet: .. First-strand cDNA was generated with 1 μg of total RNA and oligo(dT)18 primer using Advantage RT-for PCR kit (Clontech). ..

    Formalin-fixed Paraffin-Embedded:

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway
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    Quantitative RT-PCR:

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway
    Article Snippet: .. The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression
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    TaKaRa advantage rt for pcr kit
    Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the <t>qRT-PCR</t> data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p
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    Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p

    Journal: Journal of Neuroinflammation

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

    doi: 10.1186/s12974-018-1214-5

    Figure Lengend Snippet: Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p

    Article Snippet: The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes.

    Techniques: Quantitative RT-PCR, Infection, Mouse Assay, Western Blot, Expressing, Formalin-fixed Paraffin-Embedded, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay

    HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p

    Journal: Journal of Neuroinflammation

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

    doi: 10.1186/s12974-018-1214-5

    Figure Lengend Snippet: HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p

    Article Snippet: The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes.

    Techniques: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression

    Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p

    Journal: Journal of Neuroinflammation

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

    doi: 10.1186/s12974-018-1214-5

    Figure Lengend Snippet: Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p

    Article Snippet: The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes.

    Techniques: Expressing, Quantitative RT-PCR

    HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p

    Journal: Journal of Neuroinflammation

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

    doi: 10.1186/s12974-018-1214-5

    Figure Lengend Snippet: HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p

    Article Snippet: The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, In Vivo, Over Expression, Real-time Polymerase Chain Reaction

    Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p

    Journal: Nature Communications

    Article Title: Network-based approach to prediction and population-based validation of in silico drug repurposing

    doi: 10.1038/s41467-018-05116-5

    Figure Lengend Snippet: Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p

    Article Snippet: RNA was collected from cells with the RNeasy kit (Qiagen) using the optional DNase I digestion. cDNA was synthesized from 0.5 μg of RNA using oligo dT primers and the Advantage RT-for-PCR kit (Clontech).

    Techniques: Expressing, RNA Sequencing Assay, In Vitro, In Vivo, Activation Assay, Inhibition, Incubation, Quantitative RT-PCR

    Exclusive expression of Gab1 mRNA led by LSP sequence in 10W−1, especially in 10W−1L cells. One microgram of RNA from 10W+8, 10W−1E, and 10W−1L cells was subjected to reverse transcription reaction using oligo(dT) 18 primer. For some samples, reverse transcriptase (RT) was not included in the reaction (lanes 1, 3, 5, 7, 9, and 11). cDNA, in turn, was provided for PCR with either −14F or LSP as 5′ primer and 2104R as 3′ primer. PCR products were analyzed by 1.2% agarose gel and visualized with ethidium bromide staining and UV exposure.

    Journal: Molecular and Cellular Biology

    Article Title: Expression of Gab1 Lacking the Pleckstrin Homology Domain Is Associated with Neoplastic Progression

    doi: 10.1128/MCB.21.20.6895-6905.2001

    Figure Lengend Snippet: Exclusive expression of Gab1 mRNA led by LSP sequence in 10W−1, especially in 10W−1L cells. One microgram of RNA from 10W+8, 10W−1E, and 10W−1L cells was subjected to reverse transcription reaction using oligo(dT) 18 primer. For some samples, reverse transcriptase (RT) was not included in the reaction (lanes 1, 3, 5, 7, 9, and 11). cDNA, in turn, was provided for PCR with either −14F or LSP as 5′ primer and 2104R as 3′ primer. PCR products were analyzed by 1.2% agarose gel and visualized with ethidium bromide staining and UV exposure.

    Article Snippet: First-strand cDNA was generated with 1 μg of total RNA and oligo(dT)18 primer using Advantage RT-for PCR kit (Clontech).

    Techniques: Expressing, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Proteasome inhibitor LLnV induces Bax accumulation, cytochrome c release, and PARP cleavage in Bcl-2-overexpressing Jurkat T cells. ( A – C ) Jurkat T cells overexpressing Bcl-2 (0 h) were treated with 50 μM LLnV for up to 8 h, followed by preparation of cytosol and mitochondrial fractions. Both fractions were immunoblotted first by an antibody to cytochrome c (Cyto C, 17 kDa; A and C ) and then reblotted by anticytochrome oxidase subunit II (COX, 26 kDa; B ). Note: 20 μg protein from the cytosol, and 40 μg protein from the mitochondrial, preparation was used in each lane. ( D–G ) Whole-cell extracts (70 μg per lane) of the above treated cells were immunoblotted with specific antibodies to PARP ( D ), Bax (clone N-20; E ), Bcl-2 ( F ), or actin ( G ). Molecular masses of PARP, the PARP cleavage fragment (p85), Bax, Bcl-2, and actin are 113, 85, 21, 26, and 40 kDa, respectively. Positions of protein markers are indicated at right. ( H ) Bcl-2-expressing Jurkat cells (Control) were treated with 50 μM LLnV for 8 h, followed by reverse transcription–PCR (RT-PCR). For the first-strand cDNA synthesis, 0.2 (lanes 1 and 4), 0.6 (lanes 2 and 5), and 1.8 μg (lanes 3 and 6) of the total RNA were used. The positions of Bax (538 bp) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA (983 bp) are indicated. Lane M is DNA molecular weight marker.

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

    Article Title: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression

    doi:

    Figure Lengend Snippet: Proteasome inhibitor LLnV induces Bax accumulation, cytochrome c release, and PARP cleavage in Bcl-2-overexpressing Jurkat T cells. ( A – C ) Jurkat T cells overexpressing Bcl-2 (0 h) were treated with 50 μM LLnV for up to 8 h, followed by preparation of cytosol and mitochondrial fractions. Both fractions were immunoblotted first by an antibody to cytochrome c (Cyto C, 17 kDa; A and C ) and then reblotted by anticytochrome oxidase subunit II (COX, 26 kDa; B ). Note: 20 μg protein from the cytosol, and 40 μg protein from the mitochondrial, preparation was used in each lane. ( D–G ) Whole-cell extracts (70 μg per lane) of the above treated cells were immunoblotted with specific antibodies to PARP ( D ), Bax (clone N-20; E ), Bcl-2 ( F ), or actin ( G ). Molecular masses of PARP, the PARP cleavage fragment (p85), Bax, Bcl-2, and actin are 113, 85, 21, 26, and 40 kDa, respectively. Positions of protein markers are indicated at right. ( H ) Bcl-2-expressing Jurkat cells (Control) were treated with 50 μM LLnV for 8 h, followed by reverse transcription–PCR (RT-PCR). For the first-strand cDNA synthesis, 0.2 (lanes 1 and 4), 0.6 (lanes 2 and 5), and 1.8 μg (lanes 3 and 6) of the total RNA were used. The positions of Bax (538 bp) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA (983 bp) are indicated. Lane M is DNA molecular weight marker.

    Article Snippet: To perform RT-PCR, total RNA was isolated from Jurkat T cells by an Advantage RT-for-PCR kit (CLONTECH).

    Techniques: Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker