rna isolation  (Thermo Fisher)


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    Structured Review

    Thermo Fisher rna isolation
    Influence of overexpression and knockdown of microRNA-218-5p (miR-218) on the expression of roundabout1 (ROBO1). Expression of ROBO1 in rheumatoid arthritis (RA)-fibroblast-like synovial cells (FLS) was determined relative to the controls transfected with scrambled <t>RNA,</t> which was defined as 1. a Transfection of RA-FLS ( n = 5) with precursor miR-218 (pre-miR-218) for 48 h decreased the levels of ROBO1 compared to scrambled RNA-transfected controls, as determined by SYBR green real-time <t>PCR</t> analysis. b Knockdown of miR-218 for 48 h in RA-FLS ( n = 5) increased the level of ROBO1 compared to scrambled RNA-transfected controls, as determined by SYBR green real-time PCR analysis. Values are presented as means ± SEM: * p
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    1) Product Images from "Osteogenic differentiation of fibroblast-like synovial cells in rheumatoid arthritis is induced by microRNA-218 through a ROBO/Slit pathway"

    Article Title: Osteogenic differentiation of fibroblast-like synovial cells in rheumatoid arthritis is induced by microRNA-218 through a ROBO/Slit pathway

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1703-z

    Influence of overexpression and knockdown of microRNA-218-5p (miR-218) on the expression of roundabout1 (ROBO1). Expression of ROBO1 in rheumatoid arthritis (RA)-fibroblast-like synovial cells (FLS) was determined relative to the controls transfected with scrambled RNA, which was defined as 1. a Transfection of RA-FLS ( n = 5) with precursor miR-218 (pre-miR-218) for 48 h decreased the levels of ROBO1 compared to scrambled RNA-transfected controls, as determined by SYBR green real-time PCR analysis. b Knockdown of miR-218 for 48 h in RA-FLS ( n = 5) increased the level of ROBO1 compared to scrambled RNA-transfected controls, as determined by SYBR green real-time PCR analysis. Values are presented as means ± SEM: * p
    Figure Legend Snippet: Influence of overexpression and knockdown of microRNA-218-5p (miR-218) on the expression of roundabout1 (ROBO1). Expression of ROBO1 in rheumatoid arthritis (RA)-fibroblast-like synovial cells (FLS) was determined relative to the controls transfected with scrambled RNA, which was defined as 1. a Transfection of RA-FLS ( n = 5) with precursor miR-218 (pre-miR-218) for 48 h decreased the levels of ROBO1 compared to scrambled RNA-transfected controls, as determined by SYBR green real-time PCR analysis. b Knockdown of miR-218 for 48 h in RA-FLS ( n = 5) increased the level of ROBO1 compared to scrambled RNA-transfected controls, as determined by SYBR green real-time PCR analysis. Values are presented as means ± SEM: * p

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

    Overexpression of microRNA-218-5p (miR-218) promotes osteogenic differentiation of rheumatoid arthritis (RA)-fibroblast-like synovial cells (FLS). a Alkaline phosphatase (ALP) staining at day 14 showed the enhanced ALP activity of osteogenic differentiation after transfection with precursor miR-218 (pre-miR-218) compared to scrambled RNA-transfected controls. Images are representative of five samples. b The morphology of RA-FLS with ALP staining. Right: RA-FLS were transfected with scrambled control. Left: RA-FLS were transfected with pre-miR-218 (magnification × 200). Images are representative of five samples. c Transfection of RA-FLS ( n = 5) with pre-miR-218 for 72 h compared with scrambled RNA transfected controls increased the level of ALP, runt related transcription factor 2 (RUNX2), catenin beta 1 (CTNNB1), cadherin 11 (CDH11) as determined by quantitative real-time PCR analysis. Values are presented as means ± SEM: * p
    Figure Legend Snippet: Overexpression of microRNA-218-5p (miR-218) promotes osteogenic differentiation of rheumatoid arthritis (RA)-fibroblast-like synovial cells (FLS). a Alkaline phosphatase (ALP) staining at day 14 showed the enhanced ALP activity of osteogenic differentiation after transfection with precursor miR-218 (pre-miR-218) compared to scrambled RNA-transfected controls. Images are representative of five samples. b The morphology of RA-FLS with ALP staining. Right: RA-FLS were transfected with scrambled control. Left: RA-FLS were transfected with pre-miR-218 (magnification × 200). Images are representative of five samples. c Transfection of RA-FLS ( n = 5) with pre-miR-218 for 72 h compared with scrambled RNA transfected controls increased the level of ALP, runt related transcription factor 2 (RUNX2), catenin beta 1 (CTNNB1), cadherin 11 (CDH11) as determined by quantitative real-time PCR analysis. Values are presented as means ± SEM: * p

    Techniques Used: Over Expression, ALP Assay, Staining, Activity Assay, Transfection, Real-time Polymerase Chain Reaction

    Suppression of Dickkopf-1 (DKK1) by overexpression of microRNA-218-5p (miR-218) or silencing of roundabout1 (ROBO1). a At the protein level, transfection of rheumatoid arthritis (RA)-fibroblast-like synovial cells (FLS) ( n = 4) with precursor miR-218 (pre-miR-218) for 48 h decreased DKK1 protein production in the culture supernatant compared to scrambled RNA transfected controls, as determined by ELISA. Graphs represent optical density (OD) value; each mean amount of DDK1 protein are as follows; scrambled control: pre-miR-218 1.17 ng/ml:0.76 ng/ml, scrambled control: anti-miR-218 1.58 ng/ml:1.65 ng/ml, respectively. b At the mRNA level, transfection of RA-FLS ( n = 4) with pre-miR-218 for 48 h decreased DKK1 expression compared to scrambled RNA transfected controls, as determined by SYBR green real-time PCR analysis. c Secretion of DKK1 from RA-FLS ( n = 4) was decreased after transfection with ROBO1-specific small interfering RNA (siRNA) compared to the scrambled RNA transfected controls, as determined by ELISA. Graphs represent OD value; each mean amount of DDK1 protein are as follows; scrambled control: siROBO1.28 ng/ml:1.09 ng/ml, respectively. Values are presented as means ± SEM: * p
    Figure Legend Snippet: Suppression of Dickkopf-1 (DKK1) by overexpression of microRNA-218-5p (miR-218) or silencing of roundabout1 (ROBO1). a At the protein level, transfection of rheumatoid arthritis (RA)-fibroblast-like synovial cells (FLS) ( n = 4) with precursor miR-218 (pre-miR-218) for 48 h decreased DKK1 protein production in the culture supernatant compared to scrambled RNA transfected controls, as determined by ELISA. Graphs represent optical density (OD) value; each mean amount of DDK1 protein are as follows; scrambled control: pre-miR-218 1.17 ng/ml:0.76 ng/ml, scrambled control: anti-miR-218 1.58 ng/ml:1.65 ng/ml, respectively. b At the mRNA level, transfection of RA-FLS ( n = 4) with pre-miR-218 for 48 h decreased DKK1 expression compared to scrambled RNA transfected controls, as determined by SYBR green real-time PCR analysis. c Secretion of DKK1 from RA-FLS ( n = 4) was decreased after transfection with ROBO1-specific small interfering RNA (siRNA) compared to the scrambled RNA transfected controls, as determined by ELISA. Graphs represent OD value; each mean amount of DDK1 protein are as follows; scrambled control: siROBO1.28 ng/ml:1.09 ng/ml, respectively. Values are presented as means ± SEM: * p

    Techniques Used: Over Expression, Transfection, Enzyme-linked Immunosorbent Assay, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA

    2) Product Images from "25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes"

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.09.003

    Effect of OSBP silencing (panels A, B, and C) or VAP-A silencing (panels D, E, and F) by short interfering RNA (siRNA) transfection on the antiviral activity of 25HC or 27HC. Cells were transfected for 72 h with 20 nM of anti-OSBP siRNA, anti-VAP-A siRNA or control non-interfering siRNA. Cells were then treated for 20 h with oxysterols at sub-optimal concentrations and finally infected with HRV Wa. Viral infections were detected as described in the Material and Methods section. The infectivity titers of virus in the treated samples are expressed as a percentage of the titer obtained in the absence of treatment. Error bars represent the standard error of the mean (SEM) of 3 independent experiments.*p ANOVA
    Figure Legend Snippet: Effect of OSBP silencing (panels A, B, and C) or VAP-A silencing (panels D, E, and F) by short interfering RNA (siRNA) transfection on the antiviral activity of 25HC or 27HC. Cells were transfected for 72 h with 20 nM of anti-OSBP siRNA, anti-VAP-A siRNA or control non-interfering siRNA. Cells were then treated for 20 h with oxysterols at sub-optimal concentrations and finally infected with HRV Wa. Viral infections were detected as described in the Material and Methods section. The infectivity titers of virus in the treated samples are expressed as a percentage of the titer obtained in the absence of treatment. Error bars represent the standard error of the mean (SEM) of 3 independent experiments.*p ANOVA

    Techniques Used: Small Interfering RNA, Transfection, Activity Assay, Infection

    3) Product Images from "Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy"

    Article Title: Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06267-1

    CB1 and PAX7 expression and 2-AG levels in dystrophic muscles. a mRNA expression levels of CB1 and PAX7 in muscle samples of healthy children (HT, controls n = 3) or DMD donors of 3 ( n = 4) and 7 ( n = 4) years. b Measurement of the endogenous levels of 2-AG in muscle samples of the same healthy and DMD donors. Time course of the endogenous levels of 2-AG in the quadriceps ( c ) and gastrocnemius ( d ) muscle of control (dark yellow; n = 8) and mdx (red columns; n = 8) mice. The levels of 2-AG are expressed as pmol mg −1 of wet tissue weight. * P ≤ 0.05 vs. control group, determined by Student’s t test. e The bar graphs show RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalized values for the Daglα and Magl genes obtained from RNA-seq analysis in satellite cells isolated from 8-week-old wt ( n = 4) and mdx mice ( n = 4)
    Figure Legend Snippet: CB1 and PAX7 expression and 2-AG levels in dystrophic muscles. a mRNA expression levels of CB1 and PAX7 in muscle samples of healthy children (HT, controls n = 3) or DMD donors of 3 ( n = 4) and 7 ( n = 4) years. b Measurement of the endogenous levels of 2-AG in muscle samples of the same healthy and DMD donors. Time course of the endogenous levels of 2-AG in the quadriceps ( c ) and gastrocnemius ( d ) muscle of control (dark yellow; n = 8) and mdx (red columns; n = 8) mice. The levels of 2-AG are expressed as pmol mg −1 of wet tissue weight. * P ≤ 0.05 vs. control group, determined by Student’s t test. e The bar graphs show RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalized values for the Daglα and Magl genes obtained from RNA-seq analysis in satellite cells isolated from 8-week-old wt ( n = 4) and mdx mice ( n = 4)

    Techniques Used: Expressing, Mouse Assay, RNA Sequencing Assay, Isolation

    4) Product Images from "Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts"

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1689-6

    colIα1 and mmp-1 mRNA expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p
    Figure Legend Snippet: colIα1 and mmp-1 mRNA expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p

    Techniques Used: Expressing, Papanicolaou Stain, Positive Control

    TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p
    Figure Legend Snippet: TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p

    Techniques Used: Incubation, Inhibition

    TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p
    Figure Legend Snippet: TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

    Techniques Used: Expressing, Positive Control

    TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. *** p
    Figure Legend Snippet: TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. *** p

    Techniques Used: Incubation, Inhibition

    5) Product Images from "Molecular profiling of ALDH1+ colorectal cancer stem cells reveals preferential activation of MAPK, FAK, and oxidative stress pro-survival signalling pathways"

    Article Title: Molecular profiling of ALDH1+ colorectal cancer stem cells reveals preferential activation of MAPK, FAK, and oxidative stress pro-survival signalling pathways

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24420

    Preferential activation of multiple signalling pathways in ALDH + cells ( A ) Hierarchical clustering of ALDH + vs ALDH − subpopulations based on differentially expressed mRNA levels. Each column represents one replica and each row represents a transcript. Expression level of each gene in a single sample is depicted according to the colour scale. ( B ) Pie chart illustrating the distribution of the top 20 pathways designations for the differentially expressed genes in ALDH + cells. The pie size corresponds to the number of matched entities. ( C ) Expression levels of selected genes from the microarray data were validated using qRT-PCR in ALDH1 + compared to ALDH − cells. Data are presented as the means ± S.D, n = 3. * p
    Figure Legend Snippet: Preferential activation of multiple signalling pathways in ALDH + cells ( A ) Hierarchical clustering of ALDH + vs ALDH − subpopulations based on differentially expressed mRNA levels. Each column represents one replica and each row represents a transcript. Expression level of each gene in a single sample is depicted according to the colour scale. ( B ) Pie chart illustrating the distribution of the top 20 pathways designations for the differentially expressed genes in ALDH + cells. The pie size corresponds to the number of matched entities. ( C ) Expression levels of selected genes from the microarray data were validated using qRT-PCR in ALDH1 + compared to ALDH − cells. Data are presented as the means ± S.D, n = 3. * p

    Techniques Used: Activation Assay, Expressing, Microarray, Quantitative RT-PCR

    6) Product Images from "Deciphering the complex role of thrombospondin-1 in glioblastoma development"

    Article Title: Deciphering the complex role of thrombospondin-1 in glioblastoma development

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08480-y

    THBS1/CD47 interaction in P3 tumour invasion and growth. a P3 cells were included into collagen I gels and then incubated in normoxia (21 % O 2 ) or hypoxia (1 % O 2 ). P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph represents the results as means ± s.d. of three independent experiments, each done in 6–8 replicates for each condition. * P
    Figure Legend Snippet: THBS1/CD47 interaction in P3 tumour invasion and growth. a P3 cells were included into collagen I gels and then incubated in normoxia (21 % O 2 ) or hypoxia (1 % O 2 ). P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph represents the results as means ± s.d. of three independent experiments, each done in 6–8 replicates for each condition. * P

    Techniques Used: Incubation

    Proposed model for GBM invasion TGFβ1 is expressed in both the core and the invasive areas in GBM. THBS1 is transcriptionally regulated via SMAD3, which binds to regulatory elements in the THBS1 gene. THBS1 will then be released and act on tumour cell invasion and expansion. The interaction with CD47 is critical in this process
    Figure Legend Snippet: Proposed model for GBM invasion TGFβ1 is expressed in both the core and the invasive areas in GBM. THBS1 is transcriptionally regulated via SMAD3, which binds to regulatory elements in the THBS1 gene. THBS1 will then be released and act on tumour cell invasion and expansion. The interaction with CD47 is critical in this process

    Techniques Used: Activated Clotting Time Assay

    Tumour-associated CD47 controls glioma cell invasion and motility. a Immunoblots of protein extracts from control or CD47-1/-2 shRNA-transduced P3 cells probed with anti-CD47 or anti-Tubulin antibodies. b P3 cells were transduced with control or CD47 (−1 and −2) shRNAs. P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph below represents the results as means ± s.d. of three independent experiments each done in 6–8 replicates for each condition. ** P
    Figure Legend Snippet: Tumour-associated CD47 controls glioma cell invasion and motility. a Immunoblots of protein extracts from control or CD47-1/-2 shRNA-transduced P3 cells probed with anti-CD47 or anti-Tubulin antibodies. b P3 cells were transduced with control or CD47 (−1 and −2) shRNAs. P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph below represents the results as means ± s.d. of three independent experiments each done in 6–8 replicates for each condition. ** P

    Techniques Used: Western Blot, shRNA, Transduction

    7) Product Images from "CD24 isoform a promotes cell proliferation, migration and invasion and is downregulated by EGR1 in hepatocellular carcinoma"

    Article Title: CD24 isoform a promotes cell proliferation, migration and invasion and is downregulated by EGR1 in hepatocellular carcinoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S196506

    EGR1 is downregulated in HCC tissues and represses CD24A expression in human HCC cells. Notes: ( A ) mRNA expression of EGR1 in 90 pairs of HCC tissues and corresponding adjacent non-cancerous liver tissues was detected by qPCR; ( B ) mRNA expression of EGR1 in 50 pairs of HCC tissues and corresponding adjacent non-cancerous liver tissues in the TCGA database was analyzed; ( C ) Western blotting was performed to detect the expression of EGR1 in four pairs of HCC tissues (K) and corresponding adjacent non-cancerous liver tissues (N); ( D , E ) the expression of EGR1 in human HCC cell lines was assessed via qPCR and Western blotting; ( F ) qPCR (top panel) and Western blotting (bottom panel) analyses of EGR1 and CD24A in EGR1-overexpressing SMMC-7721 and Hep3B cells; ( G ) tumor xenografts were collected from BALB/c (nu/nu) mice inoculated with Li7 cell lines stably overexpressing EGR1 or control (pWPXL) vectors (left panel). Xenografts were weighed and analyzed (right panel); n=10; ( H ) the EGR1 protein level was detected by Western blotting in xenograft tissue samples. β-Actin was used as the loading control. * P
    Figure Legend Snippet: EGR1 is downregulated in HCC tissues and represses CD24A expression in human HCC cells. Notes: ( A ) mRNA expression of EGR1 in 90 pairs of HCC tissues and corresponding adjacent non-cancerous liver tissues was detected by qPCR; ( B ) mRNA expression of EGR1 in 50 pairs of HCC tissues and corresponding adjacent non-cancerous liver tissues in the TCGA database was analyzed; ( C ) Western blotting was performed to detect the expression of EGR1 in four pairs of HCC tissues (K) and corresponding adjacent non-cancerous liver tissues (N); ( D , E ) the expression of EGR1 in human HCC cell lines was assessed via qPCR and Western blotting; ( F ) qPCR (top panel) and Western blotting (bottom panel) analyses of EGR1 and CD24A in EGR1-overexpressing SMMC-7721 and Hep3B cells; ( G ) tumor xenografts were collected from BALB/c (nu/nu) mice inoculated with Li7 cell lines stably overexpressing EGR1 or control (pWPXL) vectors (left panel). Xenografts were weighed and analyzed (right panel); n=10; ( H ) the EGR1 protein level was detected by Western blotting in xenograft tissue samples. β-Actin was used as the loading control. * P

