β tubulin detection  (Millipore)


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    Structured Review

    Millipore β tubulin detection
    Reduction of CENPA overexpression by RNAi in pRb depleted cells . A) Western blot showing CENP-A and CENP-F protein levels in HCT116 cells, wild type (HCT, lane 1), p53-knockout (HCTp53KO, lane 3) and after pRb acute loss (siRB+, lanes 2 and 4), <t>β-tubulin</t> was used as a loading control. B) Real-time RT-PCR showed CENPA decreased transcript levels after simultaneous RB/CENPA post-transcriptional silencing (siRB/CenpA), in comparison to CENPA expression in RB-depleted cells (siRB). RB transcripts were reduced in both pRb- and pRb/CenpA- depleted cells. Untransfected HCT116 cells were used as a calibrator. C) Western blot showing reduction of CENP-A protein levels, in comparison to CENPA expression in RB-depleted cells (siRB+), in cells simultaneously transfected with siRNAs specific for both RB and CENPA (lane 3). Protein extracts of HCT116 wild type and pRb-depleted cells were loaded in lanes 1 and 2 respectively. β-tubulin was used as a loading control. D) Real-time RT-PCR showing CENPA transcript levels in HCT116 cells transfected with a control siRNA targeting GFP (siGFP) and after transfection of siRNAs targeting CENPA (two different doses siCenpA 60 nM, siCenpA 80 nM). E) Western blot showing reduction of CENP-A protein levels after CenpA post-transcriptional silencing (lane 3: 60 nM, lane 4: 80 nM). Normal levels of CENPA were present in untransfected cells (lane 1) and in cells transfected with a control siRNA (lane 2: siGFP).
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    Images

    1) Product Images from "CENPA overexpression promotes genome instability in pRb-depleted human cells"

    Article Title: CENPA overexpression promotes genome instability in pRb-depleted human cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-8-119

    Reduction of CENPA overexpression by RNAi in pRb depleted cells . A) Western blot showing CENP-A and CENP-F protein levels in HCT116 cells, wild type (HCT, lane 1), p53-knockout (HCTp53KO, lane 3) and after pRb acute loss (siRB+, lanes 2 and 4), β-tubulin was used as a loading control. B) Real-time RT-PCR showed CENPA decreased transcript levels after simultaneous RB/CENPA post-transcriptional silencing (siRB/CenpA), in comparison to CENPA expression in RB-depleted cells (siRB). RB transcripts were reduced in both pRb- and pRb/CenpA- depleted cells. Untransfected HCT116 cells were used as a calibrator. C) Western blot showing reduction of CENP-A protein levels, in comparison to CENPA expression in RB-depleted cells (siRB+), in cells simultaneously transfected with siRNAs specific for both RB and CENPA (lane 3). Protein extracts of HCT116 wild type and pRb-depleted cells were loaded in lanes 1 and 2 respectively. β-tubulin was used as a loading control. D) Real-time RT-PCR showing CENPA transcript levels in HCT116 cells transfected with a control siRNA targeting GFP (siGFP) and after transfection of siRNAs targeting CENPA (two different doses siCenpA 60 nM, siCenpA 80 nM). E) Western blot showing reduction of CENP-A protein levels after CenpA post-transcriptional silencing (lane 3: 60 nM, lane 4: 80 nM). Normal levels of CENPA were present in untransfected cells (lane 1) and in cells transfected with a control siRNA (lane 2: siGFP).
    Figure Legend Snippet: Reduction of CENPA overexpression by RNAi in pRb depleted cells . A) Western blot showing CENP-A and CENP-F protein levels in HCT116 cells, wild type (HCT, lane 1), p53-knockout (HCTp53KO, lane 3) and after pRb acute loss (siRB+, lanes 2 and 4), β-tubulin was used as a loading control. B) Real-time RT-PCR showed CENPA decreased transcript levels after simultaneous RB/CENPA post-transcriptional silencing (siRB/CenpA), in comparison to CENPA expression in RB-depleted cells (siRB). RB transcripts were reduced in both pRb- and pRb/CenpA- depleted cells. Untransfected HCT116 cells were used as a calibrator. C) Western blot showing reduction of CENP-A protein levels, in comparison to CENPA expression in RB-depleted cells (siRB+), in cells simultaneously transfected with siRNAs specific for both RB and CENPA (lane 3). Protein extracts of HCT116 wild type and pRb-depleted cells were loaded in lanes 1 and 2 respectively. β-tubulin was used as a loading control. D) Real-time RT-PCR showing CENPA transcript levels in HCT116 cells transfected with a control siRNA targeting GFP (siGFP) and after transfection of siRNAs targeting CENPA (two different doses siCenpA 60 nM, siCenpA 80 nM). E) Western blot showing reduction of CENP-A protein levels after CenpA post-transcriptional silencing (lane 3: 60 nM, lane 4: 80 nM). Normal levels of CENPA were present in untransfected cells (lane 1) and in cells transfected with a control siRNA (lane 2: siGFP).

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

    pRb depleted cells overrode mitosis even when chromosome mis-segregation occurred . A) Immunodetection of β-tubulin (green) in pRb depleted cells, wild-type (top) and p53-KO (bottom), showing mitotic defects (white arrows). Nuclei were counterstained with DAPI (blue). B) Centromere immunostaining with a CREST antibody detected the presence of centromeres (green) in micronuclei (white arrow) after pRb acute loss in both HCT116 wild-type (top) and p53-KO (bottom) cells. Nuclei were counterstained with DAPI (blue). C) Graph showing the percentage of HCT116 cells with 1, 2 or more than 2 centrosomes at 72 hours post-transfection of siRNAs targeting p53 , alone or in combination with siRNAs targeting RB . D) Graph summarizing the percentages of diploid (2N), hypodiploid (
    Figure Legend Snippet: pRb depleted cells overrode mitosis even when chromosome mis-segregation occurred . A) Immunodetection of β-tubulin (green) in pRb depleted cells, wild-type (top) and p53-KO (bottom), showing mitotic defects (white arrows). Nuclei were counterstained with DAPI (blue). B) Centromere immunostaining with a CREST antibody detected the presence of centromeres (green) in micronuclei (white arrow) after pRb acute loss in both HCT116 wild-type (top) and p53-KO (bottom) cells. Nuclei were counterstained with DAPI (blue). C) Graph showing the percentage of HCT116 cells with 1, 2 or more than 2 centrosomes at 72 hours post-transfection of siRNAs targeting p53 , alone or in combination with siRNAs targeting RB . D) Graph summarizing the percentages of diploid (2N), hypodiploid (

    Techniques Used: Immunodetection, Immunostaining, Transfection

    pRb acute loss induced differential expression of several centrosome and mitotic genes . A) Real-time RT-PCR showed increased expression levels of genes involved in centrosome duplication ( PLK1, AURKA, and CYCE ), as well in the SAC and mitosis ( BRCA1, PTTG1, CDC20, BUBR1, MAD2, CENPA, and CENPF ) after pRb acute loss in both HCT116wt (HCT) and p53-knockout (HCTp53KO) cells. The x-axis indicates the genes and the y-axis the relative quantification in pRb-depleted cells in respect to the gene expression level in control cells, (untransfected HCT116wt and HCT116p53KO were used as calibrator). B) Western blot showing Mad2, AurkA, Plk1, p53 and BRCA1 protein levels in HCT116 cells both wild-type (HCT, lane 1) and p53-knockout (HCTp53KO, lane 3) and after pRb acute loss (siRB+, lane 2 and lane 4 respectively). β-tubulin was used as a loading control. C) Real-time RT-PCR showing that changes in MAD2 transcript depended on BRCA1 gene expression levels. BRCA1 transcripts were reduced more than 80% at 72 hours post-transfection of siRNAs targeting BRCA1 (siBrca1 60 nM). Modulation of BRCA1 transcript levels in RB-depleted cells using different siRNA concentration (50 nM and 60 nM) reduced MAD2 expression accordingly. D) Graph showing mitotic indices in both wild type and p53-knockout HCT116 cells after pRb acute loss (siRB) and in released cells in comparison with untransfected cells (untr). E) Western blot showing similar BubR1 protein levels in untransfected cells (lanes 1 and 4) and in RB depleted cells (lanes 3 and 6). As expected HCT116 and HCTp53KO cell types after colcemid treatment (lanes 2 and 5) showed increase in BubR1 protein levels.
    Figure Legend Snippet: pRb acute loss induced differential expression of several centrosome and mitotic genes . A) Real-time RT-PCR showed increased expression levels of genes involved in centrosome duplication ( PLK1, AURKA, and CYCE ), as well in the SAC and mitosis ( BRCA1, PTTG1, CDC20, BUBR1, MAD2, CENPA, and CENPF ) after pRb acute loss in both HCT116wt (HCT) and p53-knockout (HCTp53KO) cells. The x-axis indicates the genes and the y-axis the relative quantification in pRb-depleted cells in respect to the gene expression level in control cells, (untransfected HCT116wt and HCT116p53KO were used as calibrator). B) Western blot showing Mad2, AurkA, Plk1, p53 and BRCA1 protein levels in HCT116 cells both wild-type (HCT, lane 1) and p53-knockout (HCTp53KO, lane 3) and after pRb acute loss (siRB+, lane 2 and lane 4 respectively). β-tubulin was used as a loading control. C) Real-time RT-PCR showing that changes in MAD2 transcript depended on BRCA1 gene expression levels. BRCA1 transcripts were reduced more than 80% at 72 hours post-transfection of siRNAs targeting BRCA1 (siBrca1 60 nM). Modulation of BRCA1 transcript levels in RB-depleted cells using different siRNA concentration (50 nM and 60 nM) reduced MAD2 expression accordingly. D) Graph showing mitotic indices in both wild type and p53-knockout HCT116 cells after pRb acute loss (siRB) and in released cells in comparison with untransfected cells (untr). E) Western blot showing similar BubR1 protein levels in untransfected cells (lanes 1 and 4) and in RB depleted cells (lanes 3 and 6). As expected HCT116 and HCTp53KO cell types after colcemid treatment (lanes 2 and 5) showed increase in BubR1 protein levels.