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

    Expression of CD24 in human HCC clinical specimens and cell lines. Notes: ( A ) Amino acid sequence alignment of CD24A with CD24B; ( B ) qPCR was carried out to evaluate the mRNA expression of CD24A in 90 pairs of HCC tissues and corresponding adjacent non-cancerous liver tissues; ( C ) Western blotting was performed to detect the expression of CD24 in 4 pairs of HCC tissues (K) and corresponding adjacent non-cancerous liver tissues (N); ( D ) mRNA expression levels of CD24A and CD24B in human HCC cell lines were detected by qPCR; ( E ) the protein expression level of CD24 in human HCC cell lines was determined by Western blotting; ( F ) the mRNA expression levels of CD24A and CD24B in SK-Hep1 and Li7 cells stably transfected with CD24A, CD24B or control (pWPXL) vectors; ( G ) Western blot analysis of CD24 protein in SK-Hep1 and Li7 cells stably transfected with CD24A, CD24B or control (pWPXL) vectors. ** P
    Figure Legend Snippet: Expression of CD24 in human HCC clinical specimens and cell lines. Notes: ( A ) Amino acid sequence alignment of CD24A with CD24B; ( B ) qPCR was carried out to evaluate the mRNA expression of CD24A in 90 pairs of HCC tissues and corresponding adjacent non-cancerous liver tissues; ( C ) Western blotting was performed to detect the expression of CD24 in 4 pairs of HCC tissues (K) and corresponding adjacent non-cancerous liver tissues (N); ( D ) mRNA expression levels of CD24A and CD24B in human HCC cell lines were detected by qPCR; ( E ) the protein expression level of CD24 in human HCC cell lines was determined by Western blotting; ( F ) the mRNA expression levels of CD24A and CD24B in SK-Hep1 and Li7 cells stably transfected with CD24A, CD24B or control (pWPXL) vectors; ( G ) Western blot analysis of CD24 protein in SK-Hep1 and Li7 cells stably transfected with CD24A, CD24B or control (pWPXL) vectors. ** P

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

    EGR1 suppresses HCC cell proliferation by repressing CD24A expression. Notes: ( A ) qPCR (top panel) and Western blotting (bottom panel) analysis of EGR1 and CD24A in EGR1-overexpressing Li7 and SK-Hep1 cells after restoration of CD24A expression; ( B ) overexpression of CD24A reversed the inhibitory effect of EGR1 on cell proliferation in vitro based on MTT assays; ( C ) overexpression of CD24A reversed the inhibitory effect of EGR1 on cell colony formation in vitro. * P
    Figure Legend Snippet: EGR1 suppresses HCC cell proliferation by repressing CD24A expression. Notes: ( A ) qPCR (top panel) and Western blotting (bottom panel) analysis of EGR1 and CD24A in EGR1-overexpressing Li7 and SK-Hep1 cells after restoration of CD24A expression; ( B ) overexpression of CD24A reversed the inhibitory effect of EGR1 on cell proliferation in vitro based on MTT assays; ( C ) overexpression of CD24A reversed the inhibitory effect of EGR1 on cell colony formation in vitro. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, In Vitro, MTT Assay

    8) Product Images from "α2 δ1 may be a potential marker for cancer stem cell in laryngeal squamous cell carcinoma"

    Article Title: α2 δ1 may be a potential marker for cancer stem cell in laryngeal squamous cell carcinoma

    Journal: Cancer Biomarkers

    doi: 10.3233/CBM-181947

    Chemoresistance ofTU686 cells. The percentage of sorted α 2 δ 1 + TU686 cells after exposure to cisplatin (6 μ mol/L) or paclitaxel (0.2 μ mol/L) for 72 hours.
    Figure Legend Snippet: Chemoresistance ofTU686 cells. The percentage of sorted α 2 δ 1 + TU686 cells after exposure to cisplatin (6 μ mol/L) or paclitaxel (0.2 μ mol/L) for 72 hours.

    Techniques Used:

    Differentiation properties of α 2 δ 1 + TU212 and TU686 cells. Flow cytometry shows the percentage of α 2 δ 1 + cells in TU212 and TU686 cells, FACS-purified α 2 δ 1 + cells and purified α 2 δ 1 + cells cultured in 10% serum-containing medium for 1 week (cultured).
    Figure Legend Snippet: Differentiation properties of α 2 δ 1 + TU212 and TU686 cells. Flow cytometry shows the percentage of α 2 δ 1 + cells in TU212 and TU686 cells, FACS-purified α 2 δ 1 + cells and purified α 2 δ 1 + cells cultured in 10% serum-containing medium for 1 week (cultured).

    Techniques Used: Flow Cytometry, Cytometry, FACS, Purification, Cell Culture

    Expression of α 2 δ 1 in primary LSCC tissues. (A) Representative images of immunofluorescence expression of α 2 δ 1 in laryngeal tissue, paracancerous tissue and normal adjacent tissue in a patient with T3N0M0 laryngeal cancer are shown. α 2 δ 1 (green) was stained with specific monoclonal antibody and nuclei (blue) were stained with DAPI. Arrows indicate α 2 δ 1-positive cells. (B) Summary of α 2 δ 1 expression in LSCC tissues, paracancerous tissues and normal adjacent tissues.
    Figure Legend Snippet: Expression of α 2 δ 1 in primary LSCC tissues. (A) Representative images of immunofluorescence expression of α 2 δ 1 in laryngeal tissue, paracancerous tissue and normal adjacent tissue in a patient with T3N0M0 laryngeal cancer are shown. α 2 δ 1 (green) was stained with specific monoclonal antibody and nuclei (blue) were stained with DAPI. Arrows indicate α 2 δ 1-positive cells. (B) Summary of α 2 δ 1 expression in LSCC tissues, paracancerous tissues and normal adjacent tissues.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Migration and invasive potential of α 2 δ 1 + cells. Sorted α 2 δ 1 + and α 2 δ 1 - cells were assayed for their invasive ability on Matrigel using a Boyden chamber assay. The invasive and migratory potential of α 2 δ 1 + cells (A, C) and α 2 δ 1 - cells (B, D) were detected. Statistical results of invasive cells were calculated (E). Data represent the mean ± SD of three independent experiments. *Student’s t -test.
    Figure Legend Snippet: Migration and invasive potential of α 2 δ 1 + cells. Sorted α 2 δ 1 + and α 2 δ 1 - cells were assayed for their invasive ability on Matrigel using a Boyden chamber assay. The invasive and migratory potential of α 2 δ 1 + cells (A, C) and α 2 δ 1 - cells (B, D) were detected. Statistical results of invasive cells were calculated (E). Data represent the mean ± SD of three independent experiments. *Student’s t -test.

    Techniques Used: Migration, Boyden Chamber Assay

    Sphere-forming efficiency of α 2 δ 1 + and α 2 δ 1 - cells. (A, B) The sphere-forming ability of α 2 δ 1 + and α 2 δ 1 - cells was examined by sphere formation assays as detailed in Methods. (C) Histograms show the spheroid forming efficiency of FACS-sorted α 2 δ 1 + and α 2 δ 1 - fractions from indicated sources. The ability of the spheres formed by α 2 δ 1 + TU 212 cells (D) and TU 686 cells (E) to form secondary spheroids is also shown ( α 2 δ 1 + /2 nd passage). (F) The sphere forming ability of purified α 2 δ 1 + TU212 cells was reduced when α 2 δ 1 + cells were incubated with indicated lentivirus for 4 hours, and were plated at 100 cells per well ( n = 5). One hundred cells per well were plated( n = 5). Spheroids ( ⩾ 100 μ m) were counted under a stereomicroscope. Statistical analysis was carried out using data on sphere formation in 100 cells (C, F). Data are represented as mean ± SD.
    Figure Legend Snippet: Sphere-forming efficiency of α 2 δ 1 + and α 2 δ 1 - cells. (A, B) The sphere-forming ability of α 2 δ 1 + and α 2 δ 1 - cells was examined by sphere formation assays as detailed in Methods. (C) Histograms show the spheroid forming efficiency of FACS-sorted α 2 δ 1 + and α 2 δ 1 - fractions from indicated sources. The ability of the spheres formed by α 2 δ 1 + TU 212 cells (D) and TU 686 cells (E) to form secondary spheroids is also shown ( α 2 δ 1 + /2 nd passage). (F) The sphere forming ability of purified α 2 δ 1 + TU212 cells was reduced when α 2 δ 1 + cells were incubated with indicated lentivirus for 4 hours, and were plated at 100 cells per well ( n = 5). One hundred cells per well were plated( n = 5). Spheroids ( ⩾ 100 μ m) were counted under a stereomicroscope. Statistical analysis was carried out using data on sphere formation in 100 cells (C, F). Data are represented as mean ± SD.

    Techniques Used: FACS, Purification, Incubation

    Expression of stem cell and drug efflux and resistance genes in α 2 δ 1 - versus α 2 δ 1 + subpopulation of TU686 cells. qRT-PCR analysis of the expression of stem cell markers and drug-resistance-related genes in purified α 2 δ 1 + and α 2 δ 1 - subpopulations of TU686 cells. Data are presented as fold difference over α 2 δ 1 - cells for each gene. Compared with α 2 δ 1 - cells, α 2 δ 1 + cells consistently expressed 1 to 4.5 fold higher levels of BMI1 , SOX2 , EPCAM , OCT4 , CNTTB KLF4 , NANOG , ABCG2 and MDR1 . *Error bars indicate SD.
    Figure Legend Snippet: Expression of stem cell and drug efflux and resistance genes in α 2 δ 1 - versus α 2 δ 1 + subpopulation of TU686 cells. qRT-PCR analysis of the expression of stem cell markers and drug-resistance-related genes in purified α 2 δ 1 + and α 2 δ 1 - subpopulations of TU686 cells. Data are presented as fold difference over α 2 δ 1 - cells for each gene. Compared with α 2 δ 1 - cells, α 2 δ 1 + cells consistently expressed 1 to 4.5 fold higher levels of BMI1 , SOX2 , EPCAM , OCT4 , CNTTB KLF4 , NANOG , ABCG2 and MDR1 . *Error bars indicate SD.

    Techniques Used: Expressing, Quantitative RT-PCR, Purification

    Tumorigenesis of α 2 δ 1 + vs. α 2 δ 1 - subpopulation. (A) TU212 cells were transplanted subcutaneously in NOD/SCID mice as indicated. (B, C) Representative photographs showing dissected tumors by FACS-purified α 2 δ 1 + (red arrows) and α 2 δ 1 - (blue arrows) TU212 cells. (D) TU686 cells were inoculated subcutaneously in NOD/SCID mice as indicated. (E) Photographs showing dissected tumors formed by FACS-purified α 2 δ 1 + and α 2 δ 1 - TU686 cells. (F, G) Serial inoculation of 10 2 and 10 3 TU686 cells into NOD/SCID mice from tumors formed by α 2 δ 1 + TU686 cells. (H, I) Tumorigenicity of TU686 α 2 δ 1 + cells FACS-purified from indicated sources after α 2 δ 1 shRNA53/shRNA56 knockdown. TU686 α 2 δ 1 + cells were incubated with indicated lentiviruses for 4 hours, and 10 2 /10 3 cells per mouse were injected. The statistical results of tumorigenic formation in 5 mice were calculated. (J) The volume and weight of tumors by α 2 δ 1 + cells, and α 2 δ 1 + cells knockdown with shRNA53and shRNA56 were detected.
    Figure Legend Snippet: Tumorigenesis of α 2 δ 1 + vs. α 2 δ 1 - subpopulation. (A) TU212 cells were transplanted subcutaneously in NOD/SCID mice as indicated. (B, C) Representative photographs showing dissected tumors by FACS-purified α 2 δ 1 + (red arrows) and α 2 δ 1 - (blue arrows) TU212 cells. (D) TU686 cells were inoculated subcutaneously in NOD/SCID mice as indicated. (E) Photographs showing dissected tumors formed by FACS-purified α 2 δ 1 + and α 2 δ 1 - TU686 cells. (F, G) Serial inoculation of 10 2 and 10 3 TU686 cells into NOD/SCID mice from tumors formed by α 2 δ 1 + TU686 cells. (H, I) Tumorigenicity of TU686 α 2 δ 1 + cells FACS-purified from indicated sources after α 2 δ 1 shRNA53/shRNA56 knockdown. TU686 α 2 δ 1 + cells were incubated with indicated lentiviruses for 4 hours, and 10 2 /10 3 cells per mouse were injected. The statistical results of tumorigenic formation in 5 mice were calculated. (J) The volume and weight of tumors by α 2 δ 1 + cells, and α 2 δ 1 + cells knockdown with shRNA53and shRNA56 were detected.

    Techniques Used: Mouse Assay, FACS, Purification, Incubation, Injection

    9) Product Images from "Follicle-Stimulating Hormone Is an Autocrine Regulator of the Ovarian Cancer Metastatic Niche Through Notch Signaling"

    Article Title: Follicle-Stimulating Hormone Is an Autocrine Regulator of the Ovarian Cancer Metastatic Niche Through Notch Signaling

    Journal: Journal of the Endocrine Society

    doi: 10.1210/js.2018-00272

    Expression of FSH and FSHR in primary tumor and spheroids obtained from patients with ovarian cancer. Total RNA was isolated from the primary tumors and spheroids obtained from the ascites of patients with ovarian cancer, and cDNA was prepared from 1 µg total RNA. Cycle threshold values of (A) FSH α subunit, (B) FSH β subunit, and (C) FSHR were examined by RT-PCR, and RNA levels were interpolated from the dilution curves of the respective genes.
    Figure Legend Snippet: Expression of FSH and FSHR in primary tumor and spheroids obtained from patients with ovarian cancer. Total RNA was isolated from the primary tumors and spheroids obtained from the ascites of patients with ovarian cancer, and cDNA was prepared from 1 µg total RNA. Cycle threshold values of (A) FSH α subunit, (B) FSH β subunit, and (C) FSHR were examined by RT-PCR, and RNA levels were interpolated from the dilution curves of the respective genes.

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

    10) Product Images from "Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice"

    Article Title: Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36864-5

    Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p
    Figure Legend Snippet: Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    11) Product Images from "A determining factor for insect feeding preference in the silkworm, Bombyx mori"

    Article Title: A determining factor for insect feeding preference in the silkworm, Bombyx mori

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000162

    GR66 expression in tissues and cellular localization of GR66. (A) Scanning electron micrographs of first-instar heads. The area inside the white box is enlarged on the right. (B) Relative mRNA levels of BmGR66 in Bombyx mori tissues as determined by qRT-PCR. Total RNA was isolated from the antennae, labra, mandibles, maxillae, labia, thoracic legs, and midguts. The RNA was converted to cDNA, which was used as a template to quantify BmGR66 mRNA levels using Bmrp49 as a reference gene. The data shown was the mean ± SEM ( n = 3). Underlying data can be found in S1 Data . (C) Photographs of HEK293T cells expressing the EGFP-GR66 fusion protein and stained with DAPI. n = 32. Scale bars: 10 μm. cDNA, complementary DNA; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; GR66, gustatory receptor 66; HEK293T, human embryonic kidney 239T; Ls, lateral sensilla; Mp, maxillary palp; Ms, medial sensilla; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean.
    Figure Legend Snippet: GR66 expression in tissues and cellular localization of GR66. (A) Scanning electron micrographs of first-instar heads. The area inside the white box is enlarged on the right. (B) Relative mRNA levels of BmGR66 in Bombyx mori tissues as determined by qRT-PCR. Total RNA was isolated from the antennae, labra, mandibles, maxillae, labia, thoracic legs, and midguts. The RNA was converted to cDNA, which was used as a template to quantify BmGR66 mRNA levels using Bmrp49 as a reference gene. The data shown was the mean ± SEM ( n = 3). Underlying data can be found in S1 Data . (C) Photographs of HEK293T cells expressing the EGFP-GR66 fusion protein and stained with DAPI. n = 32. Scale bars: 10 μm. cDNA, complementary DNA; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; GR66, gustatory receptor 66; HEK293T, human embryonic kidney 239T; Ls, lateral sensilla; Mp, maxillary palp; Ms, medial sensilla; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean.