    Techniques Used: Expressing, Quantitative RT-PCR, Knock-Out, Western Blot, Transfection, Concentration Assay

    Supernumerary centrosomes and chromosomal instability promoted by pRb acute loss were not affected by p53 status . A) RT-PCR (top panel) and Western blot (bottom panel) showing RB mRNA and protein levels, respectively, at 72 hours post-transfection in both wild type and p53 knockout HCT116 cells. RT-PCR revealed the presence of RB mRNA in both untransfected cell lines, (lane 2: HCT116-wt, lane 4: HCT116p53KO) but not in RB -depleted cells (lane 3: HCT116-wt, lane 5: HCT116p53KO). Amplification of GAPDH (330 bp) was used as control of the quality of the cDNA. The 100 bp DNA-ladder was loaded in lane 1 as a size marker. Western blot confirmed selective depletion of pRb in siRNA-transfected cells (lane 2: HCT116-wt, lane 4: HCT116p53KO) in comparison with untransfected cells (lane 1: HCT116-wt, lane 3: HCT116p53KO). β-tubulin was used as a loading control. B) Graphs (top panels) showing percentage of cells with 1, 2 or more than 2 ( > 2) centrosomes in untransfected (untr.), RB -depleted (siRB) and released (release) HCT116-wt and HCT116p53KO. Presence of supernumerary centrosomes (bottom panels, white arrow) detected by γ-tubulin immunostaining (green) in both HCT116-wt and HCT116p53KO cells after pRb acute loss (a, b, respectively). A magnification for each cell type is reported on the right. Nuclei were stained with DAPI (blue). C) Representative pictures of diploid (2N), hypodiploid (
    Figure Legend Snippet: Supernumerary centrosomes and chromosomal instability promoted by pRb acute loss were not affected by p53 status . A) RT-PCR (top panel) and Western blot (bottom panel) showing RB mRNA and protein levels, respectively, at 72 hours post-transfection in both wild type and p53 knockout HCT116 cells. RT-PCR revealed the presence of RB mRNA in both untransfected cell lines, (lane 2: HCT116-wt, lane 4: HCT116p53KO) but not in RB -depleted cells (lane 3: HCT116-wt, lane 5: HCT116p53KO). Amplification of GAPDH (330 bp) was used as control of the quality of the cDNA. The 100 bp DNA-ladder was loaded in lane 1 as a size marker. Western blot confirmed selective depletion of pRb in siRNA-transfected cells (lane 2: HCT116-wt, lane 4: HCT116p53KO) in comparison with untransfected cells (lane 1: HCT116-wt, lane 3: HCT116p53KO). β-tubulin was used as a loading control. B) Graphs (top panels) showing percentage of cells with 1, 2 or more than 2 ( > 2) centrosomes in untransfected (untr.), RB -depleted (siRB) and released (release) HCT116-wt and HCT116p53KO. Presence of supernumerary centrosomes (bottom panels, white arrow) detected by γ-tubulin immunostaining (green) in both HCT116-wt and HCT116p53KO cells after pRb acute loss (a, b, respectively). A magnification for each cell type is reported on the right. Nuclei were stained with DAPI (blue). C) Representative pictures of diploid (2N), hypodiploid (

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Knock-Out, Amplification, Marker, Immunostaining, Staining

    2) Product Images from "PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells"

    Article Title: PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells

    Journal: Scientific Reports

    doi: 10.1038/srep25268

    miR-29b targeting of PATZ1 in rat thyroid cells. ( a ) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( b ) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P
    Figure Legend Snippet: miR-29b targeting of PATZ1 in rat thyroid cells. ( a ) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( b ) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P

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

    Validation of PATZ1 as a target of miR-29b. ( a ) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. ( b ) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( c ) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. ( d ) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P
    Figure Legend Snippet: Validation of PATZ1 as a target of miR-29b. ( a ) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. ( b ) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( c ) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. ( d ) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P

    Techniques Used: Western Blot, Transfection, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay

    3) Product Images from "Changes in Parasite Virulence Induced by the Disruption of a Single Member of the 235 kDa Rhoptry Protein Multigene Family of Plasmodium yoelii"

    Article Title: Changes in Parasite Virulence Induced by the Disruption of a Single Member of the 235 kDa Rhoptry Protein Multigene Family of Plasmodium yoelii

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020170

    Comparison of growth behavior of YM and PYΔpy01365. A- Parasitaemia of BALB/c mice infected with 10 4 parasites on day 0 was taken daily. The average parasitaemia of 5 mice for both YM and PYΔpy01365 is represented. Error bars are given for each time point. † Indicates death of animals. B- Average Selective index of 5 BALB/c mice infected with either YM or PYΔpy01365. Parasites smears were analyzed when parasitaemia was in the range of 5–15%. Differences in SI between YM and PYΔpy01365 were significant (p
    Figure Legend Snippet: Comparison of growth behavior of YM and PYΔpy01365. A- Parasitaemia of BALB/c mice infected with 10 4 parasites on day 0 was taken daily. The average parasitaemia of 5 mice for both YM and PYΔpy01365 is represented. Error bars are given for each time point. † Indicates death of animals. B- Average Selective index of 5 BALB/c mice infected with either YM or PYΔpy01365. Parasites smears were analyzed when parasitaemia was in the range of 5–15%. Differences in SI between YM and PYΔpy01365 were significant (p

    Techniques Used: Mouse Assay, Infection

    4) Product Images from "Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition"

    Article Title: Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

    Journal: Scientific Reports

    doi: 10.1038/srep32888

    ( a ) Stage-specific protein expressions of hiPSC-EBs during the differentiation process. SOX17 and FOXA2 for the definitive endoderm stage; HHEX and GATA4 for the foregut endoderm; AFP and HNF-4α for the hepatic progenitor cells; ALBUMIN and CK-18 for the mature HLCs. DAPI stains for cell nuclei. Scale bar 100 μm. ( b ) Stage-specific gene expression analysis by Real-Time PCR. The relative quantities of stage-specific genes were measured at the mRNA level to follow the progression of the differentiation process. Sox17 as the definitive endoderm marker; Gata4 as the foregut endoderm marker; HNF-4α as the hepatic progenitor cells marker; Albumin was used to determine the final maturation for the hepatocyte-like cells (HLCs). Undifferentiated cells were used as negative control. ( c ) quantitative RT-PCR displayed the presence of mRNA for AFP, five P450 isoforms (Cyp3A4, Cyp2C9, Cyp3A7, Cyp1B1, and Cyp2B6), Albumin, and CK18 in the terminally differentiated hiPSC-EB-HLCs with and without inhibitors. Gene expression for the condition with inhibitors was greater compared with the one without inhibitors for any gene tested; ( d,e ) Following the differentiation program, terminally differentiated hiPSC-EB-HLCs expressed mature hepatocyte-specific markers, as evidenced by co-staining of ALBUMIN and HNF-1α, and ALBUMIN and C-MET. Scale bar 100 μm. ( f ) FACS analysis for albumin positive cells showed a higher percentage of albumin producing cells in the condition with inhibitors compared with the one without inhibitors (80% vs 68%).
    Figure Legend Snippet: ( a ) Stage-specific protein expressions of hiPSC-EBs during the differentiation process. SOX17 and FOXA2 for the definitive endoderm stage; HHEX and GATA4 for the foregut endoderm; AFP and HNF-4α for the hepatic progenitor cells; ALBUMIN and CK-18 for the mature HLCs. DAPI stains for cell nuclei. Scale bar 100 μm. ( b ) Stage-specific gene expression analysis by Real-Time PCR. The relative quantities of stage-specific genes were measured at the mRNA level to follow the progression of the differentiation process. Sox17 as the definitive endoderm marker; Gata4 as the foregut endoderm marker; HNF-4α as the hepatic progenitor cells marker; Albumin was used to determine the final maturation for the hepatocyte-like cells (HLCs). Undifferentiated cells were used as negative control. ( c ) quantitative RT-PCR displayed the presence of mRNA for AFP, five P450 isoforms (Cyp3A4, Cyp2C9, Cyp3A7, Cyp1B1, and Cyp2B6), Albumin, and CK18 in the terminally differentiated hiPSC-EB-HLCs with and without inhibitors. Gene expression for the condition with inhibitors was greater compared with the one without inhibitors for any gene tested; ( d,e ) Following the differentiation program, terminally differentiated hiPSC-EB-HLCs expressed mature hepatocyte-specific markers, as evidenced by co-staining of ALBUMIN and HNF-1α, and ALBUMIN and C-MET. Scale bar 100 μm. ( f ) FACS analysis for albumin positive cells showed a higher percentage of albumin producing cells in the condition with inhibitors compared with the one without inhibitors (80% vs 68%).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Marker, Negative Control, Quantitative RT-PCR, Staining, FACS