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

    12) Product Images from "Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming"

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

    Journal: Cell Discovery

    doi: 10.1038/s41421-018-0074-6

    Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )
    Figure Legend Snippet: Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )

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

    Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i
    Figure Legend Snippet: Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i

    Techniques Used: Expressing, RNA Sequencing Assay, Methylation, Generated, Real-time Polymerase Chain Reaction

    13) Product Images from "Inhibition of Human Dendritic Cell ER Stress Response Reduces T Cell Alloreactivity Yet Spares Donor Anti-tumor Immunity"

    Article Title: Inhibition of Human Dendritic Cell ER Stress Response Reduces T Cell Alloreactivity Yet Spares Donor Anti-tumor Immunity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02887

    XBP-1s is dispensable for anti-tumor activity by CD8 + cytotoxic T lymphocytes and NK cells. (A) Replicate mean specific lysis by human CD8 + CTL generated in vitro using PBMCs stimulated by irradiated U937cells (1:1) on days 0 and +7 of a 10–12 day culture. B-I09 or DMSO was added once on days 0 and +7. Tumor-specific killing by purified CD8 + T cells was determined using fresh U937 cells. U937 lysis was measured by a colorimetric assay after 4 h. Triplicate means from 3 independent experiments are shown. (B) T cell proliferation stimulated by autologous moDCs loaded with CMV, EBV, influenza, or tetanus peptides is shown. B-I09 or DMSO was added once on day 0. T cell proliferation (MTS colormetric assay) was measured on day +3. Replicate means from 4 independent experiments are shown, Dunnett's test. (C) Mean specific lysis by human NK cells against K562 targets is shown. B-I09 or DMSO was added at the outset of the culture. K562 lysis was measured by a colorimetric assay after 4 h. Replicate means from 3 independent experiments are shown. (D) NK cell proliferation stimulated by allogeneic moDCs (moDC:NK cell ratio 1:10) or IL-2 plus IL-15. NK cell proliferation (MTS colormetric assay) was measured on day +5. Replicate means from 3 independent experiments are shown.
    Figure Legend Snippet: XBP-1s is dispensable for anti-tumor activity by CD8 + cytotoxic T lymphocytes and NK cells. (A) Replicate mean specific lysis by human CD8 + CTL generated in vitro using PBMCs stimulated by irradiated U937cells (1:1) on days 0 and +7 of a 10–12 day culture. B-I09 or DMSO was added once on days 0 and +7. Tumor-specific killing by purified CD8 + T cells was determined using fresh U937 cells. U937 lysis was measured by a colorimetric assay after 4 h. Triplicate means from 3 independent experiments are shown. (B) T cell proliferation stimulated by autologous moDCs loaded with CMV, EBV, influenza, or tetanus peptides is shown. B-I09 or DMSO was added once on day 0. T cell proliferation (MTS colormetric assay) was measured on day +3. Replicate means from 4 independent experiments are shown, Dunnett's test. (C) Mean specific lysis by human NK cells against K562 targets is shown. B-I09 or DMSO was added at the outset of the culture. K562 lysis was measured by a colorimetric assay after 4 h. Replicate means from 3 independent experiments are shown. (D) NK cell proliferation stimulated by allogeneic moDCs (moDC:NK cell ratio 1:10) or IL-2 plus IL-15. NK cell proliferation (MTS colormetric assay) was measured on day +5. Replicate means from 3 independent experiments are shown.

    Techniques Used: Activity Assay, Lysis, CTL Assay, Generated, In Vitro, Irradiation, Purification, Colorimetric Assay

    XBP-1s inhibited moDCs reduce Th1 differentiation . (A,B) ELISAs were used to determine the concentration of IL-12/23 p40 cytokines or TNFα in the supernatants of B-I09- or DMSO-treated moDCs after 24 h of LPS stimulation. Replicate means from 8 (IL-12/23 p40) and 2 (TNFα) independent experiments are shown, Dunnett's test. (C–G) T cells were cultured with B-I09 or DMSO pre-treated moDCs (moDC:T cell ratio of 1:30), and additional B-I09 (20 μM) or DMSO (0.1%) was added once on day 0. Harvested T cells were evaluated on day +5 for Th1 (CD4 + , IFNγ + ) and Th2 (CD4 + , IL-4 + ) phenotype. Percentage or absolute numbers of Th1 (C,D) and Th2 (E,F) are shown. (G) Representative contour plots show CD4 + Th1 vs. Th2 cells on day +5 of the allogeneic co-culture. Means of 8 independent experiments are shown, paired t-test.
    Figure Legend Snippet: XBP-1s inhibited moDCs reduce Th1 differentiation . (A,B) ELISAs were used to determine the concentration of IL-12/23 p40 cytokines or TNFα in the supernatants of B-I09- or DMSO-treated moDCs after 24 h of LPS stimulation. Replicate means from 8 (IL-12/23 p40) and 2 (TNFα) independent experiments are shown, Dunnett's test. (C–G) T cells were cultured with B-I09 or DMSO pre-treated moDCs (moDC:T cell ratio of 1:30), and additional B-I09 (20 μM) or DMSO (0.1%) was added once on day 0. Harvested T cells were evaluated on day +5 for Th1 (CD4 + , IFNγ + ) and Th2 (CD4 + , IL-4 + ) phenotype. Percentage or absolute numbers of Th1 (C,D) and Th2 (E,F) are shown. (G) Representative contour plots show CD4 + Th1 vs. Th2 cells on day +5 of the allogeneic co-culture. Means of 8 independent experiments are shown, paired t-test.

    Techniques Used: Concentration Assay, Cell Culture, Co-Culture Assay

    Targeting XBP-1s abrogates human moDC production of IL-1β, TGFβ, and diminishes alloresponder Th17 differentiation . (A–C) Supernatant concentrations of IL-1β, TGFβ, or IL-6 from LPS-stimulated moDCs exposed to B-I09 or DMSO after 24 h of culture are shown. Replicate means from 5 (IL-1 β), 3 (TGFβ), and 4 (IL-6) independent experiments are shown, paired t -test. (D–H) T cells were cultured with B-I09 or DMSO pre-treated moDCs (moDC:T cell ratio of 1:30), and additional B-I09 (20 μM) or DMSO (0.1%) was added once on day 0. pSTAT3 + CD4 + T cells were analyzed at day +5 by flow cytometry (D) and representative histograms are shown (E) . Means from 3 independent experiments are shown, paired t -test. (F) Total STAT3 was measured in T cells from co-cultures of DCs and T cells treated with B-I09 or DMSO. Means from 3 experiments are shown. In similarly treated co-cultures, the supernatant concentration of IL-17 was quantified (G) and Th17 (CD4 + , CCR6 + , IL-17A + ) differentiation was evaluated by flow cytometry (H,I) . Replicate means from 3 (IL-17 ELISA) and 4 (Th17) independent experiments, paired t -test. (I) Representative contour plots are shown.
    Figure Legend Snippet: Targeting XBP-1s abrogates human moDC production of IL-1β, TGFβ, and diminishes alloresponder Th17 differentiation . (A–C) Supernatant concentrations of IL-1β, TGFβ, or IL-6 from LPS-stimulated moDCs exposed to B-I09 or DMSO after 24 h of culture are shown. Replicate means from 5 (IL-1 β), 3 (TGFβ), and 4 (IL-6) independent experiments are shown, paired t -test. (D–H) T cells were cultured with B-I09 or DMSO pre-treated moDCs (moDC:T cell ratio of 1:30), and additional B-I09 (20 μM) or DMSO (0.1%) was added once on day 0. pSTAT3 + CD4 + T cells were analyzed at day +5 by flow cytometry (D) and representative histograms are shown (E) . Means from 3 independent experiments are shown, paired t -test. (F) Total STAT3 was measured in T cells from co-cultures of DCs and T cells treated with B-I09 or DMSO. Means from 3 experiments are shown. In similarly treated co-cultures, the supernatant concentration of IL-17 was quantified (G) and Th17 (CD4 + , CCR6 + , IL-17A + ) differentiation was evaluated by flow cytometry (H,I) . Replicate means from 3 (IL-17 ELISA) and 4 (Th17) independent experiments, paired t -test. (I) Representative contour plots are shown.

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

    moDC XBP-1s direct human iTreg differentiation via TGFβ. T cells were cultured with B-I09 or DMSO pre-treated moDCs (moDC:T cell ratio of 1:30), and additional B-I09 (20μM) or DMSO (0.1%) was added once on day 0. (A) Percentage of Tregs (CD4 + , CD127 − , CD25 + , Foxp3 + ) in the 5-day co-cultures, with representative contour plots shown (B) . Means from 6 independent experiments are shown. (C) The suppressive capacity of harvested moDC-allostimulated Tregs was tested at different ratios of Treg to T cell responders stimulated by fresh allogeneic moDCs (moDC:responder T cell ratio 1:30) in alloMLRs. No additional B-I09 or DMSO was added. Graph shows mean percent T effector (Teff) proliferation measured by Ki-67. Means from 3 independent experiments are shown. To generate inducible Tregs, Treg-depleted CD4 + Tconv were stimulated by B-I09 or DMSO-pretreated allogeneic moDCs at a moDC:T cell ratio of 1:30. B-I09 or DMSO was added once on day 0. Magnetic bead enriched natural Tregs (CD4 + , CD25 + ) were similarly treated in allogeneic Treg:moDC co-cultures. Treg populations were evaluated by flow cytometry on day +5. Means from 4 iTreg and 4 nTreg independent experiments are shown, paired t -test. Percentage and absolute number of iTreg (D,E) and nTreg (F,G) are shown. (H) Representative contour plots are shown for iTreg and nTreg. (I) Representative contour plots show that adding recombinant human TGFβ rescues iTreg generation in Treg-depleted alloMLRs treated with B-I09 vs. DMSO. 1 representative experiment of 2 independent studies is shown. (J) T cell proliferation (MTS colormetric assay) at day +5 among Treg-depleted alloMLRs treated with B-I09 or DMSO, with recombinant human TGFβ added as indicated. Replicate means from 4 independent experiments are shown, Dunnett's test.
    Figure Legend Snippet: moDC XBP-1s direct human iTreg differentiation via TGFβ. T cells were cultured with B-I09 or DMSO pre-treated moDCs (moDC:T cell ratio of 1:30), and additional B-I09 (20μM) or DMSO (0.1%) was added once on day 0. (A) Percentage of Tregs (CD4 + , CD127 − , CD25 + , Foxp3 + ) in the 5-day co-cultures, with representative contour plots shown (B) . Means from 6 independent experiments are shown. (C) The suppressive capacity of harvested moDC-allostimulated Tregs was tested at different ratios of Treg to T cell responders stimulated by fresh allogeneic moDCs (moDC:responder T cell ratio 1:30) in alloMLRs. No additional B-I09 or DMSO was added. Graph shows mean percent T effector (Teff) proliferation measured by Ki-67. Means from 3 independent experiments are shown. To generate inducible Tregs, Treg-depleted CD4 + Tconv were stimulated by B-I09 or DMSO-pretreated allogeneic moDCs at a moDC:T cell ratio of 1:30. B-I09 or DMSO was added once on day 0. Magnetic bead enriched natural Tregs (CD4 + , CD25 + ) were similarly treated in allogeneic Treg:moDC co-cultures. Treg populations were evaluated by flow cytometry on day +5. Means from 4 iTreg and 4 nTreg independent experiments are shown, paired t -test. Percentage and absolute number of iTreg (D,E) and nTreg (F,G) are shown. (H) Representative contour plots are shown for iTreg and nTreg. (I) Representative contour plots show that adding recombinant human TGFβ rescues iTreg generation in Treg-depleted alloMLRs treated with B-I09 vs. DMSO. 1 representative experiment of 2 independent studies is shown. (J) T cell proliferation (MTS colormetric assay) at day +5 among Treg-depleted alloMLRs treated with B-I09 or DMSO, with recombinant human TGFβ added as indicated. Replicate means from 4 independent experiments are shown, Dunnett's test.

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Recombinant

    XBP-1s inhibition reduces the stimulatory potency of moDCs toward allogeneic T cells. Human moDCs were stimulated with LPS (1 μg/ml) for 24 h in the presence of XBP-1 or control siRNA. (A) Representative histograms show XBP-1s expression in siRNA-treated moDCs. (B) T cells were stimulated by XBP-1- or control-siRNA-treated moDCs in alloMLRs. T cell proliferation (MTS colormetric assay) after 5 days is shown. AlloMLRs were plated in replicates of 5 at a moDC: T cell ratio of 1:30. 1 representative experiment of 2 independent studies is shown, Dunnett's test. (C) Human moDCs were stimulated with LPS (1 μg/ml) for 24 h in the presence of B-I09 (20 μM) or DMSO (0.1%). Bar graph shows triplicate mean XBP-1s or total XBP-1 mRNA, vs. the RIDD components BlocS1 and CD59 in B-I09- or DMSO-treated moDCs after 24 h of LPS stimulation as measured by RT-PCR. 1 representative experiment of 4 independent studies is shown, Tukey's test. (D) T cell proliferation (MTS colormetric assay) measured in 5-day MLRs using B-I09- or DMSO-treated allogeneic, moDCs. Table depicts whether moDCs were pre-treated with DMSO (0.1%) or B-I09 (20 μM), or if DMSO (0.1%) or B-I09 (20 μM) was added to the MLR medium. Replicate means from 4 independent experiments are shown, Dunnett's test. (E,F) Human moDCs were LPS-stimulated for 24 h with B-I09 or DMSO. Bar graphs show proportion of migrating moDCs in transwell assays testing CCL19 or CCL21 (300 ng/ml, 3 h) chemotaxis. Replicate means from 4 independent experiments are shown for each, Dunnett's test. (G) Human moDCs were stimulated with LPS (1 μg/ml) for 24 h in the presence of B-I09 (20 μM) or DMSO (0.1%), and then used to activate allogeneic T cells for 5 days. T cells were rested for 24 h at 37°C, loaded with Fluo-4 dye for 30 min, then restimulated with fresh B-I09- or DMSO-treated moDCs during live cell imaging to monitor calcium flux in real time. Replicate means from 3 independent studies are shown. P values are shown for early (
    Figure Legend Snippet: XBP-1s inhibition reduces the stimulatory potency of moDCs toward allogeneic T cells. Human moDCs were stimulated with LPS (1 μg/ml) for 24 h in the presence of XBP-1 or control siRNA. (A) Representative histograms show XBP-1s expression in siRNA-treated moDCs. (B) T cells were stimulated by XBP-1- or control-siRNA-treated moDCs in alloMLRs. T cell proliferation (MTS colormetric assay) after 5 days is shown. AlloMLRs were plated in replicates of 5 at a moDC: T cell ratio of 1:30. 1 representative experiment of 2 independent studies is shown, Dunnett's test. (C) Human moDCs were stimulated with LPS (1 μg/ml) for 24 h in the presence of B-I09 (20 μM) or DMSO (0.1%). Bar graph shows triplicate mean XBP-1s or total XBP-1 mRNA, vs. the RIDD components BlocS1 and CD59 in B-I09- or DMSO-treated moDCs after 24 h of LPS stimulation as measured by RT-PCR. 1 representative experiment of 4 independent studies is shown, Tukey's test. (D) T cell proliferation (MTS colormetric assay) measured in 5-day MLRs using B-I09- or DMSO-treated allogeneic, moDCs. Table depicts whether moDCs were pre-treated with DMSO (0.1%) or B-I09 (20 μM), or if DMSO (0.1%) or B-I09 (20 μM) was added to the MLR medium. Replicate means from 4 independent experiments are shown, Dunnett's test. (E,F) Human moDCs were LPS-stimulated for 24 h with B-I09 or DMSO. Bar graphs show proportion of migrating moDCs in transwell assays testing CCL19 or CCL21 (300 ng/ml, 3 h) chemotaxis. Replicate means from 4 independent experiments are shown for each, Dunnett's test. (G) Human moDCs were stimulated with LPS (1 μg/ml) for 24 h in the presence of B-I09 (20 μM) or DMSO (0.1%), and then used to activate allogeneic T cells for 5 days. T cells were rested for 24 h at 37°C, loaded with Fluo-4 dye for 30 min, then restimulated with fresh B-I09- or DMSO-treated moDCs during live cell imaging to monitor calcium flux in real time. Replicate means from 3 independent studies are shown. P values are shown for early (

    Techniques Used: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Chemotaxis Assay, Live Cell Imaging

    14) Product Images from "Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice"

    Article Title: Preclinical Evaluation of Long-Term Neuroprotective Effects of BDNF-Engineered Mesenchymal Stromal Cells as Intravitreal Therapy for Chronic Retinal Degeneration in Rd6 Mutant Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20030777

    The expression profile of selected apoptosis-related molecules in retinas treated with MSC-BDNF or MSC alone at different time points (at 28 days and three months post transplantation) and compared to WT and rd6 mice. The mRNA expression of Bcl-xL and BAX genes was determined by the quantitative PCR and the relative ratio Bcl-xL/BAX was calculated ( A ). The concentration of Bcl-xL and Bak protein dimer was determined by specific Luminex ( B ) Similarly, the concentration of Mcl-1/Bak dimer protein was measured ( C ). Caspase-3 protein expression was determined by Western blot, which revealed a lack of expression of this form in WT mice and rd6 after cell transplantation compared to rd6 mice with no treatment ( D ). GAPDH served as loading. A representative image is shown. Mean values ± SDs are presented in the diagrams, * p
    Figure Legend Snippet: The expression profile of selected apoptosis-related molecules in retinas treated with MSC-BDNF or MSC alone at different time points (at 28 days and three months post transplantation) and compared to WT and rd6 mice. The mRNA expression of Bcl-xL and BAX genes was determined by the quantitative PCR and the relative ratio Bcl-xL/BAX was calculated ( A ). The concentration of Bcl-xL and Bak protein dimer was determined by specific Luminex ( B ) Similarly, the concentration of Mcl-1/Bak dimer protein was measured ( C ). Caspase-3 protein expression was determined by Western blot, which revealed a lack of expression of this form in WT mice and rd6 after cell transplantation compared to rd6 mice with no treatment ( D ). GAPDH served as loading. A representative image is shown. Mean values ± SDs are presented in the diagrams, * p

    Techniques Used: Expressing, Transplantation Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Luminex, Western Blot

    Long-term follow-up of BDNF production and its biological function at different time points post-intravitreal MSC-BDNF transplantation. BDNF mRNA ( A ), BDNF, phosphoAkt, totalAkt, phosphoMAPK and totalMAPK protein ( B ) expression was detected in retinas from eyes treated with MSC-BDNF, and their levels were significantly increased post-transplantation compared to other groups: at 28 days in case of mRNA and protein and at three months in the case of BDNF protein only. We also observed increased TrkB gene expression in retinas at 28 days and three months after MSC-BDNF and MSC alone transplantation compared to rd6 and wild type (WT) ( C ). The follow-up of retinal cell proliferation at different time points post-intravitreal transplantation in rd6 mice was also performed. Quantitative analysis of proliferating cell nuclear antigen (PCNA) mRNA expression revealed that their levels were significantly increased in retinas from eyes treated with MSC-BDNF at 28 days post transplantation compared with those in eyes treated with the PBS and MSCs alone ( D ). Double-stained sections for PCNA and GFP (endogenous marker of transplanted MSC) used to visualize and localize proliferating cells revealed the extraordinary PCNA protein concentration in MSC-BDNF transplanted 28 days previously ( E ). Representative images of the performed analyses are shown. Scale bar: 20 µm. Reference gene used for qRT-PCR analysis was glyceraldehyde 3-phospate dehydrogenase (GAPDH). Mean values ± SD are presented in the diagrams, * p
    Figure Legend Snippet: Long-term follow-up of BDNF production and its biological function at different time points post-intravitreal MSC-BDNF transplantation. BDNF mRNA ( A ), BDNF, phosphoAkt, totalAkt, phosphoMAPK and totalMAPK protein ( B ) expression was detected in retinas from eyes treated with MSC-BDNF, and their levels were significantly increased post-transplantation compared to other groups: at 28 days in case of mRNA and protein and at three months in the case of BDNF protein only. We also observed increased TrkB gene expression in retinas at 28 days and three months after MSC-BDNF and MSC alone transplantation compared to rd6 and wild type (WT) ( C ). The follow-up of retinal cell proliferation at different time points post-intravitreal transplantation in rd6 mice was also performed. Quantitative analysis of proliferating cell nuclear antigen (PCNA) mRNA expression revealed that their levels were significantly increased in retinas from eyes treated with MSC-BDNF at 28 days post transplantation compared with those in eyes treated with the PBS and MSCs alone ( D ). Double-stained sections for PCNA and GFP (endogenous marker of transplanted MSC) used to visualize and localize proliferating cells revealed the extraordinary PCNA protein concentration in MSC-BDNF transplanted 28 days previously ( E ). Representative images of the performed analyses are shown. Scale bar: 20 µm. Reference gene used for qRT-PCR analysis was glyceraldehyde 3-phospate dehydrogenase (GAPDH). Mean values ± SD are presented in the diagrams, * p

    Techniques Used: Transplantation Assay, Expressing, Mouse Assay, Staining, Marker, Protein Concentration, Quantitative RT-PCR

    15) Product Images from "Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells"

    Article Title: Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20030551

    Effect of Mettl3 knockdown on the osteogenic differentiation potential of BMSCs. ( A ) A green fluorescence protein marker was used to determine the transfer efficiency of Mettl3 knockdown in BMSCs. After transfection for 72 h, the cells were observed under a microscope (on the left). The right image is an immunofluorescence image taken at the same time. The “black” scale bars represent 100 μm (original magnification ×100). ( B ) The expression level of Mettl3 was determined using Western blotting in the Mettl3 -shRNA and Mettl3 -shCtrl groups. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( C ) The mRNA expression levels of Alp and Ocn in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using qRT-PCR after 7 and 14 days of osteogenic induction. Gapdh was used as an internal control. ( D ) The protein levels of Runx2 and Osterix in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using Western blotting after 7 and 14 days of osteogenic induction. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( E ) ALP activity was determined in Mettl3 -shRNA and Mettl3 -shCtrl cells cultured in osteogenic differentiation medium for seven days. ( F ) The formation of mineralized nodules was analyzed in the Mettl3 -shCtrl and Mettl3 -shRNA groups undergoing osteogenic induction on Days 7, 14, and 21. Mineralization was analyzed using Alizarin Red S staining. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p
    Figure Legend Snippet: Effect of Mettl3 knockdown on the osteogenic differentiation potential of BMSCs. ( A ) A green fluorescence protein marker was used to determine the transfer efficiency of Mettl3 knockdown in BMSCs. After transfection for 72 h, the cells were observed under a microscope (on the left). The right image is an immunofluorescence image taken at the same time. The “black” scale bars represent 100 μm (original magnification ×100). ( B ) The expression level of Mettl3 was determined using Western blotting in the Mettl3 -shRNA and Mettl3 -shCtrl groups. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( C ) The mRNA expression levels of Alp and Ocn in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using qRT-PCR after 7 and 14 days of osteogenic induction. Gapdh was used as an internal control. ( D ) The protein levels of Runx2 and Osterix in the Mettl3 -shRNA and Mettl3 -shCtrl groups were assessed using Western blotting after 7 and 14 days of osteogenic induction. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( E ) ALP activity was determined in Mettl3 -shRNA and Mettl3 -shCtrl cells cultured in osteogenic differentiation medium for seven days. ( F ) The formation of mineralized nodules was analyzed in the Mettl3 -shCtrl and Mettl3 -shRNA groups undergoing osteogenic induction on Days 7, 14, and 21. Mineralization was analyzed using Alizarin Red S staining. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p

    Techniques Used: Fluorescence, Marker, Transfection, Microscopy, Immunofluorescence, Expressing, Western Blot, shRNA, Software, ALP Assay, Quantitative RT-PCR, Activity Assay, Cell Culture, Staining, Standard Deviation

    Osteogenic differentiation of BMSCs and expression of m 6 A methyltransferase and demethylases. ( A , B ) The formation of mineralized nodules was analyzed in BMSCs undergoing osteogenic differentiation on Days 7, 14, and 21. The “white” scale bars represent 100 μm (original magnification ×100). ( C ) ALP activity assays. ALP activity was significantly higher in the cells cultured in osteogenic differentiation medium for seven days. ( D ) The expression of Runx2 , Alp and Ocn was assessed using qRT-PCR on Days 7 and 14 of culture in osteogenic induction medium. Gapdh was used as an internal control. ( E ) The expression of Runx2 and Osterix was examined using Western blotting. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( F , G ) RNA methylation modification-related enzymes were detected using qRT-PCR and Western blotting in cells cultured in osteogenic induction medium for 7 and 14 days. Gapdh was used as an internal control for qRT-PCR. Vinculin was used as an internal control in Western blotting. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p
    Figure Legend Snippet: Osteogenic differentiation of BMSCs and expression of m 6 A methyltransferase and demethylases. ( A , B ) The formation of mineralized nodules was analyzed in BMSCs undergoing osteogenic differentiation on Days 7, 14, and 21. The “white” scale bars represent 100 μm (original magnification ×100). ( C ) ALP activity assays. ALP activity was significantly higher in the cells cultured in osteogenic differentiation medium for seven days. ( D ) The expression of Runx2 , Alp and Ocn was assessed using qRT-PCR on Days 7 and 14 of culture in osteogenic induction medium. Gapdh was used as an internal control. ( E ) The expression of Runx2 and Osterix was examined using Western blotting. Vinculin was used as an internal control. The band intensities were analyzed using ImageJ software. ( F , G ) RNA methylation modification-related enzymes were detected using qRT-PCR and Western blotting in cells cultured in osteogenic induction medium for 7 and 14 days. Gapdh was used as an internal control for qRT-PCR. Vinculin was used as an internal control in Western blotting. All of the results represent the mean ± standard deviation of three independent experiments ( n = 3). Significant difference compared with the control (* p

    Techniques Used: Expressing, ALP Assay, Activity Assay, Cell Culture, Quantitative RT-PCR, Western Blot, Software, Methylation, Modification, Standard Deviation

    16) Product Images from "CD36 mediates palmitate acid-induced metastasis of gastric cancer via AKT/GSK-3β/β-catenin pathway"

    Article Title: CD36 mediates palmitate acid-induced metastasis of gastric cancer via AKT/GSK-3β/β-catenin pathway

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1049-7

    PA promotes GC cell migration and invasion via the AKT/GSK-3β/ β-catenin signaling pathway. a and c Effect of PA on protein levels of p-AKT, p-GSK-3β, AKT, GSK-3β and global β-catenin at different time points. b and d Densitometry showing effect of PA on protein levels of p-AKT, p-GSK-3β, AKT, GSK-3β and global β-catenin. e and f Effect of PA on cellular mRNA level of β-catenin at different time points. g and h Effects of PA on nuclear-transport of β-catenin at selected time points. Densitometry showing effect of PA on nuclear protein level of β-catenin. i and j IF showing effect of PA on cellular location of β-catenin in GC cells at selected time points. Data are shown as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: PA promotes GC cell migration and invasion via the AKT/GSK-3β/ β-catenin signaling pathway. a and c Effect of PA on protein levels of p-AKT, p-GSK-3β, AKT, GSK-3β and global β-catenin at different time points. b and d Densitometry showing effect of PA on protein levels of p-AKT, p-GSK-3β, AKT, GSK-3β and global β-catenin. e and f Effect of PA on cellular mRNA level of β-catenin at different time points. g and h Effects of PA on nuclear-transport of β-catenin at selected time points. Densitometry showing effect of PA on nuclear protein level of β-catenin. i and j IF showing effect of PA on cellular location of β-catenin in GC cells at selected time points. Data are shown as mean ± SD of three independent experiments. * P

    Techniques Used: Migration

    17) Product Images from "Prefrontal cortex miR-29b-3p plays a key role in the antidepressant-like effect of ketamine in rats"

    Article Title: Prefrontal cortex miR-29b-3p plays a key role in the antidepressant-like effect of ketamine in rats

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0164-4

    Overexpression of miR-29b-3p improved depressive-like behaviors in CUMS rats. The prefrontal cortex of rats infected by rAAV-miRNA-29b-3p (immunofluorescence microscope) ( a ). The sucrose preference rate ( b ) and immobility time ( c ) of depressed rats 28 days after rAAV-miRNA-29b-3p infection. RELs of miR-29b-3p ( d ), GRM4 mRNA ( e ), and GRM4 protein ( f ) 28 days after rAAV-miRNA-29b-3p infection. RELs relative expression levels, NS CUMS + 0.9% saline group, AAV-GPF CUMS + rAAV-GFP infection group, AAV-miR-29b-3p CUMS + rAAV-miRNA-29b-3p infection group. The data represent eight sets of independent experiments and are shown as the means ± SD. * p
    Figure Legend Snippet: Overexpression of miR-29b-3p improved depressive-like behaviors in CUMS rats. The prefrontal cortex of rats infected by rAAV-miRNA-29b-3p (immunofluorescence microscope) ( a ). The sucrose preference rate ( b ) and immobility time ( c ) of depressed rats 28 days after rAAV-miRNA-29b-3p infection. RELs of miR-29b-3p ( d ), GRM4 mRNA ( e ), and GRM4 protein ( f ) 28 days after rAAV-miRNA-29b-3p infection. RELs relative expression levels, NS CUMS + 0.9% saline group, AAV-GPF CUMS + rAAV-GFP infection group, AAV-miR-29b-3p CUMS + rAAV-miRNA-29b-3p infection group. The data represent eight sets of independent experiments and are shown as the means ± SD. * p

    Techniques Used: Over Expression, Infection, Immunofluorescence, Microscopy, Expressing

    Change in GRM4 expression levels in the prefrontal cortex of depressed rats following ketamine treatment. RELs of GRM4 mRNA in the prefrontal cortex of depressed rats 24 h after a 10 mg/kg ketamine treatment ( a ). RELs of GRM4 protein in the prefrontal cortex of depressed rats 24 h after a 10 mg/kg ketamine treatment ( b , c ). C control group, CUMS chronic unpredictable mild stress, CUMS + NS CUMS + 0.9% saline group, CUMS + K CUMS + ketamine group, RELs relative expression levels. The data represent eight sets of independent experiments and are shown as the means ± SD. * p
    Figure Legend Snippet: Change in GRM4 expression levels in the prefrontal cortex of depressed rats following ketamine treatment. RELs of GRM4 mRNA in the prefrontal cortex of depressed rats 24 h after a 10 mg/kg ketamine treatment ( a ). RELs of GRM4 protein in the prefrontal cortex of depressed rats 24 h after a 10 mg/kg ketamine treatment ( b , c ). C control group, CUMS chronic unpredictable mild stress, CUMS + NS CUMS + 0.9% saline group, CUMS + K CUMS + ketamine group, RELs relative expression levels. The data represent eight sets of independent experiments and are shown as the means ± SD. * p

    Techniques Used: Expressing

    Change in miR-29b-3p and GRM4 levels at different times after ketamine treatment in cultured prefrontal cortex neurons. RELs of miR-29b-3p ( a ) and GRM4 mRNA ( b ) in the prefrontal cortex neurons 1, 3, 6, and 12 h after a 50 µM ketamine treatment. RELs of GRM4 protein in the prefrontal cortex neurons 1, 3, 6, and 12 h after a 50 µM ketamine treatment ( c ). RELs relative expression levels. The data represent three sets of independent experiments and are shown as the means ± SD. * p
    Figure Legend Snippet: Change in miR-29b-3p and GRM4 levels at different times after ketamine treatment in cultured prefrontal cortex neurons. RELs of miR-29b-3p ( a ) and GRM4 mRNA ( b ) in the prefrontal cortex neurons 1, 3, 6, and 12 h after a 50 µM ketamine treatment. RELs of GRM4 protein in the prefrontal cortex neurons 1, 3, 6, and 12 h after a 50 µM ketamine treatment ( c ). RELs relative expression levels. The data represent three sets of independent experiments and are shown as the means ± SD. * p

    Techniques Used: Cell Culture, Expressing

    18) Product Images from "Expression of a cyclo-oxygenase-2 transgene in murine liver causes hepatitis"

    Article Title: Expression of a cyclo-oxygenase-2 transgene in murine liver causes hepatitis

    Journal: Gut

    doi: 10.1136/gut.2006.097923

    Increased liver cell apoptosis is evident in the TG livers
    Figure Legend Snippet: Increased liver cell apoptosis is evident in the TG livers

    Techniques Used:

    19) Product Images from "Rat heart cannot synthesize docosahexaenoic acid from circulating α-linolenic acid because it lacks elongase-2"

    Article Title: Rat heart cannot synthesize docosahexaenoic acid from circulating α-linolenic acid because it lacks elongase-2

    Journal: Journal of lipid research

    doi: 10.1194/jlr.M800093-JLR200

    mRNA levels of Δ6 desaturase (A), Δ5 desaturase (B), elongase-5 (C), elongase-2 (D), and acyl-CoA oxidase (E) in heart of rats fed n-3 PUFA-adequate and n-3 PUFA-deficient diets. ND, not detected. Values are means ± SD (n = 10 for each group). ** P
    Figure Legend Snippet: mRNA levels of Δ6 desaturase (A), Δ5 desaturase (B), elongase-5 (C), elongase-2 (D), and acyl-CoA oxidase (E) in heart of rats fed n-3 PUFA-adequate and n-3 PUFA-deficient diets. ND, not detected. Values are means ± SD (n = 10 for each group). ** P

    Techniques Used:

    20) Product Images from "groEL Expression in gyrB Mutants of Borrelia burgdorferi"

    Article Title: groEL Expression in gyrB Mutants of Borrelia burgdorferi

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.183.21.6069-6072.2002

    Increased GroEL synthesis and groEL expression in a gyrB mutant of B. burgdorferi . Immunoblot (A) and Northern blot (B) analyses of wild-type B31 (wt), experimentally heat-shocked wild-type B31 (hs), and the gyrB mutant X32 were carried out.
    Figure Legend Snippet: Increased GroEL synthesis and groEL expression in a gyrB mutant of B. burgdorferi . Immunoblot (A) and Northern blot (B) analyses of wild-type B31 (wt), experimentally heat-shocked wild-type B31 (hs), and the gyrB mutant X32 were carried out.

    Techniques Used: Expressing, Mutagenesis, Northern Blot

    Effect of a gyrB mutation on DNA topology. Plasmid pGOΔ15 was transiently introduced into wild-type B31 and the gyrB mutant X32. The topology of the plasmid was determined by one-dimensional 0.8% agarose gel electrophoresis in the absence (native) or presence of 15 μM chloroquine (CQ). The more negatively supercoiled molecules migrate faster through the gel; chloroquine intercalates into the plasmid DNA, introducing positive supercoiling that retards migration of negatively supercoiled DNA (sc) and expedites migration of relaxed DNA (r). The migration of the linearized (l) form of the plasmid is not affected in the mutant. Molecular size standards are in kilobase pairs.
    Figure Legend Snippet: Effect of a gyrB mutation on DNA topology. Plasmid pGOΔ15 was transiently introduced into wild-type B31 and the gyrB mutant X32. The topology of the plasmid was determined by one-dimensional 0.8% agarose gel electrophoresis in the absence (native) or presence of 15 μM chloroquine (CQ). The more negatively supercoiled molecules migrate faster through the gel; chloroquine intercalates into the plasmid DNA, introducing positive supercoiling that retards migration of negatively supercoiled DNA (sc) and expedites migration of relaxed DNA (r). The migration of the linearized (l) form of the plasmid is not affected in the mutant. Molecular size standards are in kilobase pairs.