    ( a ) Ammonium metabolism assay over a 24-hour period for both conditions with and without inhibitors; Cytochrome P450 (CYP450) induction analysis comparing the two experimental conditions with and without inhibitors. Several CYP enzymes were evaluated through incubation of the cells with different inducers: ( b ) Phenobarbital for the CYP2B6, ( c ) Rifampicin for the CYP3A4 and ( d ) Omeprazole for the CYP1A2 for a period of 72 hours. DMSO was used as control to test the basal activity of different CYP450. Data presented as mean ± SD (n = 3). *p
    Figure Legend Snippet: ( a ) Ammonium metabolism assay over a 24-hour period for both conditions with and without inhibitors; Cytochrome P450 (CYP450) induction analysis comparing the two experimental conditions with and without inhibitors. Several CYP enzymes were evaluated through incubation of the cells with different inducers: ( b ) Phenobarbital for the CYP2B6, ( c ) Rifampicin for the CYP3A4 and ( d ) Omeprazole for the CYP1A2 for a period of 72 hours. DMSO was used as control to test the basal activity of different CYP450. Data presented as mean ± SD (n = 3). *p

    Techniques Used: Incubation, Activity Assay

    5) Product Images from "Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses"

    Article Title: Concurrent Overexpression of OsGS1;1 and OsGS2 Genes in Transgenic Rice (Oryza sativa L.): Impact on Tolerance to Abiotic Stresses

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00786

    Assessment of tolerance of OsGS1;1/OsGS2 co-overexpressing transgenic rice to methyl viologen (MV) induced photo-oxidative stress. ( A ; top panel) Leaf strips of wt, ns , and three T 2 transgenic lines (L1, L4, and L5) after photo-oxidative stress treatment (incubation in 10 μM MV). Untreated (–MV) wt was used as a control ( A ; middle panel) Histochemical assessment of in vivo H 2 O 2 formation following MV treatment by DAB staining. ( A ; bottom panel) In vivo generation of O 2 - in leaf strips after MV treatment as detected by NBT staining. (B) Total chlorophyll contents (in mg/g FW) after MV treatment as compared to untreated controls. Data represented are means ± SD ( n = 3). Asterisks above bars indicate significant differences from wt (* p -value ≤ 0.05 and ** p -value ≤ 0.01).
    Figure Legend Snippet: Assessment of tolerance of OsGS1;1/OsGS2 co-overexpressing transgenic rice to methyl viologen (MV) induced photo-oxidative stress. ( A ; top panel) Leaf strips of wt, ns , and three T 2 transgenic lines (L1, L4, and L5) after photo-oxidative stress treatment (incubation in 10 μM MV). Untreated (–MV) wt was used as a control ( A ; middle panel) Histochemical assessment of in vivo H 2 O 2 formation following MV treatment by DAB staining. ( A ; bottom panel) In vivo generation of O 2 - in leaf strips after MV treatment as detected by NBT staining. (B) Total chlorophyll contents (in mg/g FW) after MV treatment as compared to untreated controls. Data represented are means ± SD ( n = 3). Asterisks above bars indicate significant differences from wt (* p -value ≤ 0.05 and ** p -value ≤ 0.01).

    Techniques Used: Transgenic Assay, Incubation, In Vivo, Staining

    Assessment of tolerance of OsGS1;1/OsGS2 co-overexpressing transgenic rice to phosphinothricin (PPT). (A) Phenotypes of wt, ns , and three T 2 transgenic (L1, L4, and L5) rice seedlings after 0.5% (v/v) Basta herbicide (Glufosinate/PPT) spraying. Survival rates (SR) after spraying are indicated as percentages. (B) Mature leaves of wt, ns , and three T 2 transgenic lines (L1, L4, and L5) painted with a solution of 0.5% Basta (v/v) (Bayer, 13.5% ai) supplemented with 0.01% Tween-20. (C) Mean NH 4 + liberation from leaves before and after PPT treatment. Data represented are the means ± SD ( n = 3). Different letters above bars indicate significant difference among means of PPT treated group (Tukey-Kramer tests, p -value
    Figure Legend Snippet: Assessment of tolerance of OsGS1;1/OsGS2 co-overexpressing transgenic rice to phosphinothricin (PPT). (A) Phenotypes of wt, ns , and three T 2 transgenic (L1, L4, and L5) rice seedlings after 0.5% (v/v) Basta herbicide (Glufosinate/PPT) spraying. Survival rates (SR) after spraying are indicated as percentages. (B) Mature leaves of wt, ns , and three T 2 transgenic lines (L1, L4, and L5) painted with a solution of 0.5% Basta (v/v) (Bayer, 13.5% ai) supplemented with 0.01% Tween-20. (C) Mean NH 4 + liberation from leaves before and after PPT treatment. Data represented are the means ± SD ( n = 3). Different letters above bars indicate significant difference among means of PPT treated group (Tukey-Kramer tests, p -value

    Techniques Used: Transgenic Assay

    Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).
    Figure Legend Snippet: Molecular and biochemical analysis of transgenic rice lines co-overexpressing OsGS1;1 and OsGS2 . (A) PCR amplification of hygromycin phosphotransferase ( hpt ), OsGS1;1 and OSGS2 genes using specific primers in wild type ( wt ), null segregant ( ns ), and five positive T 2 transgenic lines (L1-L5). M: 1Kb DNA ladder (+): positive PCR control (pMDC99) and (–) water blank. (B) Southern blot analysis of wt and five T 2 transgenic lines (L1, L2, L3, L4, and L5), probed with hpt gene probe showing single copy insertion. (C) Semi quantitative RT-PCR showing overexpression of OsGS1;1 and OsGS2 in transgenic lines (L1, L4, and L5) as compared to wt . The rice eEF1α gene was used as a reference gene and rRNA was used as loading control. (D ; top panel) Immunoblot analysis of three transgenic rice lines (L1, L4, and L5) and wt using a recombinant antibody which detects both OsGS1;1 and OsGS2 isoforms (black lines separate spliced regions from same blot) ( D ; bottom panel) Coomassie blue stained Rubisco large subunit (RubL) was used as loading control. (E) Total GS activity of three transgenic rice lines (L1, L4, and L5) in comparison to wt ). One unit of GS activity represents 1.0 μmol of γ-glutamylhydroxamate produced in 20 min. Asterisks above bars indicate significant differences from wt (* at p ≤ 0.05 and ** at p ≤ 0.01).

    Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Amplification, Southern Blot, Quantitative RT-PCR, Over Expression, Recombinant, Staining, Activity Assay, Produced

    Phenotype of OsGS1;1/OsGS2 co-overexpressing transgenic rice under moderate osmotic and salinity stress at seedling stage. Phenotype of 2-week-old seedlings of wt, ns and three transgenic rice lines (L1, L4, and L5), grown hydroponically in (A) normal Yoshida solution (untreated control) or (B) Yoshida solution supplemented with 15% PEG (osmotic stress) or (C) 150 mM NaCl (EC ~ 12 dS/m) (salinity stress) before and after 7 days of treatment. Visual phenotypic variation amongst seedlings of wt, ns and three transgenic rice lines (L1, L4, and L5), following 4 days of recovery after being grown hydroponically for 4 days in (D) Yoshida solution supplemented with 20% PEG (osmotic stress) or (E) 200 mM NaCl (EC ~ 19 dS/m) (salt stress). Scale bar = 1 cm.
    Figure Legend Snippet: Phenotype of OsGS1;1/OsGS2 co-overexpressing transgenic rice under moderate osmotic and salinity stress at seedling stage. Phenotype of 2-week-old seedlings of wt, ns and three transgenic rice lines (L1, L4, and L5), grown hydroponically in (A) normal Yoshida solution (untreated control) or (B) Yoshida solution supplemented with 15% PEG (osmotic stress) or (C) 150 mM NaCl (EC ~ 12 dS/m) (salinity stress) before and after 7 days of treatment. Visual phenotypic variation amongst seedlings of wt, ns and three transgenic rice lines (L1, L4, and L5), following 4 days of recovery after being grown hydroponically for 4 days in (D) Yoshida solution supplemented with 20% PEG (osmotic stress) or (E) 200 mM NaCl (EC ~ 19 dS/m) (salt stress). Scale bar = 1 cm.

    Techniques Used: Transgenic Assay

    Various biochemical and physiological parameters of 2-week-old seedlings of wt, ns , and three OsGS1;1/OsGS2 co-overexpressing transgenic rice lines (L1, L4, and L5) assessed after 2 days of osmotic (20% PEG) or salinity stress (200 mM NaCl) treatments as compared to untreated control conditions. (A) Fresh weight (FW) (in g). (B) Total chlorophyll content (in mg/g FW). (C) Relative water content (RWC) (in %). (D) Proline content (in μmol/g FW). (E) Electrolyte leakage (in %) (F) Malondialdehyde (MDA) content (in nmol/g FW). All data represented are means ± SD ( n = 3). Asterisks above bars indicate significant differences from wt (* p -value ≤ 0.05 and ** p -value ≤ 0.01).
    Figure Legend Snippet: Various biochemical and physiological parameters of 2-week-old seedlings of wt, ns , and three OsGS1;1/OsGS2 co-overexpressing transgenic rice lines (L1, L4, and L5) assessed after 2 days of osmotic (20% PEG) or salinity stress (200 mM NaCl) treatments as compared to untreated control conditions. (A) Fresh weight (FW) (in g). (B) Total chlorophyll content (in mg/g FW). (C) Relative water content (RWC) (in %). (D) Proline content (in μmol/g FW). (E) Electrolyte leakage (in %) (F) Malondialdehyde (MDA) content (in nmol/g FW). All data represented are means ± SD ( n = 3). Asterisks above bars indicate significant differences from wt (* p -value ≤ 0.05 and ** p -value ≤ 0.01).

    Techniques Used: Transgenic Assay, Multiple Displacement Amplification

    Agronomic and physiological performance of OsGS1;1/OsGS2 co-overexpressing transgenic rice plants under abiotic stresses at reproductive stage. (A) Phenotypes of wild type ( wt ), null segregant ( ns ), and three transgenic lines (L1, L4, and L5) at reproductive stage under untreated control conditions. (B) Phenotypes after recovery for 15 days following drought stress treatment imposed by water withdrawal for 12 days post panicle initiation. (C) Phenotypes after recovery following moderate salinity stress imposed on ~2-month-old plants by irrigating pots every fortnight with water supplemented with 50 mM NaCl (EC~6 dS/m) until booting stage. Various physiological parameters such as (D) net photosynthetic rate (P N ) (in μmol CO 2 /m 2 /s) (E) chlorophyll content (in SPAD values) and (F) chlorophyll fluorescence (F v /F m ) assessed under control, drought and salinity stress conditions. All data represented are means ± SD ( n = 3). Asterisks above bars indicate significant differences from wt (* p -value ≤ 0.05 and ** p -value ≤ 0.01). Spider plots of agronomic traits of three independent T 2 transgenic lines (L1, L4, and L5) and corresponding ns and wt controls under (G) untreated control (H) drought and, (I) salinity stress conditions respectively. Data plotted are percentages of mean values ( n = 5). Mean values from wt plants were set at 100% as reference. (J) Grain filling phenotypes in wt, ns , and three transgenic lines (L1, L4, and L5) after recovery from drought and salinity stress as compared to untreated wt control.
    Figure Legend Snippet: Agronomic and physiological performance of OsGS1;1/OsGS2 co-overexpressing transgenic rice plants under abiotic stresses at reproductive stage. (A) Phenotypes of wild type ( wt ), null segregant ( ns ), and three transgenic lines (L1, L4, and L5) at reproductive stage under untreated control conditions. (B) Phenotypes after recovery for 15 days following drought stress treatment imposed by water withdrawal for 12 days post panicle initiation. (C) Phenotypes after recovery following moderate salinity stress imposed on ~2-month-old plants by irrigating pots every fortnight with water supplemented with 50 mM NaCl (EC~6 dS/m) until booting stage. Various physiological parameters such as (D) net photosynthetic rate (P N ) (in μmol CO 2 /m 2 /s) (E) chlorophyll content (in SPAD values) and (F) chlorophyll fluorescence (F v /F m ) assessed under control, drought and salinity stress conditions. All data represented are means ± SD ( n = 3). Asterisks above bars indicate significant differences from wt (* p -value ≤ 0.05 and ** p -value ≤ 0.01). Spider plots of agronomic traits of three independent T 2 transgenic lines (L1, L4, and L5) and corresponding ns and wt controls under (G) untreated control (H) drought and, (I) salinity stress conditions respectively. Data plotted are percentages of mean values ( n = 5). Mean values from wt plants were set at 100% as reference. (J) Grain filling phenotypes in wt, ns , and three transgenic lines (L1, L4, and L5) after recovery from drought and salinity stress as compared to untreated wt control.

    Techniques Used: Transgenic Assay, Fluorescence

    6) Product Images from "Phosphorylation of GSK-3? by cGMP-dependent protein kinase II promotes hypertrophic differentiation of murine chondrocytes"

    Article Title: Phosphorylation of GSK-3? by cGMP-dependent protein kinase II promotes hypertrophic differentiation of murine chondrocytes

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI35243

    Subcellular localization of Sox9. ( A ) Effect of cGKII on subcellular localization of Sox9 and the phosphorylation-deficient mutants at putative phosphorylation sites at Ser64 (Sox9 S64A ), Ser181 (Sox9 S181A ), or both (Sox9 S64A+S181A ). HeLa cells were transfected with GFP, GFP-tagged Sox9 (GFP-Sox9), or the GFP-tagged mutants in combination with cGKII or empty vector. Subcellular localization of Sox9 or the mutants was determined by a fluorescent microscope. ( B ) Effect of LiCl treatment or GSK-3β S9A transfection on Sox9 subcellular localization in HeLa cells cotransfected with GFP-tagged Sox9 in combination with cGKII or empty vector. Scale bars: 10 μm ( A ); 20 μm ( B , top); 5 μm ( B , bottom).
    Figure Legend Snippet: Subcellular localization of Sox9. ( A ) Effect of cGKII on subcellular localization of Sox9 and the phosphorylation-deficient mutants at putative phosphorylation sites at Ser64 (Sox9 S64A ), Ser181 (Sox9 S181A ), or both (Sox9 S64A+S181A ). HeLa cells were transfected with GFP, GFP-tagged Sox9 (GFP-Sox9), or the GFP-tagged mutants in combination with cGKII or empty vector. Subcellular localization of Sox9 or the mutants was determined by a fluorescent microscope. ( B ) Effect of LiCl treatment or GSK-3β S9A transfection on Sox9 subcellular localization in HeLa cells cotransfected with GFP-tagged Sox9 in combination with cGKII or empty vector. Scale bars: 10 μm ( A ); 20 μm ( B , top); 5 μm ( B , bottom).

    Techniques Used: Transfection, Plasmid Preparation, Microscopy

    Genetic rescue of growth plate abnormality in Prkg2 –/– mice by GSK-3β insufficiency. ( A ) H E staining, Safranin-O staining, BrdU labeling, and immunohistochemical staining of COL10 in the tibial growth plates of 3-week-old mice of the 3 genotypes. Blue, red, green, and yellow bars indicate proliferative zone, abnormal intermediate layer, hypertrophic zone, and primary spongiosa, respectively. Boxed regions in COL10 panels are shown at higher magnification to the right. Scale bars: 50 μm. ( B ) Height of the growth plates of the 3 genotypes. The percentage recovery by the GSK-3β insufficiency was 36.0%. Data are mean ± SD of 4 mice per genotype. * P
    Figure Legend Snippet: Genetic rescue of growth plate abnormality in Prkg2 –/– mice by GSK-3β insufficiency. ( A ) H E staining, Safranin-O staining, BrdU labeling, and immunohistochemical staining of COL10 in the tibial growth plates of 3-week-old mice of the 3 genotypes. Blue, red, green, and yellow bars indicate proliferative zone, abnormal intermediate layer, hypertrophic zone, and primary spongiosa, respectively. Boxed regions in COL10 panels are shown at higher magnification to the right. Scale bars: 50 μm. ( B ) Height of the growth plates of the 3 genotypes. The percentage recovery by the GSK-3β insufficiency was 36.0%. Data are mean ± SD of 4 mice per genotype. * P

    Techniques Used: Mouse Assay, Staining, Labeling, Immunohistochemistry

    Identification of GSK-3β as a principal phosphorylation target of cGKII during chondrocyte hypertrophy. ( A ) RT-PCR of 8 candidate genes that were identified by the serine/threonine kinase substrate array (Supplemental Table 1) in cultured ATDC5 cells in the prehypertrophic or hypertrophic differentiation stage. ( B ) COL10 promoter activity, as assessed by transfection of the 8 candidate genes or the empty vector (EV) in HuH-7 cells with the luciferase reporter gene construct containing a cloned 4.5-kb promoter fragment of COL10. Data are mean ± SD fold change relative to empty vector. * P
    Figure Legend Snippet: Identification of GSK-3β as a principal phosphorylation target of cGKII during chondrocyte hypertrophy. ( A ) RT-PCR of 8 candidate genes that were identified by the serine/threonine kinase substrate array (Supplemental Table 1) in cultured ATDC5 cells in the prehypertrophic or hypertrophic differentiation stage. ( B ) COL10 promoter activity, as assessed by transfection of the 8 candidate genes or the empty vector (EV) in HuH-7 cells with the luciferase reporter gene construct containing a cloned 4.5-kb promoter fragment of COL10. Data are mean ± SD fold change relative to empty vector. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Construct, Clone Assay

    Regulation of chondrocyte hypertrophy by GSK-3β. ( A ) Effects of LiCl on mRNA levels of the hypertrophic markers COL10, ALP, and MMP-13, as assessed by real-time RT-PCR in ATDC5 cells cultured in 3-dimensional alginate beads. ( B ) Effects of LiCl on the hypertrophic markers, as assessed by immunocytochemistry in primary costal chondrocytes cultured in 3-dimensional alginate beads. For morphological comparison, sections of the representative colonies containing 4 cells were selected. Scale bars: 10 μm. ( C ) mRNA levels of the hypertrophic markers, as assessed by real-time RT-PCR in cultured costal chondrocytes from WT and Gsk3b +/– mice. ( D ) mRNA levels of the hypertrophic markers in stable lines of ATDC5 cells retrovirally transfected with the constitutively active form of cGKII (CA-cGKII), GSK-3β S9A , or the control GFP (–). Data are mean ± SD of the relative amount compared with control or WT. * P
    Figure Legend Snippet: Regulation of chondrocyte hypertrophy by GSK-3β. ( A ) Effects of LiCl on mRNA levels of the hypertrophic markers COL10, ALP, and MMP-13, as assessed by real-time RT-PCR in ATDC5 cells cultured in 3-dimensional alginate beads. ( B ) Effects of LiCl on the hypertrophic markers, as assessed by immunocytochemistry in primary costal chondrocytes cultured in 3-dimensional alginate beads. For morphological comparison, sections of the representative colonies containing 4 cells were selected. Scale bars: 10 μm. ( C ) mRNA levels of the hypertrophic markers, as assessed by real-time RT-PCR in cultured costal chondrocytes from WT and Gsk3b +/– mice. ( D ) mRNA levels of the hypertrophic markers in stable lines of ATDC5 cells retrovirally transfected with the constitutively active form of cGKII (CA-cGKII), GSK-3β S9A , or the control GFP (–). Data are mean ± SD of the relative amount compared with control or WT. * P