    Techniques Used: Mutagenesis, Plasmid Preparation, Agarose Gel Electrophoresis, Migration

    21) Product Images from "Alternatively Spliced Caspase-6B Isoform Inhibits the Activation of Caspase-6A *"

    Article Title: Alternatively Spliced Caspase-6B Isoform Inhibits the Activation of Caspase-6A *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.152744

    Pro-Casp6b inhibits p20p10Casp6a-mediated processing and activation in HCT116 cells. A , VEIDase activity and Western blot analysis with anti-p10Casp6 and anti-His antibodies of protein extracts from HCT116 cells transfected with vector alone (pCep4β), pCep4βEGFP, pCep4βpro-Casp6b, or pCep4βp20p10Casp6a. B , VEIDase activity and Western blot analysis with anti-p10Casp6 and anti-p20 neoepitope antiserum of protein extracts from HCT116 cells transfected with vector alone (pCep4β)-, pCep4βp20p10-, pCep4βp20p10/pCep4βpro-Casp6b (1:3 cDNA ratio)-, or pCep4βp20p10/pCep4βpro-Casp6b (1:9 cDNA ratio)-transfected HCT116 cells. C , ethidium bromide-stained agarose gel of RT-PCR products from HCT116-transfected cells. D , Western blot analysis with anti-p10Casp6 antibody ( top ), anti-neoepitope p20 antiserum ( middle ), and anti-β-actin antibodies of proteins extracted from transfected HCT116 cells treated in the absence or presence of epoxomicin ( EPOX ). Error bars , S.E.
    Figure Legend Snippet: Pro-Casp6b inhibits p20p10Casp6a-mediated processing and activation in HCT116 cells. A , VEIDase activity and Western blot analysis with anti-p10Casp6 and anti-His antibodies of protein extracts from HCT116 cells transfected with vector alone (pCep4β), pCep4βEGFP, pCep4βpro-Casp6b, or pCep4βp20p10Casp6a. B , VEIDase activity and Western blot analysis with anti-p10Casp6 and anti-p20 neoepitope antiserum of protein extracts from HCT116 cells transfected with vector alone (pCep4β)-, pCep4βp20p10-, pCep4βp20p10/pCep4βpro-Casp6b (1:3 cDNA ratio)-, or pCep4βp20p10/pCep4βpro-Casp6b (1:9 cDNA ratio)-transfected HCT116 cells. C , ethidium bromide-stained agarose gel of RT-PCR products from HCT116-transfected cells. D , Western blot analysis with anti-p10Casp6 antibody ( top ), anti-neoepitope p20 antiserum ( middle ), and anti-β-actin antibodies of proteins extracted from transfected HCT116 cells treated in the absence or presence of epoxomicin ( EPOX ). Error bars , S.E.

    Techniques Used: Activation Assay, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    22) Product Images from "Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma"

    Article Title: Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma

    Journal: Blood

    doi:

    IFN-α and -β induce Bid and Bcl-2 cleavage. ) and Bcl-2 mAb. z-VAD-fmk was added every 24 hours following IFN treatment. The data shown are representative of 3 different experiments. (B) IFN-α and -β, but not -γ, induce Bcl-2 cleavage in U266 (left) and freshly isolated MM cells (right). Plasma cells were isolated from patients and treated for 48 hours, before being subjected to Western blotting for Bcl-2. (C) U266 cells were treated as indicated and then subjected to Western blotting with the appropriate antibodies for Bcl-x L , Bad, Bak, and Bax, and, as a control, β-actin.
    Figure Legend Snippet: IFN-α and -β induce Bid and Bcl-2 cleavage. ) and Bcl-2 mAb. z-VAD-fmk was added every 24 hours following IFN treatment. The data shown are representative of 3 different experiments. (B) IFN-α and -β, but not -γ, induce Bcl-2 cleavage in U266 (left) and freshly isolated MM cells (right). Plasma cells were isolated from patients and treated for 48 hours, before being subjected to Western blotting for Bcl-2. (C) U266 cells were treated as indicated and then subjected to Western blotting with the appropriate antibodies for Bcl-x L , Bad, Bak, and Bax, and, as a control, β-actin.

    Techniques Used: Isolation, Western Blot

    IFN-α and -β induce Apo2L expression in MM. , and detailed in “Materials and methods.” Apo2L protein levels were determined for U266 cells (B) and plasma cells of MM patients (C) treated with IFNs for 24 hours and then subjected to Western blotting using antibodies against Apo2L, and as a control, β-actin.
    Figure Legend Snippet: IFN-α and -β induce Apo2L expression in MM. , and detailed in “Materials and methods.” Apo2L protein levels were determined for U266 cells (B) and plasma cells of MM patients (C) treated with IFNs for 24 hours and then subjected to Western blotting using antibodies against Apo2L, and as a control, β-actin.

    Techniques Used: Expressing, Western Blot

    DR5Δ blocks IFN-induced apoptosis. DR5Δ also contains a FLAG epitope-tag that facilitates examination of its expression levels (A). Parental and DR5Δ-FLAG–containing U266 cells were treated with Apo2L (100 ng/mL), IFN-α, and -β (200 U/mL), and apoptosis was determined at 72 hours by Hoechst 33258 staining and expressed as the percentage of the total cells. At least 200 cells were counted for each sample. Expression of DR5Δ was determined by Western blotting using an anti-FLAG antibody and β-actin as a protein loading control (inset). In a parallel experiment (B), the cells were also lysed and subjected to Western blotting for caspase 8 and Bcl-2, upper and lower panel, respectively.
    Figure Legend Snippet: DR5Δ blocks IFN-induced apoptosis. DR5Δ also contains a FLAG epitope-tag that facilitates examination of its expression levels (A). Parental and DR5Δ-FLAG–containing U266 cells were treated with Apo2L (100 ng/mL), IFN-α, and -β (200 U/mL), and apoptosis was determined at 72 hours by Hoechst 33258 staining and expressed as the percentage of the total cells. At least 200 cells were counted for each sample. Expression of DR5Δ was determined by Western blotting using an anti-FLAG antibody and β-actin as a protein loading control (inset). In a parallel experiment (B), the cells were also lysed and subjected to Western blotting for caspase 8 and Bcl-2, upper and lower panel, respectively.

    Techniques Used: FLAG-tag, Expressing, Staining, Western Blot

    IFN-α and -β induce caspase activation in MM cells. U266 cells were collected at the indicated times (T) after IFN treatment and analyzed by protease activity assays for caspase 8 and caspase 3 by using the specific IETD and DEVD-pNA substrates (A) and Western blotting for caspase 3 cleavage (B). Data were representative of 3 separate experiments. (C) IFN-α and -β, but not -γ, induced caspase 3 cleavage in freshly isolated MM plasma cells (patient No. 2). Primary MM cells were isolated from bone marrow aspirates of patients, as described in “Materials and methods,” treated for 48 hours with IFNs, then subjected to Western blotting for caspase 3. The p32 kD band represents procaspase 3, whereas p17 represents its activated form.
    Figure Legend Snippet: IFN-α and -β induce caspase activation in MM cells. U266 cells were collected at the indicated times (T) after IFN treatment and analyzed by protease activity assays for caspase 8 and caspase 3 by using the specific IETD and DEVD-pNA substrates (A) and Western blotting for caspase 3 cleavage (B). Data were representative of 3 separate experiments. (C) IFN-α and -β, but not -γ, induced caspase 3 cleavage in freshly isolated MM plasma cells (patient No. 2). Primary MM cells were isolated from bone marrow aspirates of patients, as described in “Materials and methods,” treated for 48 hours with IFNs, then subjected to Western blotting for caspase 3. The p32 kD band represents procaspase 3, whereas p17 represents its activated form.

    Techniques Used: Activation Assay, Activity Assay, Western Blot, Isolation

    IFN-α and -β induce cyt c release and Δψ m reduction. (A) Cyt c levels were determined in cellular fractions isolated by differential centrifugation from control or IFN-treated U266 cells as described in “Materials and methods.” Immuno-blotting with anti–cyt c mAb and β-actin as a control were used to determine the levels of cyt c in the S100 fractions, representing the cytosolic cyt c in 20 μg of cellular protein. (B) The Δψ m was determined by using 10 nM DIOC 6 (3) as described in “Materials and methods.” The percentage of Δψ m (low) after the cell debris were excluded was determined, and comparative experiments were performed on the same day. The data shown are representative of 3 separate duplicate experiments.
    Figure Legend Snippet: IFN-α and -β induce cyt c release and Δψ m reduction. (A) Cyt c levels were determined in cellular fractions isolated by differential centrifugation from control or IFN-treated U266 cells as described in “Materials and methods.” Immuno-blotting with anti–cyt c mAb and β-actin as a control were used to determine the levels of cyt c in the S100 fractions, representing the cytosolic cyt c in 20 μg of cellular protein. (B) The Δψ m was determined by using 10 nM DIOC 6 (3) as described in “Materials and methods.” The percentage of Δψ m (low) after the cell debris were excluded was determined, and comparative experiments were performed on the same day. The data shown are representative of 3 separate duplicate experiments.

    Techniques Used: Isolation, Centrifugation

    23) Product Images from "A Mutation in the 5? Untranslated Region Increases Stability of norA mRNA, Encoding a Multidrug Resistance Transporter of Staphylococcus aureus"

    Article Title: A Mutation in the 5? Untranslated Region Increases Stability of norA mRNA, Encoding a Multidrug Resistance Transporter of Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.183.7.2367-2371.2001

    Determination of norA mRNA stability. (A) Agarose gel with RT-PCR, which were performed using total cellular RNA extracted from strains ISP794 (wild type) and MT23142 ( flqB mutant) after addition of rifampin (200 μg/ml) at time zero (cells grown to OD 600 of 0.6) for both strains. Primers for norA mRNA (5′-AGA AAC TTT TTA CGA ATA TT-3′ and 5′-TGA CAC TGA AAA CAA AAT TA-3′) generated a 250-bp fragment. We used 0.2 μM each primer, 2 U of reverse transcriptase- Taq mix, and 4 μg of total RNA for ISP794 or 2 μg of total RNA for MT23142 for norA ). (B) Semilogarithmic graph of mRNA concentration as a function of time (expressed as percent of amount at time of addition of rifampin). Densitometric analysis was performed by scanning the gel pictures and performing analysis using the public-domain National Institutes of Health Image program (version 6.1). (C) Northern hybridization of norA mRNA. Total RNA was extracted using the SNAP Total RNA kit (Invitrogen). Times indicate time after the addition of rifampin at time 0. The norA ).
    Figure Legend Snippet: Determination of norA mRNA stability. (A) Agarose gel with RT-PCR, which were performed using total cellular RNA extracted from strains ISP794 (wild type) and MT23142 ( flqB mutant) after addition of rifampin (200 μg/ml) at time zero (cells grown to OD 600 of 0.6) for both strains. Primers for norA mRNA (5′-AGA AAC TTT TTA CGA ATA TT-3′ and 5′-TGA CAC TGA AAA CAA AAT TA-3′) generated a 250-bp fragment. We used 0.2 μM each primer, 2 U of reverse transcriptase- Taq mix, and 4 μg of total RNA for ISP794 or 2 μg of total RNA for MT23142 for norA ). (B) Semilogarithmic graph of mRNA concentration as a function of time (expressed as percent of amount at time of addition of rifampin). Densitometric analysis was performed by scanning the gel pictures and performing analysis using the public-domain National Institutes of Health Image program (version 6.1). (C) Northern hybridization of norA mRNA. Total RNA was extracted using the SNAP Total RNA kit (Invitrogen). Times indicate time after the addition of rifampin at time 0. The norA ).

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Cellular Antioxidant Activity Assay, Generated, Concentration Assay, Northern Blot, Hybridization

    24) Product Images from "Chronic Administration of Mood Stabilizers Upregulates BDNF and Bcl-2 Expression Levels in Rat Frontal Cortex"

    Article Title: Chronic Administration of Mood Stabilizers Upregulates BDNF and Bcl-2 Expression Levels in Rat Frontal Cortex

    Journal: Neurochemical research

    doi: 10.1007/s11064-008-9817-3

    Representative mRNA and protein levels of Bcl-2 in frontal cortex from rats chronically administered saline or mood-stabilizers. mRNA levels of Bcl-2 were measured using real time RT-PCR. Bar graphs are ratios of optical densities of Bcl-2 to β-actin. Data are expressed as percent of control, and compared using an unpaired t -test, mean ± SEM (*p
    Figure Legend Snippet: Representative mRNA and protein levels of Bcl-2 in frontal cortex from rats chronically administered saline or mood-stabilizers. mRNA levels of Bcl-2 were measured using real time RT-PCR. Bar graphs are ratios of optical densities of Bcl-2 to β-actin. Data are expressed as percent of control, and compared using an unpaired t -test, mean ± SEM (*p

    Techniques Used: Quantitative RT-PCR

    25) Product Images from "Trp63 is regulated by STAT5 in mammary tissue and subject to differentiation in cancer"

    Article Title: Trp63 is regulated by STAT5 in mammary tissue and subject to differentiation in cancer

    Journal: Endocrine-related cancer

    doi: 10.1530/ERC-14-0032

    Expression levels of different Trp63 isoforms and basal markers in mammary carcinomas developing in Brca1 f11/f11/MMTV-Cre/Trp53+/− mice classified as either non-basal or basal (A) Relative expression levels of TA Trp63α (alpha) (2043bp), TA Trp63β (beta) (1664bp). TA Trp63γ (gamma) (1407bp), ΔN Trp63α (alpha) (1761 bp) , ΔN Trp63β (beta) (1382 bp), ΔN Trp63γ (gamma) (1125bp) and Actb (beta actin control) (244bp) in mammary carcinomas classified as non-basal (1–5) or basal (6–10) carcinomas by cDNA array analysis. Size of DNA ladder bands indicated in M (marker) lane. (B) Representative images of histology (H E) and expression patterns of TRP63 and ΔNTRP63 in the same non-basal (1–5) and non-basal carcinomas analyzed in (A). (C) Representative images illustrating expression patterns of cytokeratin 5 (KRT5) and smooth muscle actin (ACTA2) from basal and non-basal carcinomas corresponding to lanes illustrated in panel A. H E: large panels taken at 10X magnification, insets at 40X magnification. Scale bar = 50 μM. TRP63 and ΔNTRP63 panels taken 40X magnification. Scale bar = 10uM. Arrows indicate cells with representative immunohistochemical staining.
    Figure Legend Snippet: Expression levels of different Trp63 isoforms and basal markers in mammary carcinomas developing in Brca1 f11/f11/MMTV-Cre/Trp53+/− mice classified as either non-basal or basal (A) Relative expression levels of TA Trp63α (alpha) (2043bp), TA Trp63β (beta) (1664bp). TA Trp63γ (gamma) (1407bp), ΔN Trp63α (alpha) (1761 bp) , ΔN Trp63β (beta) (1382 bp), ΔN Trp63γ (gamma) (1125bp) and Actb (beta actin control) (244bp) in mammary carcinomas classified as non-basal (1–5) or basal (6–10) carcinomas by cDNA array analysis. Size of DNA ladder bands indicated in M (marker) lane. (B) Representative images of histology (H E) and expression patterns of TRP63 and ΔNTRP63 in the same non-basal (1–5) and non-basal carcinomas analyzed in (A). (C) Representative images illustrating expression patterns of cytokeratin 5 (KRT5) and smooth muscle actin (ACTA2) from basal and non-basal carcinomas corresponding to lanes illustrated in panel A. H E: large panels taken at 10X magnification, insets at 40X magnification. Scale bar = 50 μM. TRP63 and ΔNTRP63 panels taken 40X magnification. Scale bar = 10uM. Arrows indicate cells with representative immunohistochemical staining.

    Techniques Used: Expressing, Mouse Assay, Marker, Immunohistochemistry, Staining

    Immunohistochemical staining of distinct biological phases of post-pubertal mammary gland development and involution Representative images illustrating expression patterns of total TRP63, ΔNTRP63, KRT5 and ss (single-stranded) DNA in mammary gland from nulliparous, P17 (Pregnancy day 17), L1 (Lactation day 1), L10 (Lactation day 10), In1 (Involution day 1), In3 (Involution day 3) and In7 (Involution day 7). Images shown in large panels are small ducts (nulliparous, In7) and alveoli (P17, L1, L10, In1, In3). Images shown in insets are large ducts. TRP63, ΔNTRP63: Arrows point to representative cells with nuclear-localized TRP63 and ΔNTRP63 expression. Closed arrowheads point to representative cells shed into the lumen demonstrating TRP63 and ΔNTRP63 expression. KRT5: Open arrowheads point to representative cells with KRT5 expression. Closed arrowhead points to representative cell shed into the lumen demonstrating KRT5 expression. ss DNA: Arrows point to representative cells with a myoepithelial cell localization with reactivity for ss DNA. Closed arrowheads point to representative cells shed into the lumen with reactivity for ss DNA. Large panels taken at 60x magnification, insets at 20x magnification. Scale bar = 10 μm
    Figure Legend Snippet: Immunohistochemical staining of distinct biological phases of post-pubertal mammary gland development and involution Representative images illustrating expression patterns of total TRP63, ΔNTRP63, KRT5 and ss (single-stranded) DNA in mammary gland from nulliparous, P17 (Pregnancy day 17), L1 (Lactation day 1), L10 (Lactation day 10), In1 (Involution day 1), In3 (Involution day 3) and In7 (Involution day 7). Images shown in large panels are small ducts (nulliparous, In7) and alveoli (P17, L1, L10, In1, In3). Images shown in insets are large ducts. TRP63, ΔNTRP63: Arrows point to representative cells with nuclear-localized TRP63 and ΔNTRP63 expression. Closed arrowheads point to representative cells shed into the lumen demonstrating TRP63 and ΔNTRP63 expression. KRT5: Open arrowheads point to representative cells with KRT5 expression. Closed arrowhead points to representative cell shed into the lumen demonstrating KRT5 expression. ss DNA: Arrows point to representative cells with a myoepithelial cell localization with reactivity for ss DNA. Closed arrowheads point to representative cells shed into the lumen with reactivity for ss DNA. Large panels taken at 60x magnification, insets at 20x magnification. Scale bar = 10 μm

    Techniques Used: Immunohistochemistry, Staining, Expressing

    Immunohistochemical staining of TRP63 and KRT5 in mammary tissue from mice with and without expression of full-length Brca1 Representative images illustrating expression patterns of total TRP63 and KRT5 in mammary gland from nulliparous Brca1 -null ( Brca1 f11/f11/MMTV-Cre/Trp53+/+ ) and WT mice at ages 2.5, 6 and 10 weeks and 4 and 6 months. Images taken at 40X magnification. Scale bar = 25 μm.
    Figure Legend Snippet: Immunohistochemical staining of TRP63 and KRT5 in mammary tissue from mice with and without expression of full-length Brca1 Representative images illustrating expression patterns of total TRP63 and KRT5 in mammary gland from nulliparous Brca1 -null ( Brca1 f11/f11/MMTV-Cre/Trp53+/+ ) and WT mice at ages 2.5, 6 and 10 weeks and 4 and 6 months. Images taken at 40X magnification. Scale bar = 25 μm.