    Techniques Used: ALP Assay, Quantitative RT-PCR, Cell Culture, Immunocytochemistry, Mouse Assay, Transfection

    Mechanism underlying cGKII/GSK-3β signaling in chondrocyte hypertrophy. ( A ) Localization of β-catenin, Ser9-phosphorylated GSK-3β, and total GSK-3β, as assessed by immunohistochemistry in the growth plates of the proximal tibias of WT and Prkg2 –/– mice at 2 weeks of age. Blue, red, green, and yellow bars indicate proliferative zone, abnormal intermediate layer, hypertrophic zone, and primary spongiosa, respectively. Scale bars: 50 μm. ( B ) Time course of β-catenin protein level after stimulation by 8-bromo-cGMP, as assessed by IB in the cytosolic fraction of ATDC5 cells with retroviral introduction of cGKII or cGKII-Δkinase. ( C ) Promoter activity of the β-catenin target TCF, as assessed by luciferase (Luc) assay using TOPflash and FOPflash reporter plasmids in HEK293 cells transfected with constitutively active cGKII, GSK-3β S9A , or the control GFP (–). Data are mean ± SD fold change compared with control (–/–). * P
    Figure Legend Snippet: Mechanism underlying cGKII/GSK-3β signaling in chondrocyte hypertrophy. ( A ) Localization of β-catenin, Ser9-phosphorylated GSK-3β, and total GSK-3β, as assessed by immunohistochemistry in the growth plates of the proximal tibias of WT and Prkg2 –/– mice at 2 weeks of age. Blue, red, green, and yellow bars indicate proliferative zone, abnormal intermediate layer, hypertrophic zone, and primary spongiosa, respectively. Scale bars: 50 μm. ( B ) Time course of β-catenin protein level after stimulation by 8-bromo-cGMP, as assessed by IB in the cytosolic fraction of ATDC5 cells with retroviral introduction of cGKII or cGKII-Δkinase. ( C ) Promoter activity of the β-catenin target TCF, as assessed by luciferase (Luc) assay using TOPflash and FOPflash reporter plasmids in HEK293 cells transfected with constitutively active cGKII, GSK-3β S9A , or the control GFP (–). Data are mean ± SD fold change compared with control (–/–). * P

    Techniques Used: Immunohistochemistry, Mouse Assay, Activity Assay, Luciferase, Transfection

    Genetic rescue of growth retardation in Prkg2 –/– mice by GSK-3β insufficiency. ( A ) Radiographs of WT, Prkg2 –/– , and Prkg2 –/– Gsk3b +/– littermates at 8 weeks of age. ( B ) Time course of total axial length (from nose to tail end) of the 3 genotypes from 3 to 16 weeks of age. The recovery by the GSK-3β insufficiency in the Prkg2 –/– mice was 43.2%, 31.4%, and 41.9% at 8, 12, and 16 weeks, respectively. ( C ) Length of bones of the 3 genotypes at 8 weeks of age. Percent recovery was 21.7%, 18.3%, 24.3%, 16.2%, 24.3%, and 42.6% in femur, tibia, humerus, ulna, vertebra, and skull length, respectively. Data are mean ± SD for 4–9 mice per genotype. * P
    Figure Legend Snippet: Genetic rescue of growth retardation in Prkg2 –/– mice by GSK-3β insufficiency. ( A ) Radiographs of WT, Prkg2 –/– , and Prkg2 –/– Gsk3b +/– littermates at 8 weeks of age. ( B ) Time course of total axial length (from nose to tail end) of the 3 genotypes from 3 to 16 weeks of age. The recovery by the GSK-3β insufficiency in the Prkg2 –/– mice was 43.2%, 31.4%, and 41.9% at 8, 12, and 16 weeks, respectively. ( C ) Length of bones of the 3 genotypes at 8 weeks of age. Percent recovery was 21.7%, 18.3%, 24.3%, 16.2%, 24.3%, and 42.6% in femur, tibia, humerus, ulna, vertebra, and skull length, respectively. Data are mean ± SD for 4–9 mice per genotype. * P

    Techniques Used: Mouse Assay

    7) Product Images from "Congenital Cataract in Gpr161vl/vl Mice Is Modified by Proximal Chromosome 15"

    Article Title: Congenital Cataract in Gpr161vl/vl Mice Is Modified by Proximal Chromosome 15

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170724

    Sub1 MOLF congenic rescues Gpr161 vl/vl lens fiber defects. (A-H) Nissl stained transverse sections of E16.5 lens (A, C, E, G; 20X magnification), as well as magnified images of nasal bow and posterior medial region (B, D, F, H; 40X magnification) are shown (pm: posterior-medial region; nbr: nasal bow region). Normal (A, B) as well as mild (C, D), moderate (E, F) and severe (G, H) phenotypes are depicted. Red dashed boxes mark the region with abnormal lens fiber orientation and vacuoles. (I) The number and percentages (in parenthesis) of sections with the above phenotypes are shown for Gpr161 +/+ Sub1 C/C , Gpr161 vl/vl Sub1 C/C , Gpr161 vl/vl Sub1 C/M and Gpr161 vl/vl Sub1 M/M . (J) Pairwise comparisons between genotypes were performed using Mantel-Haenszel test with rank score. (K) The distribution of lens fiber phenotypes along the A-P axis is shown by representative illustrations for each of the four genotypes (green: normal; yellow: mild defect; orange: moderate defect and red: severe defect). In Gpr161 vl/vl mutant background, transverse sections that are closer to the lens equatorial region display more severe defects. In the Modvl4 MOLF congenic background, a partial rescue of the defect is observed by the reduction in severe lens fiber defect, and the expansion of normal and mild phenotypes.
    Figure Legend Snippet: Sub1 MOLF congenic rescues Gpr161 vl/vl lens fiber defects. (A-H) Nissl stained transverse sections of E16.5 lens (A, C, E, G; 20X magnification), as well as magnified images of nasal bow and posterior medial region (B, D, F, H; 40X magnification) are shown (pm: posterior-medial region; nbr: nasal bow region). Normal (A, B) as well as mild (C, D), moderate (E, F) and severe (G, H) phenotypes are depicted. Red dashed boxes mark the region with abnormal lens fiber orientation and vacuoles. (I) The number and percentages (in parenthesis) of sections with the above phenotypes are shown for Gpr161 +/+ Sub1 C/C , Gpr161 vl/vl Sub1 C/C , Gpr161 vl/vl Sub1 C/M and Gpr161 vl/vl Sub1 M/M . (J) Pairwise comparisons between genotypes were performed using Mantel-Haenszel test with rank score. (K) The distribution of lens fiber phenotypes along the A-P axis is shown by representative illustrations for each of the four genotypes (green: normal; yellow: mild defect; orange: moderate defect and red: severe defect). In Gpr161 vl/vl mutant background, transverse sections that are closer to the lens equatorial region display more severe defects. In the Modvl4 MOLF congenic background, a partial rescue of the defect is observed by the reduction in severe lens fiber defect, and the expansion of normal and mild phenotypes.

    Techniques Used: Staining, Mutagenesis

    Expression of the candidate genes at E16.5 eye. (A) The in vivo expression of all 20 genes within the 15 Mb interval was assessed by performing RT-PCR using E16.5 eye cDNA. A total of 15 genes display detectable expression and PCR results are represented by gel electrophoresis (two biological replicates per gene). (B) QRT-PCR compared the expression level of the 15 genes and Gpr161 between Gpr161 +/+ Sub1 C/C and Gpr161 vl/vl Sub1 C/C E16.5 eyes. Ten of them (highlighted in red) showed reduced expression in Gpr161 vl/vl Sub1 C/C . (C) QRT-PCR further compared the expression level of the 10 genes highlighted in (B) among Gpr161 +/+ Sub1 C/C , Gpr161 vl/vl Sub1 C/C and Gpr161 vl/vl Sub1 M/M E16.5 eyes. The expression level of 5 genes is fully ( Cdh6 , Cdh12 , Fbxl7 and Ank ) or partially ( Trio ) restored by Gpr161 vl/vl Sub1 M/M . (D) For Gpr161 , a 70% reduced expression is observed in Gpr161 vl/vl Sub1 C/C , which is not rescued by Gpr161 vl/vl Sub1 M/M . All qRTPCR data in (B), (C) and (D) represent averages of six biological replicates per genotype. (*: P
    Figure Legend Snippet: Expression of the candidate genes at E16.5 eye. (A) The in vivo expression of all 20 genes within the 15 Mb interval was assessed by performing RT-PCR using E16.5 eye cDNA. A total of 15 genes display detectable expression and PCR results are represented by gel electrophoresis (two biological replicates per gene). (B) QRT-PCR compared the expression level of the 15 genes and Gpr161 between Gpr161 +/+ Sub1 C/C and Gpr161 vl/vl Sub1 C/C E16.5 eyes. Ten of them (highlighted in red) showed reduced expression in Gpr161 vl/vl Sub1 C/C . (C) QRT-PCR further compared the expression level of the 10 genes highlighted in (B) among Gpr161 +/+ Sub1 C/C , Gpr161 vl/vl Sub1 C/C and Gpr161 vl/vl Sub1 M/M E16.5 eyes. The expression level of 5 genes is fully ( Cdh6 , Cdh12 , Fbxl7 and Ank ) or partially ( Trio ) restored by Gpr161 vl/vl Sub1 M/M . (D) For Gpr161 , a 70% reduced expression is observed in Gpr161 vl/vl Sub1 C/C , which is not rescued by Gpr161 vl/vl Sub1 M/M . All qRTPCR data in (B), (C) and (D) represent averages of six biological replicates per genotype. (*: P

    Techniques Used: Expressing, In Vivo, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Quantitative RT-PCR

    8) Product Images from "Metformin-repressed miR-381-YAP-snail axis activity disrupts NSCLC growth and metastasis"

    Article Title: Metformin-repressed miR-381-YAP-snail axis activity disrupts NSCLC growth and metastasis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1503-6

    Metformin decreased cell growth, migration, invasion and EMT by regulating miR-381/YAP activity. A549 cells were treated with Metformin or co-treated with Metformin and miR-381 inhibitors or YAP, respectively. a The RNA levels of miR-381 and YAP were analyzed by qPCR. b , c The cellular growth ( b ) and viability ( c ) were analyzed by CCK8 assay. d Ki67 positive cells were analyzed by the immunofluorescent staining assay. e Colony formation ability was a nalyzed by colony formation assay. f Cellular migration growth was analyzed by scratch assay. g The cellular invasion growth was analyzed by transwell assay. h The protein level of Caspase 3 Cleaved (Caspase 3-Cl) was analyzed by immunoblotting. i , j The expressions of E-cadherin and Vimentin were analyzed by RT-PCR, western blot, qPCR ( i ) and immunofluorescent staining ( j ) assays. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P
    Figure Legend Snippet: Metformin decreased cell growth, migration, invasion and EMT by regulating miR-381/YAP activity. A549 cells were treated with Metformin or co-treated with Metformin and miR-381 inhibitors or YAP, respectively. a The RNA levels of miR-381 and YAP were analyzed by qPCR. b , c The cellular growth ( b ) and viability ( c ) were analyzed by CCK8 assay. d Ki67 positive cells were analyzed by the immunofluorescent staining assay. e Colony formation ability was a nalyzed by colony formation assay. f Cellular migration growth was analyzed by scratch assay. g The cellular invasion growth was analyzed by transwell assay. h The protein level of Caspase 3 Cleaved (Caspase 3-Cl) was analyzed by immunoblotting. i , j The expressions of E-cadherin and Vimentin were analyzed by RT-PCR, western blot, qPCR ( i ) and immunofluorescent staining ( j ) assays. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P

    Techniques Used: Migration, Activity Assay, Real-time Polymerase Chain Reaction, CCK-8 Assay, Staining, Colony Assay, Wound Healing Assay, Transwell Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Metformin inhibits xenograft tumor growth and metastasis in vivo and in vitro . a , b Xenografted A549 cell tumors with stably expressing Scramble miRNA and miR-381 inhibitors (miR-381 i) in mice with treatment of vehicle and metformin. The weights ( a ) and sizes ( b ) measured at regular intervals. c Kaplan-Meier overall survival (OS) curves of vehicle- and metformin-treated mice injected A549 cells with stably expressing Scramble miRNA and miR-381 inhibitors. d The expressions of Ki67, Cleaved Caspase3, YAP, Snail, E-cadherin and Vimentin were analyzed by immunohistochemistry assay from tumor tissues injected A549 cells with stably expressing Scramble miRNA and miR-381 inhibitors in mice with treatment of vehicle and metformin ( n = 5). e-h Representative H E stained microscopic images of the metastatic lung tumors originated from xenografted A549 cells with stably expressing Scramble miRNA and miR-381 inhibitors in mice with treatment of vehicle and metformin by subcutaneous injection. i-o 95-D cells were separately treated with metformin or co-treated with metformin and Snail, respectively. i The expressions of YAP and Snail were analyzed by RT-PCR and qPCR. j The cellular viability was analyzed by CCK8. k The cellular migration growth was analyzed by scratch assay. l The cell migration and invasion were analyzed by transwell assay. m-o The expressions of YAP, Snail, E-cadherin and Vimentin were analyzed by RT-PCR ( m ), qPCR ( n ) and western blot ( o ) assays. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. ** P
    Figure Legend Snippet: Metformin inhibits xenograft tumor growth and metastasis in vivo and in vitro . a , b Xenografted A549 cell tumors with stably expressing Scramble miRNA and miR-381 inhibitors (miR-381 i) in mice with treatment of vehicle and metformin. The weights ( a ) and sizes ( b ) measured at regular intervals. c Kaplan-Meier overall survival (OS) curves of vehicle- and metformin-treated mice injected A549 cells with stably expressing Scramble miRNA and miR-381 inhibitors. d The expressions of Ki67, Cleaved Caspase3, YAP, Snail, E-cadherin and Vimentin were analyzed by immunohistochemistry assay from tumor tissues injected A549 cells with stably expressing Scramble miRNA and miR-381 inhibitors in mice with treatment of vehicle and metformin ( n = 5). e-h Representative H E stained microscopic images of the metastatic lung tumors originated from xenografted A549 cells with stably expressing Scramble miRNA and miR-381 inhibitors in mice with treatment of vehicle and metformin by subcutaneous injection. i-o 95-D cells were separately treated with metformin or co-treated with metformin and Snail, respectively. i The expressions of YAP and Snail were analyzed by RT-PCR and qPCR. j The cellular viability was analyzed by CCK8. k The cellular migration growth was analyzed by scratch assay. l The cell migration and invasion were analyzed by transwell assay. m-o The expressions of YAP, Snail, E-cadherin and Vimentin were analyzed by RT-PCR ( m ), qPCR ( n ) and western blot ( o ) assays. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. ** P

    Techniques Used: In Vivo, In Vitro, Stable Transfection, Expressing, Mouse Assay, Injection, Immunohistochemistry, Staining, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Migration, Wound Healing Assay, Transwell Assay, Western Blot

    YAP promotes cell migration, invasion and EMT by regulating snail. a IHC assay of the expression of YAP and Snail in the human lung cancer tissues from pro-metastasis (Pro-M) and after-metastasis (After-M) ( n = 10). b IHC assay of the expression of YAP and Snail from the tumor in situ (TIS) and tumor in metastasis (TIM) of lung cancers ( n = 10). c , d The expression of Snail was detected in the HBEC and 95-D cells by RT-PCR, western blot, qPCR ( c ) and immunofluorescent staining ( d ) assays. e -g HBEC cells were transfected with snail or 95-D cells were transfected with si-snail , respectively. The expression of Snail was analyzed by RT-PCR, qPCR and western blot assays ( e ). The cellular viability was analyzed by CCK8 assay ( f ). The cellular migration growth was analyzed by scratch assay ( g ). h-p HBEC cells were transfected with YAP or co-transfected with YAP and si-snail, meanwhile 95-D cells were transfected with siYAP or co-transfected with siYAP and snail, respectively. The expressions of YAP and Snail were analyzed by RT-PCR, western blot and qPCR in HEBC and 95-D cells ( h , i ). The cellular viability was analyzed by CCK8 assay in HEBC ( j ) and 95-D cells ( k ). The cellular migration growth was analyzed by scratch assay in HBEC cells ( l ). The cell migration and invasion were analyzed by transwell assay in HBEC cells ( m ). The expression of Annexin V was analyzed by immunofluorescent staining assays in 95-D cells ( n ). The expressions of Vimentin and E-cadherin were analyzed by RT-PCR, western blot and qPCR in HEBC and 95-D cells ( o , p ). Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P
    Figure Legend Snippet: YAP promotes cell migration, invasion and EMT by regulating snail. a IHC assay of the expression of YAP and Snail in the human lung cancer tissues from pro-metastasis (Pro-M) and after-metastasis (After-M) ( n = 10). b IHC assay of the expression of YAP and Snail from the tumor in situ (TIS) and tumor in metastasis (TIM) of lung cancers ( n = 10). c , d The expression of Snail was detected in the HBEC and 95-D cells by RT-PCR, western blot, qPCR ( c ) and immunofluorescent staining ( d ) assays. e -g HBEC cells were transfected with snail or 95-D cells were transfected with si-snail , respectively. The expression of Snail was analyzed by RT-PCR, qPCR and western blot assays ( e ). The cellular viability was analyzed by CCK8 assay ( f ). The cellular migration growth was analyzed by scratch assay ( g ). h-p HBEC cells were transfected with YAP or co-transfected with YAP and si-snail, meanwhile 95-D cells were transfected with siYAP or co-transfected with siYAP and snail, respectively. The expressions of YAP and Snail were analyzed by RT-PCR, western blot and qPCR in HEBC and 95-D cells ( h , i ). The cellular viability was analyzed by CCK8 assay in HEBC ( j ) and 95-D cells ( k ). The cellular migration growth was analyzed by scratch assay in HBEC cells ( l ). The cell migration and invasion were analyzed by transwell assay in HBEC cells ( m ). The expression of Annexin V was analyzed by immunofluorescent staining assays in 95-D cells ( n ). The expressions of Vimentin and E-cadherin were analyzed by RT-PCR, western blot and qPCR in HEBC and 95-D cells ( o , p ). Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P