    Techniques Used: Immunohistochemistry, Staining, Mouse Assay, Expressing

    Expression of Trp63 isoforms in primary cell lines established from mouse mammary cancers with mutated Brca1 and Trp53 genes (A) Normalized read coverage across the Trp63 locus viewed through the integrative genomics viewer illustrates the relative expression levels of different exons of the Trp63 gene in primary cancer cells from mice with two intact Brca1 alleles ( Brca1+/+Trp53+/−; Brca1 WT/WT/MMTV-Cre/Trp53+/−/tet-op-CYP19A1/MMTV-rtTA mice), one intact Brca1 allele ( Brca1+/−Trp53+/−; Brca1 f11/WT/MMTV-Cre/Trp53+/− mice), and two disrupted Brca1 alleles ( Brca−/−Trp53+/−; Brca1 f11/f11/MMTV-Cre/Trp53+/− mice) cultured under conditions maintaining epithelial cell differentiation (E) or permissive for epithelial mesenchymal transition (EMT). Note that exons demonstrating expression are all contained within ΔN Trp63 spliced forms. (B) Bar graphs illustrating relative FPKM (Fragments Per Kilobase of transcript per Million mapped) Trp63 reads in the primary cancer cells with varying numbers of Brca1 alleles (A) cultured under conditions favoring epithelial cell differentiation (dark grey) or permissive for EMT (light grey).
    Figure Legend Snippet: Expression of Trp63 isoforms in primary cell lines established from mouse mammary cancers with mutated Brca1 and Trp53 genes (A) Normalized read coverage across the Trp63 locus viewed through the integrative genomics viewer illustrates the relative expression levels of different exons of the Trp63 gene in primary cancer cells from mice with two intact Brca1 alleles ( Brca1+/+Trp53+/−; Brca1 WT/WT/MMTV-Cre/Trp53+/−/tet-op-CYP19A1/MMTV-rtTA mice), one intact Brca1 allele ( Brca1+/−Trp53+/−; Brca1 f11/WT/MMTV-Cre/Trp53+/− mice), and two disrupted Brca1 alleles ( Brca−/−Trp53+/−; Brca1 f11/f11/MMTV-Cre/Trp53+/− mice) cultured under conditions maintaining epithelial cell differentiation (E) or permissive for epithelial mesenchymal transition (EMT). Note that exons demonstrating expression are all contained within ΔN Trp63 spliced forms. (B) Bar graphs illustrating relative FPKM (Fragments Per Kilobase of transcript per Million mapped) Trp63 reads in the primary cancer cells with varying numbers of Brca1 alleles (A) cultured under conditions favoring epithelial cell differentiation (dark grey) or permissive for EMT (light grey).

    Techniques Used: Expressing, Mouse Assay, Cell Culture, Cell Differentiation

    Histone modification and transcription factor binding to the Trp63 locus (A) Available STAT5, PGR, H3K4me2 and H3K4me3 ChIP-seq data of mammary gland and liver were reanalyzed to unveil usage of proximal and distal Trp63 promoters. The seven tracks illustrated are (top to bottom): H3K4me3: MG,L1: H3K4me2: MG,L8; H3K4me2: MECs, nulliparous-diestrus; H3K4me2: Liver, L8; PGR: MG, ovariectomized nulliparous treated E 24 hrs then E+Pr 6 hrs; STAT5a: MG,L1; STAT5b: Liver, nulliparous. (B) Bar graphs illustrating relative FPKM (Fragments Per Kilobase of transcript per Million mapped) reads of Trp63 , TA Trp63 and ΔN Trp63 in mammary glands from genetically engineered STAT5 deficient ( STAT5a −/− STAT5b +/− ) (light grey) and WT ( STAT5a +/+ STAT5b +/+ ) (dark grey) mice at P6 and L1 as estimated by RNA-seq. Signal transducer and activator of transcription (STAT); Progesterone Receptor (PGR); Mammary gland (MG); Mammary epithelial cells (MECs); Gamma interferon-activated sequence (GAS); Progesterone receptor binding element (PRE); Lactation day 1 (L1); Lactation day 8 (L8); Pregnancy day 6 (P6); E (17β-Estradiol,100ng); Pr (Progesterone,2.5ug).
    Figure Legend Snippet: Histone modification and transcription factor binding to the Trp63 locus (A) Available STAT5, PGR, H3K4me2 and H3K4me3 ChIP-seq data of mammary gland and liver were reanalyzed to unveil usage of proximal and distal Trp63 promoters. The seven tracks illustrated are (top to bottom): H3K4me3: MG,L1: H3K4me2: MG,L8; H3K4me2: MECs, nulliparous-diestrus; H3K4me2: Liver, L8; PGR: MG, ovariectomized nulliparous treated E 24 hrs then E+Pr 6 hrs; STAT5a: MG,L1; STAT5b: Liver, nulliparous. (B) Bar graphs illustrating relative FPKM (Fragments Per Kilobase of transcript per Million mapped) reads of Trp63 , TA Trp63 and ΔN Trp63 in mammary glands from genetically engineered STAT5 deficient ( STAT5a −/− STAT5b +/− ) (light grey) and WT ( STAT5a +/+ STAT5b +/+ ) (dark grey) mice at P6 and L1 as estimated by RNA-seq. Signal transducer and activator of transcription (STAT); Progesterone Receptor (PGR); Mammary gland (MG); Mammary epithelial cells (MECs); Gamma interferon-activated sequence (GAS); Progesterone receptor binding element (PRE); Lactation day 1 (L1); Lactation day 8 (L8); Pregnancy day 6 (P6); E (17β-Estradiol,100ng); Pr (Progesterone,2.5ug).

    Techniques Used: Modification, Binding Assay, Chromatin Immunoprecipitation, Mouse Assay, RNA Sequencing Assay, Sequencing

    26) Product Images from "Pathway discovery in mantle cell lymphoma by integrated analysis of high-resolution gene expression and copy number profiling"

    Article Title: Pathway discovery in mantle cell lymphoma by integrated analysis of high-resolution gene expression and copy number profiling

    Journal: Blood

    doi: 10.1182/blood-2010-01-263806

    Kaplan-Meier curves visualizing MCL survival groups according to the mRNA expression of Hippo pathway associated genes in a validation series . mRNA expression of (A) MOBKL2A , (B) MOBKL2B , (C) LATS1 , and (D) LATS2 (cutoff ≤ 25th percentile) in an independent group of 32 untreated MCL patients. Expression was determined by quantitative RT-PCR and the comparative C t method.
    Figure Legend Snippet: Kaplan-Meier curves visualizing MCL survival groups according to the mRNA expression of Hippo pathway associated genes in a validation series . mRNA expression of (A) MOBKL2A , (B) MOBKL2B , (C) LATS1 , and (D) LATS2 (cutoff ≤ 25th percentile) in an independent group of 32 untreated MCL patients. Expression was determined by quantitative RT-PCR and the comparative C t method.

    Techniques Used: Expressing, Quantitative RT-PCR

    27) Product Images from "Regulation of dipeptidyl peptidase IV in the post-stroke rat brain and In vitro ischemia: Implications for chemokine mediated neural progenitor cell migration and angiogenesis"

    Article Title: Regulation of dipeptidyl peptidase IV in the post-stroke rat brain and In vitro ischemia: Implications for chemokine mediated neural progenitor cell migration and angiogenesis

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-016-0039-4

    Characterization of rat brain Neural Progenitor Cells (NPC), analysis of expression of CXCR4 in NPC exposed to in vitro ischemia and re-oxygenation, and effect of DPPIV on NPC migration in vitro. A . Photomicrographs of NPC neurospheres derived from SHR brain showing nestin positivity, and differentiated NPC showing the expression of neuronal marker MAP2 by immunofluorescence staining. B . Expression of CXCR4 mRNA in NPC exposed to 4 hour OGD, and 24 hours re-oxygenation, as shown by semi-quantitative PCR. C . Bar graph showing about 2 fold increase in CXCR4 mRNA in NPCs after OGD/re-oxygenation. Mean ± SD, n = 3. *p
    Figure Legend Snippet: Characterization of rat brain Neural Progenitor Cells (NPC), analysis of expression of CXCR4 in NPC exposed to in vitro ischemia and re-oxygenation, and effect of DPPIV on NPC migration in vitro. A . Photomicrographs of NPC neurospheres derived from SHR brain showing nestin positivity, and differentiated NPC showing the expression of neuronal marker MAP2 by immunofluorescence staining. B . Expression of CXCR4 mRNA in NPC exposed to 4 hour OGD, and 24 hours re-oxygenation, as shown by semi-quantitative PCR. C . Bar graph showing about 2 fold increase in CXCR4 mRNA in NPCs after OGD/re-oxygenation. Mean ± SD, n = 3. *p

    Techniques Used: Expressing, In Vitro, Migration, Derivative Assay, Marker, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

    In vitro oxygen glucose deprivation (OGD) upregulates DPPIV expression. A–B . The effects of OGD on DPPIV protein expression in Neuro-2a cells (A) and rat brain endothelial cells (RBEC) (B) were evaluated after 4 hours OGD, and 2 days of re-oxygenation by immunofluorescence staining. C–D . Up-regulation of DPPIV mRNA expression was confirmed by semi-quantitative PCR assay using DPPIV specific primers, and β-actin primers as internal control for RNA loading. Upregulation of DPPIV mRNA in Neuro-2a (C) and RBEC cells (D) are shown. E–F . Bar graphs indicate 2–3 fold increase in DPPIV mRNA levels in Neuro-2a (E), and RBEC (F) exposed to OGD/re-oxygenation. Mean ± SD, n = 3. * p
    Figure Legend Snippet: In vitro oxygen glucose deprivation (OGD) upregulates DPPIV expression. A–B . The effects of OGD on DPPIV protein expression in Neuro-2a cells (A) and rat brain endothelial cells (RBEC) (B) were evaluated after 4 hours OGD, and 2 days of re-oxygenation by immunofluorescence staining. C–D . Up-regulation of DPPIV mRNA expression was confirmed by semi-quantitative PCR assay using DPPIV specific primers, and β-actin primers as internal control for RNA loading. Upregulation of DPPIV mRNA in Neuro-2a (C) and RBEC cells (D) are shown. E–F . Bar graphs indicate 2–3 fold increase in DPPIV mRNA levels in Neuro-2a (E), and RBEC (F) exposed to OGD/re-oxygenation. Mean ± SD, n = 3. * p

    Techniques Used: In Vitro, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

    Expression of DPPIV is increased in the ischemic cortex after focal ischemia/reperfusion. A . Representative coronal brain sections (ipsilateral and contralateral hemisphere) of SHRs subjected to MCAO and various reperfusion times are shown. The ipsilateral and contralateral cortex stained with DPPIV antibody of ischemic rats at 1 (a and b), 3 (c and d), 7 (e and f), and 14 (g and h) days of ischemia/reperfusion. Significant up-regulation of DPPIV is seen in the ipsilateral cortex after 3 days and it persisted up to day 14. Sham operated control animal showed very low levels of DPPIV expression (i). B. Image of a schematic coronal brain section indicating the control and infarcted area from which the images were taken (square boxes). The dotted line indicates the border of the ischemic core. C . Real time PCR indicating the significant levels of DPPIV mRNA induction at day 3 and 7 of ischemia/reperfusion. Mean ± SD, n = 3. ** p
    Figure Legend Snippet: Expression of DPPIV is increased in the ischemic cortex after focal ischemia/reperfusion. A . Representative coronal brain sections (ipsilateral and contralateral hemisphere) of SHRs subjected to MCAO and various reperfusion times are shown. The ipsilateral and contralateral cortex stained with DPPIV antibody of ischemic rats at 1 (a and b), 3 (c and d), 7 (e and f), and 14 (g and h) days of ischemia/reperfusion. Significant up-regulation of DPPIV is seen in the ipsilateral cortex after 3 days and it persisted up to day 14. Sham operated control animal showed very low levels of DPPIV expression (i). B. Image of a schematic coronal brain section indicating the control and infarcted area from which the images were taken (square boxes). The dotted line indicates the border of the ischemic core. C . Real time PCR indicating the significant levels of DPPIV mRNA induction at day 3 and 7 of ischemia/reperfusion. Mean ± SD, n = 3. ** p

    Techniques Used: Expressing, Staining, Real-time Polymerase Chain Reaction

    28) Product Images from "The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways"

    Article Title: The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/1477-7827-10-58

    Effect of the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 on the expression of c-src mRNA. A , The expression of c-src mRNA (250 bp) in the ovaries by RT-PCR and semiquantitative analysis of the RT-PCR results. B , Real-time PCR was used to analyze the relative levels of c-src mRNA ( c-src mRNA/β-actin mRNA). The data are presented as the means ± SEM (n = 3).
    Figure Legend Snippet: Effect of the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 on the expression of c-src mRNA. A , The expression of c-src mRNA (250 bp) in the ovaries by RT-PCR and semiquantitative analysis of the RT-PCR results. B , Real-time PCR was used to analyze the relative levels of c-src mRNA ( c-src mRNA/β-actin mRNA). The data are presented as the means ± SEM (n = 3).

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

    The silencing efficiency of the lentivirus after transfection. A , c-src mRNA in ovaries 8 days after lentivirus transfection and a semiquantitative assay of the c-src mRNA levels 8 days after lentiviral transfection from six different replicates. B , Real-time PCR was used to analyze the relative c-src mRNA ( c-src mRNA/β-actin mRNA). Blank control corresponds to the group that was transfected with c-src-non-targeting oligonucleotides, negative control corresponds to the group that was transfected with a blank vector (lentivirus without siRNA), and c-src siRNA corresponds to the group with the lentivirus packaging c-src siRNA. The data are presented as the means ± SEM (n = 3). ** P
    Figure Legend Snippet: The silencing efficiency of the lentivirus after transfection. A , c-src mRNA in ovaries 8 days after lentivirus transfection and a semiquantitative assay of the c-src mRNA levels 8 days after lentiviral transfection from six different replicates. B , Real-time PCR was used to analyze the relative c-src mRNA ( c-src mRNA/β-actin mRNA). Blank control corresponds to the group that was transfected with c-src-non-targeting oligonucleotides, negative control corresponds to the group that was transfected with a blank vector (lentivirus without siRNA), and c-src siRNA corresponds to the group with the lentivirus packaging c-src siRNA. The data are presented as the means ± SEM (n = 3). ** P

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Negative Control, Plasmid Preparation

    29) Product Images from "Ceramide Synthases Expression and Role of Ceramide Synthase-2 in the Lung: Insight from Human Lung Cells and Mouse Models"

    Article Title: Ceramide Synthases Expression and Role of Ceramide Synthase-2 in the Lung: Insight from Human Lung Cells and Mouse Models

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062968

    Ceramide levels and CerS expression in the normal lung and human lung cells. A, Levels of ceramide species in the whole mouse lung measured by LC-MS/MS (C57BL/6 mice; female; age 3 months; mean+SEM; n = 5). B, Levels of individual CerS mRNA expressed in the whole mouse lung, measured by real time q-rtPCR; mean+SD, n = 5. C–D, Levels of ceramide species (C) and of CerS mRNA (D) in lung structural cells grown in culture: human bronchial epithelial cell line Beas2B, primary human small airway epithelial cells (SAEC), and primary human microvascular endothelial cells (HLMVEC); means+S.E.M., n = 3. Bar colors of ceramide species corresponding to the color of CerS responsible for its synthesis. E, X-Gal staining (blue) of frozen lung sections from CerS2 −/+ mice at various magnifications (size bar 100 µm in the left panels and 25 µm in the middle and right panels). Note more prominent transcriptional activity of the LacZ-promoter (blue) in the epithelial layers of the bronchi (b), rather than in the vascular (v) endothelium or alveoli (the arrow indicates an alveolar macrophage).
    Figure Legend Snippet: Ceramide levels and CerS expression in the normal lung and human lung cells. A, Levels of ceramide species in the whole mouse lung measured by LC-MS/MS (C57BL/6 mice; female; age 3 months; mean+SEM; n = 5). B, Levels of individual CerS mRNA expressed in the whole mouse lung, measured by real time q-rtPCR; mean+SD, n = 5. C–D, Levels of ceramide species (C) and of CerS mRNA (D) in lung structural cells grown in culture: human bronchial epithelial cell line Beas2B, primary human small airway epithelial cells (SAEC), and primary human microvascular endothelial cells (HLMVEC); means+S.E.M., n = 3. Bar colors of ceramide species corresponding to the color of CerS responsible for its synthesis. E, X-Gal staining (blue) of frozen lung sections from CerS2 −/+ mice at various magnifications (size bar 100 µm in the left panels and 25 µm in the middle and right panels). Note more prominent transcriptional activity of the LacZ-promoter (blue) in the epithelial layers of the bronchi (b), rather than in the vascular (v) endothelium or alveoli (the arrow indicates an alveolar macrophage).