    Techniques Used: Migration, Immunohistochemistry, Expressing, In Situ, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Staining, Transfection, CCK-8 Assay, Wound Healing Assay, Transwell Assay

    miR-381 level was lower in cancer tissues of NSCLC patients and lower miR-381 promotes cellular growth, EMT and invasion. a The miR-381 level was lower in lung cancer cells than their control cell, HBEC, by the RT-PCR and qRT-PCR assays. b FISH assay indicated that miR-381 was mainly located in the cytoplasm. c - o A549 cells were transfected with 50 nM miR-381 mimics or miR-381 inhibitors (GenePharma, Shanghai, China), respectively. The miR-381 level was analyzed by RT-PCR and qRT-PCR assays ( c , d ). The cellular growth and cell viability were analyzed by CCK8 assay ( e , f ). The protein level of Caspase 3 Cleaved (Caspase 3-Cl) was analyzed by immunoblotting ( g , h ). Colony formation ability was analyzed by colony formation assay ( i , j ). Cellular migration growth was analyzed by scratch assay ( k , l ). The expressions of E-cadherin and Vimentin were analyzed by RT-PCR ( m ), qRT-PCR ( n ) and immunofluorescent staining ( n ) assays. p , q The miR-381 level was analyzed by RT-PCR and qRT-PCR assays in human lung cancer tissues and their normal adjacent lung tissues ( p , n = 8; q , n = 16). ( r ) Kaplan Meier overall survival (OS) curves of miR-381 ( p = 0.012 by log-rank test for significance) for human lung cancers. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P
    Figure Legend Snippet: miR-381 level was lower in cancer tissues of NSCLC patients and lower miR-381 promotes cellular growth, EMT and invasion. a The miR-381 level was lower in lung cancer cells than their control cell, HBEC, by the RT-PCR and qRT-PCR assays. b FISH assay indicated that miR-381 was mainly located in the cytoplasm. c - o A549 cells were transfected with 50 nM miR-381 mimics or miR-381 inhibitors (GenePharma, Shanghai, China), respectively. The miR-381 level was analyzed by RT-PCR and qRT-PCR assays ( c , d ). The cellular growth and cell viability were analyzed by CCK8 assay ( e , f ). The protein level of Caspase 3 Cleaved (Caspase 3-Cl) was analyzed by immunoblotting ( g , h ). Colony formation ability was analyzed by colony formation assay ( i , j ). Cellular migration growth was analyzed by scratch assay ( k , l ). The expressions of E-cadherin and Vimentin were analyzed by RT-PCR ( m ), qRT-PCR ( n ) and immunofluorescent staining ( n ) assays. p , q The miR-381 level was analyzed by RT-PCR and qRT-PCR assays in human lung cancer tissues and their normal adjacent lung tissues ( p , n = 8; q , n = 16). ( r ) Kaplan Meier overall survival (OS) curves of miR-381 ( p = 0.012 by log-rank test for significance) for human lung cancers. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Transfection, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Staining

    Higher expression of YAP promotes cellular growth, migration, invasion and EMT. a The expression of YAP was higher in NSCLC cell lines compared with their control cell line, HBEC, analyzed by RT-PCR, western blot and qRT-PCR assays. b The protein level of nuclear YAP was higher in high metastasis lung cancer cell line 95-D than its control cell HBEC by the immunofluorescent staining assay. c Immunoblotting with densitometric quantitation demonstrating increased nuclear Yap and decreased p-YAP in 95-D cells than its normal cell line meanwhile YAP’s target genes, CTGF and Cyr61, were higher in 95-D cells. d - h A549 cells were transfected with siYAP or Myc-YAP, respectively. d The expression of YAP was analyzed by RT-PCR, Western blot and qPCR assays. e The cellular viability was analyzed by CCK8 assay. f Cellular migration growth was analyzed by scratch assay. g The cellular invasion growth was analyzed by transwell assay. h The expressions of E-cadherin and Vimentin were analyzed by RT-PCR, western blot assays. i The expression of YAP was higher in human lung cancer tissues compared with their normal adjacent lung tissues analyzed by RT-PCR, western blot and qRT-PCR assays ( n = 8). j Immunohistochemical (IHC) assay of the expression of YAP and Snail in the human lung cancer tissues and their normal adjacent lung tissues ( n = 15). k YAP was higher in nucleus from lung tumor tissues than their normal adjacent lung tissues ( n = 15). l Kaplan Meier overall survival (OS) curves of YAP ( p = 0.023 by log-rank test for significance) for human lung cancers. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P
    Figure Legend Snippet: Higher expression of YAP promotes cellular growth, migration, invasion and EMT. a The expression of YAP was higher in NSCLC cell lines compared with their control cell line, HBEC, analyzed by RT-PCR, western blot and qRT-PCR assays. b The protein level of nuclear YAP was higher in high metastasis lung cancer cell line 95-D than its control cell HBEC by the immunofluorescent staining assay. c Immunoblotting with densitometric quantitation demonstrating increased nuclear Yap and decreased p-YAP in 95-D cells than its normal cell line meanwhile YAP’s target genes, CTGF and Cyr61, were higher in 95-D cells. d - h A549 cells were transfected with siYAP or Myc-YAP, respectively. d The expression of YAP was analyzed by RT-PCR, Western blot and qPCR assays. e The cellular viability was analyzed by CCK8 assay. f Cellular migration growth was analyzed by scratch assay. g The cellular invasion growth was analyzed by transwell assay. h The expressions of E-cadherin and Vimentin were analyzed by RT-PCR, western blot assays. i The expression of YAP was higher in human lung cancer tissues compared with their normal adjacent lung tissues analyzed by RT-PCR, western blot and qRT-PCR assays ( n = 8). j Immunohistochemical (IHC) assay of the expression of YAP and Snail in the human lung cancer tissues and their normal adjacent lung tissues ( n = 15). k YAP was higher in nucleus from lung tumor tissues than their normal adjacent lung tissues ( n = 15). l Kaplan Meier overall survival (OS) curves of YAP ( p = 0.023 by log-rank test for significance) for human lung cancers. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P

    Techniques Used: Expressing, Migration, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Staining, Quantitation Assay, Transfection, Real-time Polymerase Chain Reaction, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Immunohistochemistry

    Metformin decreased YAP activity by regulating miR-381. a HBEC, A549 and 95-D cells were treated with 10 mM metformin for indicated times. Cell viability was assessed by CCK8 assay. b , c A549 cells were incubated with metformin at various concentrations for 24, 48, and 72 h ( b ) and indicated times at 5 mM, 10 mM and 20 mM, respectively. Cell viability was assessed by CCK8 assay. d FISH result indicated that Metformin upregulated the RNA level of MiR-381 in the A549 cells. e , f RT-PCR and qPCR results indicated that Metformin dose- and time- dependently increased the RNA level of miR-381. g Decay of miR-381 was monitored in A549, H1299 and 95-D cells cotreated with Actinomycin D and Vehicle or 10 mM Metformin. RNA levels of miR-381 were determined by qPCR. h Decay of miR-381 was monitored in A549 and 95-D cells cotreated with Actinomycin D and indicated concentrations of Metformin. RNA levels of miR-381 were determined by qPCR. i-k RT-PCR, qPCR ( i ), western blot ( j ) and immunofluorescent staining ( k ) assays indicated that Metformin dose-dependently decreased the expression of YAP. l Metformin time-dependently decreased the expression of YAP by the qPCR and western blot assays. m , n A549 cells were treated with Metformin or co-treated with Metformin and miR-381 inhibitors, respectively. The expressions of YAP, CTGF and Cyr61 were analyzed by western blot ( m ) and qPCR ( n ) assays. o , p A549 cells were treated with Metformin or co-treated with Metformin and miR-381 mimics, respectively. The expressions of YAP, CTGF and Cyr61 were analyzed by western blot ( o ) and qPCR ( p ) assays. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P
    Figure Legend Snippet: Metformin decreased YAP activity by regulating miR-381. a HBEC, A549 and 95-D cells were treated with 10 mM metformin for indicated times. Cell viability was assessed by CCK8 assay. b , c A549 cells were incubated with metformin at various concentrations for 24, 48, and 72 h ( b ) and indicated times at 5 mM, 10 mM and 20 mM, respectively. Cell viability was assessed by CCK8 assay. d FISH result indicated that Metformin upregulated the RNA level of MiR-381 in the A549 cells. e , f RT-PCR and qPCR results indicated that Metformin dose- and time- dependently increased the RNA level of miR-381. g Decay of miR-381 was monitored in A549, H1299 and 95-D cells cotreated with Actinomycin D and Vehicle or 10 mM Metformin. RNA levels of miR-381 were determined by qPCR. h Decay of miR-381 was monitored in A549 and 95-D cells cotreated with Actinomycin D and indicated concentrations of Metformin. RNA levels of miR-381 were determined by qPCR. i-k RT-PCR, qPCR ( i ), western blot ( j ) and immunofluorescent staining ( k ) assays indicated that Metformin dose-dependently decreased the expression of YAP. l Metformin time-dependently decreased the expression of YAP by the qPCR and western blot assays. m , n A549 cells were treated with Metformin or co-treated with Metformin and miR-381 inhibitors, respectively. The expressions of YAP, CTGF and Cyr61 were analyzed by western blot ( m ) and qPCR ( n ) assays. o , p A549 cells were treated with Metformin or co-treated with Metformin and miR-381 mimics, respectively. The expressions of YAP, CTGF and Cyr61 were analyzed by western blot ( o ) and qPCR ( p ) assays. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P

    Techniques Used: Activity Assay, CCK-8 Assay, Incubation, Fluorescence In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Staining, Expressing

    9) Product Images from "Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects"

    Article Title: Dose-dependent alcohol-induced alterations in chromatin structure persist beyond the window of exposure and correlate with fetal alcohol syndrome birth defects