    Techniques Used: Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Activity Assay

    30) Product Images from "Quantitative proteomics identifies a Dab2/integrin module regulating cell migration"

    Article Title: Quantitative proteomics identifies a Dab2/integrin module regulating cell migration

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200812160

    Steady-state surface levels of integrins α1 and β1 but not αv increase in Dab2-deficient cells. (A) Fixed, nonpermeabilized control and Dab2-deficient HeLa cells were analyzed by FACS. (B) Mean and standard error of fluorescent intensity from four independent experiments are shown. (C) FACS analysis of fixed, permeabilized HeLa cells shows that total integrin β1 only increases slightly on Dab2 removal. RT-PCR for integrin β1 and actin mRNA shows equal mRNA levels in control and Dab2-deficient cells. Mean and standard error from five independent experiments are shown. (D) HFFs treated with control or Dab2 siRNA. Western blot showing decreased Dab2 protein and FACS results showing increased surface integrin β1 (mean and standard error of two independent experiments). Data for total integrin β1 level in Dab2-deficient HFFs are shown in Fig. S1 B . (B and D) Dashed lines indicate the control levels. #, P
    Figure Legend Snippet: Steady-state surface levels of integrins α1 and β1 but not αv increase in Dab2-deficient cells. (A) Fixed, nonpermeabilized control and Dab2-deficient HeLa cells were analyzed by FACS. (B) Mean and standard error of fluorescent intensity from four independent experiments are shown. (C) FACS analysis of fixed, permeabilized HeLa cells shows that total integrin β1 only increases slightly on Dab2 removal. RT-PCR for integrin β1 and actin mRNA shows equal mRNA levels in control and Dab2-deficient cells. Mean and standard error from five independent experiments are shown. (D) HFFs treated with control or Dab2 siRNA. Western blot showing decreased Dab2 protein and FACS results showing increased surface integrin β1 (mean and standard error of two independent experiments). Data for total integrin β1 level in Dab2-deficient HFFs are shown in Fig. S1 B . (B and D) Dashed lines indicate the control levels. #, P

    Techniques Used: FACS, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B . (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P
    Figure Legend Snippet: Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B . (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P

    Techniques Used: FACS, Western Blot, Transfection

    31) Product Images from "Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy"

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003550

    The transmembrane domain of FAT1 is required to polarize muscle migration. ( A ) Schemes representing the main protein product expected from a wild type, a Fat1 LacZ , and a Fat1 ΔTM locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. ( B ) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 Fat1 LacZ/LacZ , wild type, and Fat1 ΔTM/ΔTM embryos using indicated antibodies, which targeted epitopes are positioned in ( A ). ( C ) Whole mount LacZ staining of E12.5 Fat1 LacZ/LacZ mutant embryo. ( D ) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 Fat1 ΔTM/ΔTM embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). ( E ) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in Figure S4A ). The profile of Fat1 RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. Fat1 ΔTM/ΔTM embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in Fat1 LacZ/LacZ embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.
    Figure Legend Snippet: The transmembrane domain of FAT1 is required to polarize muscle migration. ( A ) Schemes representing the main protein product expected from a wild type, a Fat1 LacZ , and a Fat1 ΔTM locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. ( B ) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 Fat1 LacZ/LacZ , wild type, and Fat1 ΔTM/ΔTM embryos using indicated antibodies, which targeted epitopes are positioned in ( A ). ( C ) Whole mount LacZ staining of E12.5 Fat1 LacZ/LacZ mutant embryo. ( D ) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 Fat1 ΔTM/ΔTM embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). ( E ) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in Figure S4A ). The profile of Fat1 RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. Fat1 ΔTM/ΔTM embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in Fat1 LacZ/LacZ embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.

    Techniques Used: Migration, Western Blot, Staining, Mutagenesis, In Situ Hybridization, RNA Expression, Expressing

    Fat1 controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration. ( A–C ) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. ( A ) Gdnf-lacZ staining labels myoblasts of the latissimus dorsee (LD) and cutaneous maximus (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. ( B ) At E13.5, MLC3f-lacZ staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. ( C ) Fat1 expression detected using the lacZ gene trap allele KST249 ( Fat1 LacZ ) is selectively localized within the CM and in surrounding tissue (pink arrow). ( D ) CM myoblasts express Fat1 and migrate towards an increasing gradient of Fat1 expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with Fat1 (left column) and MyoD (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of Fat1 staining color in pink (right column; Fat1 in pink, MyoD in purple). MyoD expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of Fat1 expression within and around the cutaneous maximus (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of Fat1 RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of Fat1 staining in this subcutaneous layer increases gradually in caudal sections. ( E–H ) Orientation of CM myoblast migration in whole-mounts of E12.5 Fat1 LacZ/LacZ and control embryos detected using MyoD in situ hybridization. In all panels anterior is to the left, dorsal is to the top. ( E ) The CM muscle (purple dotted line) in Fat1 LacZ/LacZ embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by Fat1 -deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). ( F ) Quantification of the abnormal orientation of Fat1 mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of myoD + nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and Fat1 LacZ/LacZ (black) embryos. ( G, H ) High magnification images of MyoD -expressing myoblasts in equivalent positions – within the chains ( G ) or at the leading edge (migration front, H ) – in the CM of mutants and controls. Scale bars: ( A–C ), 0.8 mm; ( D ) 300 µm; ( E ), left: 0.5 mm; ( E ), right: 100 µm; ( G, H ) 10 µm.
    Figure Legend Snippet: Fat1 controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration. ( A–C ) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. ( A ) Gdnf-lacZ staining labels myoblasts of the latissimus dorsee (LD) and cutaneous maximus (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. ( B ) At E13.5, MLC3f-lacZ staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. ( C ) Fat1 expression detected using the lacZ gene trap allele KST249 ( Fat1 LacZ ) is selectively localized within the CM and in surrounding tissue (pink arrow). ( D ) CM myoblasts express Fat1 and migrate towards an increasing gradient of Fat1 expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with Fat1 (left column) and MyoD (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of Fat1 staining color in pink (right column; Fat1 in pink, MyoD in purple). MyoD expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of Fat1 expression within and around the cutaneous maximus (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of Fat1 RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of Fat1 staining in this subcutaneous layer increases gradually in caudal sections. ( E–H ) Orientation of CM myoblast migration in whole-mounts of E12.5 Fat1 LacZ/LacZ and control embryos detected using MyoD in situ hybridization. In all panels anterior is to the left, dorsal is to the top. ( E ) The CM muscle (purple dotted line) in Fat1 LacZ/LacZ embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by Fat1 -deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). ( F ) Quantification of the abnormal orientation of Fat1 mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of myoD + nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and Fat1 LacZ/LacZ (black) embryos. ( G, H ) High magnification images of MyoD -expressing myoblasts in equivalent positions – within the chains ( G ) or at the leading edge (migration front, H ) – in the CM of mutants and controls. Scale bars: ( A–C ), 0.8 mm; ( D ) 300 µm; ( E ), left: 0.5 mm; ( E ), right: 100 µm; ( G, H ) 10 µm.

    Techniques Used: Migration, Expressing, Staining, Marker, In Situ Hybridization, Mutagenesis

    32) Product Images from "Keap1-Knockdown Decreases Fasting-Induced Fatty Liver via Altered Lipid Metabolism and Decreased Fatty Acid Mobilization from Adipose Tissue"

    Article Title: Keap1-Knockdown Decreases Fasting-Induced Fatty Liver via Altered Lipid Metabolism and Decreased Fatty Acid Mobilization from Adipose Tissue

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079841

    Enhanced Nrf2 activity decreases fatty acid transport and results in increased fatty acids content in white adipose tissue. ( A ) TG and ( B ) FFA content were measured in white adipose tissue of fed and fasted C57BL/6 (WT) and Keap1-KD (KD) mice (n=5-6 per group). ( C ) Nrf2 increased serum glycerol contents in Keap1-KD mice. ( D , E , F ) The induction of Pparα signaling and fatty acid transporter expression by fasting was attenuated in white adipose tissue of Keap1-KD mice. Total RNA was isolated from fed or fasted C57BL/6 (WT) and Keap1-KD (KD) mice and the relative mRNA levels were quantified by quantitative real-time PCR and normalized with β-2 microglobulin as loading control (n=4-6 per group). ( G , H , I ) Immunoblot analysis of Pparα and FATP1 in white adipose tissue from fed or fasted C57BL/6 (WT) and Keap1-KD (KD) mice. *, P
    Figure Legend Snippet: Enhanced Nrf2 activity decreases fatty acid transport and results in increased fatty acids content in white adipose tissue. ( A ) TG and ( B ) FFA content were measured in white adipose tissue of fed and fasted C57BL/6 (WT) and Keap1-KD (KD) mice (n=5-6 per group). ( C ) Nrf2 increased serum glycerol contents in Keap1-KD mice. ( D , E , F ) The induction of Pparα signaling and fatty acid transporter expression by fasting was attenuated in white adipose tissue of Keap1-KD mice. Total RNA was isolated from fed or fasted C57BL/6 (WT) and Keap1-KD (KD) mice and the relative mRNA levels were quantified by quantitative real-time PCR and normalized with β-2 microglobulin as loading control (n=4-6 per group). ( G , H , I ) Immunoblot analysis of Pparα and FATP1 in white adipose tissue from fed or fasted C57BL/6 (WT) and Keap1-KD (KD) mice. *, P

    Techniques Used: Activity Assay, Mouse Assay, Expressing, Isolation, Real-time Polymerase Chain Reaction

    33) Product Images from "New Insights into the RNA-Based Mechanism of Action of the Anticancer Drug 5?-Fluorouracil in Eukaryotic Cells"

    Article Title: New Insights into the RNA-Based Mechanism of Action of the Anticancer Drug 5?-Fluorouracil in Eukaryotic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078172

    Effect of 5FU in tRNA transcripts and in the viability of different tRNA modification mutants. ( A ) Relative levels of intronic or exonic regions within transcripts of 9 selected tRNA genes at both 0 and 240 min of 5FU exposure quantified by qPCR. Genes for tRNA Ala CGC , tRNA Arg CCU and tRNA Gly CCC only have one copy in the genome, whereas multiple identical copies are found for the other tRNA genes examined. In these cases, the amplified products detected represent a mixture of the expression levels of the individual repetitions. The values represent the relative fold change in transcripts abundance between untreated and 5FU-treated cells normalized to the initial amount of starting RNA. Note that only differences between the same group are directly comparable. The expression levels of the normalization gene myo1 were shown as a control. Bar charts show the average values ± s.d. of two independent experiments. Asterisks designate overall significance, * P
    Figure Legend Snippet: Effect of 5FU in tRNA transcripts and in the viability of different tRNA modification mutants. ( A ) Relative levels of intronic or exonic regions within transcripts of 9 selected tRNA genes at both 0 and 240 min of 5FU exposure quantified by qPCR. Genes for tRNA Ala CGC , tRNA Arg CCU and tRNA Gly CCC only have one copy in the genome, whereas multiple identical copies are found for the other tRNA genes examined. In these cases, the amplified products detected represent a mixture of the expression levels of the individual repetitions. The values represent the relative fold change in transcripts abundance between untreated and 5FU-treated cells normalized to the initial amount of starting RNA. Note that only differences between the same group are directly comparable. The expression levels of the normalization gene myo1 were shown as a control. Bar charts show the average values ± s.d. of two independent experiments. Asterisks designate overall significance, * P

    Techniques Used: Modification, Real-time Polymerase Chain Reaction, Countercurrent Chromatography, Amplification, Expressing

    Sensitivity to 5FU of different mutants affected in RNA processing pathways. Strains deleted for genes involved in the processing of mRNA ( A ), tRNA ( B ) or rRNA ( C ) were tested for 5FU sensitivity. Mutants grew at near wild-type rates on liquid medium but exhibited reduced growth rates when 5FU was added to the cultures with the exception of the strain mss1Δ . The growth of the 5FU sensitive strain dus3Δ and the control wild-type (ED668) is also shown. Data are representative of three independent experiments.
    Figure Legend Snippet: Sensitivity to 5FU of different mutants affected in RNA processing pathways. Strains deleted for genes involved in the processing of mRNA ( A ), tRNA ( B ) or rRNA ( C ) were tested for 5FU sensitivity. Mutants grew at near wild-type rates on liquid medium but exhibited reduced growth rates when 5FU was added to the cultures with the exception of the strain mss1Δ . The growth of the 5FU sensitive strain dus3Δ and the control wild-type (ED668) is also shown. Data are representative of three independent experiments.

    Techniques Used:

    Sensitivity to 5FU of different mutants affected in RNA processing pathways. Strains deleted for genes involved in the processing of mRNA ( A ), tRNA ( B ) or rRNA ( C ) were tested for 5FU sensitivity. Mutants grew at near wild-type rates on liquid medium but exhibited reduced growth rates when 5FU was added to the cultures with the exception of the strain mss1Δ . The growth of the 5FU sensitive strain dus3Δ and the control wild-type (ED668) is also shown. Data are representative of three independent experiments.
    Figure Legend Snippet: Sensitivity to 5FU of different mutants affected in RNA processing pathways. Strains deleted for genes involved in the processing of mRNA ( A ), tRNA ( B ) or rRNA ( C ) were tested for 5FU sensitivity. Mutants grew at near wild-type rates on liquid medium but exhibited reduced growth rates when 5FU was added to the cultures with the exception of the strain mss1Δ . The growth of the 5FU sensitive strain dus3Δ and the control wild-type (ED668) is also shown. Data are representative of three independent experiments.

    Techniques Used:

    34) Product Images from "BH3-only protein Noxa contributes to apoptotic control of stress-erythropoiesis"

    Article Title: BH3-only protein Noxa contributes to apoptotic control of stress-erythropoiesis

    Journal: Apoptosis

    doi: 10.1007/s10495-013-0890-y

    Noxa is induced in human hematopoietic progenitors during early erythroid development. a Purified CD34 + progenitors from peripheral blood were differentiated towards erythroblasts in 14 days. FACS analysis of cell cultures shows erythroid differentiation as exemplified by acquisition of CD71 and CD235a and loss of the stem cell marker CD34 in one representative experiment of 4 performed. b Expression profiling by RT-MLPA, reveals induction of Noxa, Bim and Bcl-XL and downregulation of Bcl-2 during erythroid development. Average of 4 independent experiments is shown, expressed as log transformed expression data in relation to day 2 of culture. The number of cells and the yield of RNA at day 0 was too low to allow reproducible analysis. c Protein lysates were prepared from CD34 + cells undergoing erythroid differentiation for 2, 8 and 12 days. Western blot analysis was performed using antibodies against Noxa, Bim, Puma, Bcl-XL, Mcl-1 and Bcl-2. β-Actin is used as loading control. Similar results were obtained in two independent experiments. To compensate for low cell numbers, protein lysates on day 2 were pooled. Asterisks mark non-specific bands. Error bars represent SEM
    Figure Legend Snippet: Noxa is induced in human hematopoietic progenitors during early erythroid development. a Purified CD34 + progenitors from peripheral blood were differentiated towards erythroblasts in 14 days. FACS analysis of cell cultures shows erythroid differentiation as exemplified by acquisition of CD71 and CD235a and loss of the stem cell marker CD34 in one representative experiment of 4 performed. b Expression profiling by RT-MLPA, reveals induction of Noxa, Bim and Bcl-XL and downregulation of Bcl-2 during erythroid development. Average of 4 independent experiments is shown, expressed as log transformed expression data in relation to day 2 of culture. The number of cells and the yield of RNA at day 0 was too low to allow reproducible analysis. c Protein lysates were prepared from CD34 + cells undergoing erythroid differentiation for 2, 8 and 12 days. Western blot analysis was performed using antibodies against Noxa, Bim, Puma, Bcl-XL, Mcl-1 and Bcl-2. β-Actin is used as loading control. Similar results were obtained in two independent experiments. To compensate for low cell numbers, protein lysates on day 2 were pooled. Asterisks mark non-specific bands. Error bars represent SEM

    Techniques Used: Purification, FACS, Marker, Expressing, Multiplex Ligation-dependent Probe Amplification, Transformation Assay, Western Blot

    35) Product Images from "Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults"

    Article Title: Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/S0100-879X2011007500157

    mRNA expression of Frizzled (Fz) receptors ( 1 - 10 ) in PC12 cells by regular RT-PCR. Gel electrophoresis of the PCR product was performed using a 2% ethidium bromide-containing agarose gel and the resulting bands were visualized under UV light. lane 1 : Fz receptor 1; lane 2 : Fz receptor 2; lane 3 : Fz receptor 3; lane 4 : Fz receptor 4; lane 5 : Fz receptor 5; lane 6 : Fz receptor 6; lane 7 : Fz receptor 7; lane 8 : Fz receptor 8; lane 9 : Fz receptor 9; lane 10 : Fz receptor 10.
    Figure Legend Snippet: mRNA expression of Frizzled (Fz) receptors ( 1 - 10 ) in PC12 cells by regular RT-PCR. Gel electrophoresis of the PCR product was performed using a 2% ethidium bromide-containing agarose gel and the resulting bands were visualized under UV light. lane 1 : Fz receptor 1; lane 2 : Fz receptor 2; lane 3 : Fz receptor 3; lane 4 : Fz receptor 4; lane 5 : Fz receptor 5; lane 6 : Fz receptor 6; lane 7 : Fz receptor 7; lane 8 : Fz receptor 8; lane 9 : Fz receptor 9; lane 10 : Fz receptor 10.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    36) Product Images from "Kinetic Characterization of PB1-F2-Mediated Immunopathology during Highly Pathogenic Avian H5N1 Influenza Virus Infection"

    Article Title: Kinetic Characterization of PB1-F2-Mediated Immunopathology during Highly Pathogenic Avian H5N1 Influenza Virus Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057894

    Characterization of the WT and ΔF2 Nig06 HPAIV. (A) Minigenome assays were used to compare polymerase activities of the WT and ΔF2 polymerase complex. Plasmids encoding the NP, PA, PB2 and PB1 or ΔF2-PB1 were co-transfected in 293T cells together with the reporter gene expressing plasmid pPolI-LUC allowing the quantification of polymerase activity. A plasmid encoding the pRSV-β-Gal was also cotransfected to control DNA uptake. Luciferase activity was measured in cell lysates 24 and 48 hours post-transfection. Data are expressed as the mean luciferase activity ± SEM of 3 replicates normalized to β-galactosidase activity. (B) Lysates from MDCK cells mock-infected, infected during 8h and 24h with the wt, ΔF2 or VN04 (A/Vietnam/1203/2004 (H5N1)) viruses (MOI = 5) were analyzed by Western blotting using anti PB1-F2, anti PB1 and anti α-tubulin antibodies. VN04 virus is used as a positive control for antibodies reactivity. Wt and VN04 viruses, but not ΔF2 virus, expressed PB1-F2 around 11 kDa as expected. Two non-specific bands also expressed in mock-infected cells are observed (*, #). The PB1 antibody is directed against the C-terminal part of PB1 and is able to recognize PB1 and N40. Wt and ΔF2 viruses expressed similar amount of PB1, indicating identical expression efficiency. No band corresponding to N40 was observed with every virus, possibly due to a weak expression of N40 which cannot be detected by the PB1 antibody. Alpha-tubulin is used as loading control (C) Lethality induced by WT and ΔF2 Nig06 viruses in C57Bl/6 mice. Mice received intranasally (n = 5) 200 TCID50 of WT or ΔF2 Nig06 virus and were observed every two days for mortality. (D) Time course of M vRNA and IFN-β mRNA expression in the airways of infected mice. Mice were infected with 200 TCID50 of WT or ΔF2 viruses and were euthanized at 2, 4 and 8 days pi. After extraction, lung total RNAs were reverse transcribed and used to quantify viral load and IFN-β transcription. Viral loads were evaluated as M vRNA copies and normalized to the amount of RNA engaged in the reaction; IFN-β mRNA levels were normalized to β-actin mRNA levels and presented as fold increase relative to mock-treated mice.
    Figure Legend Snippet: Characterization of the WT and ΔF2 Nig06 HPAIV. (A) Minigenome assays were used to compare polymerase activities of the WT and ΔF2 polymerase complex. Plasmids encoding the NP, PA, PB2 and PB1 or ΔF2-PB1 were co-transfected in 293T cells together with the reporter gene expressing plasmid pPolI-LUC allowing the quantification of polymerase activity. A plasmid encoding the pRSV-β-Gal was also cotransfected to control DNA uptake. Luciferase activity was measured in cell lysates 24 and 48 hours post-transfection. Data are expressed as the mean luciferase activity ± SEM of 3 replicates normalized to β-galactosidase activity. (B) Lysates from MDCK cells mock-infected, infected during 8h and 24h with the wt, ΔF2 or VN04 (A/Vietnam/1203/2004 (H5N1)) viruses (MOI = 5) were analyzed by Western blotting using anti PB1-F2, anti PB1 and anti α-tubulin antibodies. VN04 virus is used as a positive control for antibodies reactivity. Wt and VN04 viruses, but not ΔF2 virus, expressed PB1-F2 around 11 kDa as expected. Two non-specific bands also expressed in mock-infected cells are observed (*, #). The PB1 antibody is directed against the C-terminal part of PB1 and is able to recognize PB1 and N40. Wt and ΔF2 viruses expressed similar amount of PB1, indicating identical expression efficiency. No band corresponding to N40 was observed with every virus, possibly due to a weak expression of N40 which cannot be detected by the PB1 antibody. Alpha-tubulin is used as loading control (C) Lethality induced by WT and ΔF2 Nig06 viruses in C57Bl/6 mice. Mice received intranasally (n = 5) 200 TCID50 of WT or ΔF2 Nig06 virus and were observed every two days for mortality. (D) Time course of M vRNA and IFN-β mRNA expression in the airways of infected mice. Mice were infected with 200 TCID50 of WT or ΔF2 viruses and were euthanized at 2, 4 and 8 days pi. After extraction, lung total RNAs were reverse transcribed and used to quantify viral load and IFN-β transcription. Viral loads were evaluated as M vRNA copies and normalized to the amount of RNA engaged in the reaction; IFN-β mRNA levels were normalized to β-actin mRNA levels and presented as fold increase relative to mock-treated mice.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Activity Assay, Luciferase, Infection, Western Blot, Positive Control, Mouse Assay

    37) Product Images from "Propofol Reduces Lipopolysaccharide-Induced, NADPH Oxidase (NOX2) Mediated TNF-α and IL-6 Production in Macrophages"

    Article Title: Propofol Reduces Lipopolysaccharide-Induced, NADPH Oxidase (NOX2) Mediated TNF-α and IL-6 Production in Macrophages

    Journal: Clinical and Developmental Immunology

    doi: 10.1155/2013/325481

    Effect of NF- κ B Inhibitor on LPS-induced TNF- α and IL-6 expression. RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 20 μ M pyrrolidine dithiocarbamate (PDTC) for 1 h and stimulated with 100 ng/mL LPS for 8 h. The concentrations of TNF- α and IL-6 in culture supernatants were measured by ELISA. (c) RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 20 μ M PDTC for 1 h and then stimulated with LPS for 2 h. Steady state mRNA levels of TNF- α and IL-6 were examined by RT-PCR. (d) The levels of TNF- α and IL-6 mRNA were quantified by measuring band intensities and shown as fold increase relative to β -actin mRNA levels. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P
    Figure Legend Snippet: Effect of NF- κ B Inhibitor on LPS-induced TNF- α and IL-6 expression. RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 20 μ M pyrrolidine dithiocarbamate (PDTC) for 1 h and stimulated with 100 ng/mL LPS for 8 h. The concentrations of TNF- α and IL-6 in culture supernatants were measured by ELISA. (c) RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 20 μ M PDTC for 1 h and then stimulated with LPS for 2 h. Steady state mRNA levels of TNF- α and IL-6 were examined by RT-PCR. (d) The levels of TNF- α and IL-6 mRNA were quantified by measuring band intensities and shown as fold increase relative to β -actin mRNA levels. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P

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

    Effect of propofol on LPS-induced TNF- α and IL-6 expression. (a) RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 50 μ M propofol for 40 min and then stimulated with LPS for 2 h. Steady state mRNA levels of TNF- α and IL-6 were examined by RT-PCR. (b) The levels of TNF- α and IL-6 mRNA were quantified by measuring band intensities and shown as fold increase relative to β -actin mRNA levels. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P
    Figure Legend Snippet: Effect of propofol on LPS-induced TNF- α and IL-6 expression. (a) RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 50 μ M propofol for 40 min and then stimulated with LPS for 2 h. Steady state mRNA levels of TNF- α and IL-6 were examined by RT-PCR. (b) The levels of TNF- α and IL-6 mRNA were quantified by measuring band intensities and shown as fold increase relative to β -actin mRNA levels. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    38) Product Images from "Pre-conditioning with synthetic CpG-oligonucleotides attenuates myocardial ischemia/reperfusion injury via IL-10 up-regulation"

    Article Title: Pre-conditioning with synthetic CpG-oligonucleotides attenuates myocardial ischemia/reperfusion injury via IL-10 up-regulation

    Journal: Basic Research in Cardiology

    doi: 10.1007/s00395-013-0376-7

    Cytokine ( a – e TNF-α, IL-1β, IL-6, IL-10, IL-12) and IFNγ ( f ) protein in blood serum measured at 2, 4, 6 and 16 h after priming with 1668-thioate, H154-thioate and 1612-thioate. All investigated mediators were up-regulated during the priming phase by 1668-thioate stimulation, whereas the other CpG-ODNs did not affect protein levels in the serum ( n = 5/group, * p
    Figure Legend Snippet: Cytokine ( a – e TNF-α, IL-1β, IL-6, IL-10, IL-12) and IFNγ ( f ) protein in blood serum measured at 2, 4, 6 and 16 h after priming with 1668-thioate, H154-thioate and 1612-thioate. All investigated mediators were up-regulated during the priming phase by 1668-thioate stimulation, whereas the other CpG-ODNs did not affect protein levels in the serum ( n = 5/group, * p

    Techniques Used:

    39) Product Images from "Loss of the AE3 Cl−/HCO−3 exchanger in mice affects rate-dependent inotropy and stress-related AKT signaling in heart"

    Article Title: Loss of the AE3 Cl−/HCO−3 exchanger in mice affects rate-dependent inotropy and stress-related AKT signaling in heart

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00399

    Expression of HCO − 3 transporters in AE3-null hearts . RT-PCR analysis of cDNA generated from total RNA of WT and AE3-null (KO) hearts was carried out to determine mRNA levels the Slc4a4 and Slc4a7 Na + /HCO − 3 cotransporters and Slc26a6 , encoding the anion exchanger PAT1. When normalized to Gapdh levels, expression of Slc4a4 was downregulated in AE3-null hearts (A) , while Slc4a7 (B) and Sl26a6 (C) levels showed no change. Total cardiac homogenates of WT and KO hearts were subjected to immunoblot and densitometric analyses as described in Figure 3 ; results show that NBCe1 protein expression, when normalized to levels of sarcomeric actin (s.actin) was reduced in AE3-null hearts (D,E) . Values shown are mean ± S.E. n = at least 10 mice of each genotype for RT-PCR analysis and at least 7 mice of each genotype for immunoblot analysis. * p
    Figure Legend Snippet: Expression of HCO − 3 transporters in AE3-null hearts . RT-PCR analysis of cDNA generated from total RNA of WT and AE3-null (KO) hearts was carried out to determine mRNA levels the Slc4a4 and Slc4a7 Na + /HCO − 3 cotransporters and Slc26a6 , encoding the anion exchanger PAT1. When normalized to Gapdh levels, expression of Slc4a4 was downregulated in AE3-null hearts (A) , while Slc4a7 (B) and Sl26a6 (C) levels showed no change. Total cardiac homogenates of WT and KO hearts were subjected to immunoblot and densitometric analyses as described in Figure 3 ; results show that NBCe1 protein expression, when normalized to levels of sarcomeric actin (s.actin) was reduced in AE3-null hearts (D,E) . Values shown are mean ± S.E. n = at least 10 mice of each genotype for RT-PCR analysis and at least 7 mice of each genotype for immunoblot analysis. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Mouse Assay

    40) Product Images from "A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics"

    Article Title: A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics

    Journal: eLife

    doi: 10.7554/eLife.00762

    ( A ) Western analysis of RelA protein levels. ( B ) PolyA+ Lethe is found on the chromatin. Immunoblot of wildtype and RelA−/− MEFs. Cellular fractionation was performed, total RNA was purified and polyA+ selection was performed. The fraction polyA+ RNA found in the chromatin, nucleus and cytoplasm is shown. MEFs were treated with 20 ng/ml TNFα for 6 hr. Quantitative Taqman real time RT-PCR of the indicated RNAs is shown (mean ± SD is shown). DOI: http://dx.doi.org/10.7554/eLife.00762.006
    Figure Legend Snippet: ( A ) Western analysis of RelA protein levels. ( B ) PolyA+ Lethe is found on the chromatin. Immunoblot of wildtype and RelA−/− MEFs. Cellular fractionation was performed, total RNA was purified and polyA+ selection was performed. The fraction polyA+ RNA found in the chromatin, nucleus and cytoplasm is shown. MEFs were treated with 20 ng/ml TNFα for 6 hr. Quantitative Taqman real time RT-PCR of the indicated RNAs is shown (mean ± SD is shown). DOI: http://dx.doi.org/10.7554/eLife.00762.006

    Techniques Used: Western Blot, Cell Fractionation, Purification, Selection, Quantitative RT-PCR

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    Article Snippet: .. MTT assay To evaluate the effect of DMSO on cell viability, Vybrant® MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Proliferation Assay (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. ..

    Transfection:

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    Mutagenesis:

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    Isolation:

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    Article Snippet: .. RNA isolation and quantitative real-time PCR analysis A mir Vana miRNA Isolation kit was used for isolation of total RNA (Ambion/Applied Biosystems). .. Specific single TaqMan miRNA assays (Ambion/Applied Biosystems) were used to measure the expression levels of selected miRNA in a model light cycler 1.5 (Roche Diagnostics).

    Article Title: Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia
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    Infection:

    Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
    Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

    Real-time Polymerase Chain Reaction:

    Article Title: Osteogenic differentiation of fibroblast-like synovial cells in rheumatoid arthritis is induced by microRNA-218 through a ROBO/Slit pathway
    Article Snippet: .. RNA isolation and quantitative real-time PCR analysis A mir Vana miRNA Isolation kit was used for isolation of total RNA (Ambion/Applied Biosystems). .. Specific single TaqMan miRNA assays (Ambion/Applied Biosystems) were used to measure the expression levels of selected miRNA in a model light cycler 1.5 (Roche Diagnostics).

    Sequencing:

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma
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    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
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    other:

    Article Title: Inflammasome Proteins in Serum and Serum-Derived Extracellular Vesicles as Biomarkers of Stroke
    Article Snippet: However, the ExoQuick method was able to isolate EV with higher levels of IL-1β than the Invitrogen method (Supplementary Figure ).

    Proliferation Assay:

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    Quantitative RT-PCR:

    Article Title: Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia
    Article Snippet: .. RNA isolation and quantitative reverse transcription (qRT)-PCR To determine gene expression at the mRNA level, pneumococci in the logarithmic phase of growth (OD550 = 0.30) were exposed to the serum or human LF (6 mM and 30 mM) for the specified time periods, and the total RNA was isolated using the Trizol method (Invitrogen, USA). .. The isolated RNA (1–2 µg) was used as a template for preparing cDNA, using the manufacturer’s instructions (Enzymonics, Korea).

    Expressing:

    Article Title: Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia
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    High Throughput Screening Assay:

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    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Plasmid Preparation:

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    Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

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  • 99
    Thermo Fisher rna
    Two-stage laser model induces fibrotic gene expression. <t>RNA</t> was extracted from retina (A, C) and <t>RPE/choroid</t> (B, D) 25 days after laser/second laser and gene expression was examined by RT-PCR. (A, B) The expression of profibrotic growth factor genes (TGF-β, FGF2, and VEGF-a) in the retina and RPE/choroid. (C, D) The expression of fibrosis marker genes (COL-1, fibronectin, and α-smooth muscle actin). Mean ± SEM, N = 6, one-way ANOVA, Bonferroni corrected, * P
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/Thermo Fisher
    Average 99 stars, based on 3653 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-09
    99/100 stars
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    92
    Thermo Fisher mmp 1 mrna expression levels total rna
    colIα1 and <t>mmp-1</t> <t>mRNA</t> expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p
    Mmp 1 Mrna Expression Levels Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mmp 1 mrna expression levels total rna/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
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    mmp 1 mrna expression levels total rna - by Bioz Stars, 2020-09
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    92
    Thermo Fisher mrna levels
    colIα1 and <t>mmp-1</t> <t>mRNA</t> expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p
    Mrna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna levels/product/Thermo Fisher
    Average 92 stars, based on 1120 article reviews
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    Two-stage laser model induces fibrotic gene expression. RNA was extracted from retina (A, C) and RPE/choroid (B, D) 25 days after laser/second laser and gene expression was examined by RT-PCR. (A, B) The expression of profibrotic growth factor genes (TGF-β, FGF2, and VEGF-a) in the retina and RPE/choroid. (C, D) The expression of fibrosis marker genes (COL-1, fibronectin, and α-smooth muscle actin). Mean ± SEM, N = 6, one-way ANOVA, Bonferroni corrected, * P

    Journal: Translational Vision Science & Technology

    Article Title: A Two-Stage Laser-Induced Mouse Model of Subretinal Fibrosis Secondary to Choroidal Neovascularization

    doi: 10.1167/tvst.9.4.3

    Figure Lengend Snippet: Two-stage laser model induces fibrotic gene expression. RNA was extracted from retina (A, C) and RPE/choroid (B, D) 25 days after laser/second laser and gene expression was examined by RT-PCR. (A, B) The expression of profibrotic growth factor genes (TGF-β, FGF2, and VEGF-a) in the retina and RPE/choroid. (C, D) The expression of fibrosis marker genes (COL-1, fibronectin, and α-smooth muscle actin). Mean ± SEM, N = 6, one-way ANOVA, Bonferroni corrected, * P

    Article Snippet: Baseline levels of gene expression were tested in RNA from retina and RPE/choroid from 3-month-old C57Bl6 mice (untreated) n = 6 eyes. cDNA was generated using a reverse transcriptase kit (Thermofisher).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker

    colIα1 and mmp-1 mRNA expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p

    Journal: Arthritis Research & Therapy

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    doi: 10.1186/s13075-018-1689-6

    Figure Lengend Snippet: colIα1 and mmp-1 mRNA expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1 ; b mmp-1 ; c Pro-CollagenIα1; d MMP-2. * p

    Article Snippet: tlr2 , tlr3 , tlr4 , tlr7 , tlr8 , tlr9 , interferon-α , interferon-β , endothelin-1 , collagenIα1 and mmp-1 mRNA expression levels Total RNA from fibroblasts was purified using Trizol Reagent (ThermoFisher Scientific).

    Techniques: Expressing, Papanicolaou Stain, Positive Control

    TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

    Journal: Arthritis Research & Therapy

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    doi: 10.1186/s13075-018-1689-6

    Figure Lengend Snippet: TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

    Article Snippet: tlr2 , tlr3 , tlr4 , tlr7 , tlr8 , tlr9 , interferon-α , interferon-β , endothelin-1 , collagenIα1 and mmp-1 mRNA expression levels Total RNA from fibroblasts was purified using Trizol Reagent (ThermoFisher Scientific).

    Techniques: Expressing, Positive Control