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-015-0031-7

    Dose-dependent epigenetic signatures of EtOH exposure persist past the period of exposure. a Experimental paradigm. b Alcohol-induced epigenetic alterations in H3K4 me3, H3K9 ac, H3K9 me2, and H3K27 me3. Primary fetal cerebral cortical neuroepithelial stem cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in medium lacking EtOH. Samples were collected at days 3 and 7, and examined for changes in the indicated post-translational histone modifications using chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Heat maps represent fold change in H3K4 me3, H3K9 ac, H3K9 me2, and H3K27 me3 within the regulatory regions of the genes listed. Primers were designed to fall within 250 base pairs of the transcriptional start site. Within the three separate biological replicates ( N = 3), three ChIPs were performed, and two qPCR replicates performed on each independent ChIP. Statistical measures were conducted using the Wilcoxon signed rank nonparametric test. * p
    Figure Legend Snippet: Dose-dependent epigenetic signatures of EtOH exposure persist past the period of exposure. a Experimental paradigm. b Alcohol-induced epigenetic alterations in H3K4 me3, H3K9 ac, H3K9 me2, and H3K27 me3. Primary fetal cerebral cortical neuroepithelial stem cells were cultured in the presence of 160 or 240 mg/dL EtOH for 3 days, followed by a 4-day recovery in medium lacking EtOH. Samples were collected at days 3 and 7, and examined for changes in the indicated post-translational histone modifications using chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Heat maps represent fold change in H3K4 me3, H3K9 ac, H3K9 me2, and H3K27 me3 within the regulatory regions of the genes listed. Primers were designed to fall within 250 base pairs of the transcriptional start site. Within the three separate biological replicates ( N = 3), three ChIPs were performed, and two qPCR replicates performed on each independent ChIP. Statistical measures were conducted using the Wilcoxon signed rank nonparametric test. * p

    Techniques Used: Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    10) Product Images from "Identification of a novel C-terminal extension mutation in EPHA2 in a family affected with congenital cataract"

    Article Title: Identification of a novel C-terminal extension mutation in EPHA2 in a family affected with congenital cataract

    Journal: Molecular Vision

    doi:

    Cosegregation analysis of the identified alleles and schematic representation of EPHA2 wild-type and mutant proteins. A : Pedigree with genotype data for EPHA2 , CRYBB3 , and CRYBA2 alleles. Individuals affected with congenital cataract are indicated by shaded symbols. Genotyping results for the three alleles identified in the family are shown below each individual tested: 1 = EPHA2; 2 = CRYBB3; 3 = CRYBA2. The pathogenic allele is indicated in bold. The proband is indicated with an arrow; wt, wild-type allele at the variant position. B : Schematic drawing of the EPHA2 protein and C-terminal extension mutant sequences. The EPHA2 domain structure is shown at the top; SP = signal peptide, FN III = fibronectin III type repeats, TM = transmembrane domain, JMR = juxtamembrane region, SAM = sterile alpha motif, PDZ = PDZ-binding motif. C-terminal sequences of EPHA2 wild-type and frameshift mutants are shown at the bottom with the PDZ-motif residues indicated in blue and erroneous amino acids in red.
    Figure Legend Snippet: Cosegregation analysis of the identified alleles and schematic representation of EPHA2 wild-type and mutant proteins. A : Pedigree with genotype data for EPHA2 , CRYBB3 , and CRYBA2 alleles. Individuals affected with congenital cataract are indicated by shaded symbols. Genotyping results for the three alleles identified in the family are shown below each individual tested: 1 = EPHA2; 2 = CRYBB3; 3 = CRYBA2. The pathogenic allele is indicated in bold. The proband is indicated with an arrow; wt, wild-type allele at the variant position. B : Schematic drawing of the EPHA2 protein and C-terminal extension mutant sequences. The EPHA2 domain structure is shown at the top; SP = signal peptide, FN III = fibronectin III type repeats, TM = transmembrane domain, JMR = juxtamembrane region, SAM = sterile alpha motif, PDZ = PDZ-binding motif. C-terminal sequences of EPHA2 wild-type and frameshift mutants are shown at the bottom with the PDZ-motif residues indicated in blue and erroneous amino acids in red.

    Techniques Used: Mutagenesis, Variant Assay, Binding Assay

    11) Product Images from "Long non-coding RNA NEAT1 promotes non-small cell lung cancer progression through regulation of miR-377-3p-E2F3 pathway"

    Article Title: Long non-coding RNA NEAT1 promotes non-small cell lung cancer progression through regulation of miR-377-3p-E2F3 pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10108

    NEAT1 is a direct target of miR-377-3p A. Screen of the candidate miRNAs that interacted with NEAT1 by real-time PCR-based miRNA expression profiling and starBase (v2.0). Co-analysis of the down-regulated miRNAs in stable overexpression NEAT1/A549 cells compared to the control A549 cells and the miRNA list that potentially target NEAT1 predicted by starBase (v2.0), shown in Table 1 , we got seven candidates. B. Sequence alignment of miR-377-3p with the putative binding sites within the wild-type regions of NEAT1. C. The luciferase report assay demonstrated that overexpression of miR-377-3p could reduce the intensity of fluorescence in A549 and H1299 cells transfected with the NEAT1-WT vector, while had no effect on the NEAT1-MUT vector. D. WT and the mutated forms of miR-377-3p sequence are shown. E. Detection of NEAT1 using qRT-PCR in the sample pulled down by biotinylated miR-377-3p. F. Detection of miR-377-3p using qRT-PCR in the sample pulled down by biotinylated NEAT1 probe. G. The correlation between NEAT1 mRNA and miR-377-3p expression in 96 lung cancer tissues. H. Detection of miR-377-3p using qRT-PCR in the si-NEAT1 or NEAT1 overexpression A549 and H1299 cell lines compared with control group. Assays were performed in triplicate. * P
    Figure Legend Snippet: NEAT1 is a direct target of miR-377-3p A. Screen of the candidate miRNAs that interacted with NEAT1 by real-time PCR-based miRNA expression profiling and starBase (v2.0). Co-analysis of the down-regulated miRNAs in stable overexpression NEAT1/A549 cells compared to the control A549 cells and the miRNA list that potentially target NEAT1 predicted by starBase (v2.0), shown in Table 1 , we got seven candidates. B. Sequence alignment of miR-377-3p with the putative binding sites within the wild-type regions of NEAT1. C. The luciferase report assay demonstrated that overexpression of miR-377-3p could reduce the intensity of fluorescence in A549 and H1299 cells transfected with the NEAT1-WT vector, while had no effect on the NEAT1-MUT vector. D. WT and the mutated forms of miR-377-3p sequence are shown. E. Detection of NEAT1 using qRT-PCR in the sample pulled down by biotinylated miR-377-3p. F. Detection of miR-377-3p using qRT-PCR in the sample pulled down by biotinylated NEAT1 probe. G. The correlation between NEAT1 mRNA and miR-377-3p expression in 96 lung cancer tissues. H. Detection of miR-377-3p using qRT-PCR in the si-NEAT1 or NEAT1 overexpression A549 and H1299 cell lines compared with control group. Assays were performed in triplicate. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Over Expression, Sequencing, Binding Assay, Luciferase, Fluorescence, Transfection, Plasmid Preparation, Quantitative RT-PCR

    miR-377-3p exerts tumor suppressor function through down-regulation of E2F3 in NSCLC cell lines A-B. CCK8 and colony formation assays demonstrated E2F3 reversed the growth inhibitory role of miR-377-3p in A549 and H1299 cells. C. Protein expression of E2F3, cyclin D1, cyclin D2, CDK4, p21, and p57 after transfection. D-E. Transwell migration/invasion assays demonstrated that E2F3 reversed the inhibitory role of miR-377-3p on migration and invasion in A549 and H1299 cells. F. Protein expression of E2F3, MMP-7, and MMP-9 after transfection. G-H. Caspase-3 and caspase-7 activity assays demonstrated that E2F3 reversed the favorable role of miR-377-3p on apoptosis in A549 and H1299 cells. I. Protein expression of E2F3, Bcl2, and cleaved-caspase-3 after transfection. Assays were performed in triplicate. * P
    Figure Legend Snippet: miR-377-3p exerts tumor suppressor function through down-regulation of E2F3 in NSCLC cell lines A-B. CCK8 and colony formation assays demonstrated E2F3 reversed the growth inhibitory role of miR-377-3p in A549 and H1299 cells. C. Protein expression of E2F3, cyclin D1, cyclin D2, CDK4, p21, and p57 after transfection. D-E. Transwell migration/invasion assays demonstrated that E2F3 reversed the inhibitory role of miR-377-3p on migration and invasion in A549 and H1299 cells. F. Protein expression of E2F3, MMP-7, and MMP-9 after transfection. G-H. Caspase-3 and caspase-7 activity assays demonstrated that E2F3 reversed the favorable role of miR-377-3p on apoptosis in A549 and H1299 cells. I. Protein expression of E2F3, Bcl2, and cleaved-caspase-3 after transfection. Assays were performed in triplicate. * P

    Techniques Used: Expressing, Transfection, Migration, Activity Assay

    E2F3 proto-oncogene is a target of miR-377-3p at specific 3′-UTR sites A. miR-377-3p is lowly expressed in primary human lung cancer tissues (n=96) and NSCLC cell lines. B. Scatter plots showing the inverse association between miR-377-3p level and E2F3 mRNA expression in primary human lung cancer tissues (n=96). C. E2F3 is up-regulated in primary human lung cancer tissues (n=96). D. Left pmiR-RB-REPORT™ dual-luciferase reporter vector. Middle The 3′-UTR of E2F3 harbors two miR-377-3p cognate sites. Right Relative luciferase activity of reporter plasmids carrying wild-type or mutant E2F3 3′-UTR in A549 and H1299 cells co-transfected with negative control (NC) or miR-377-3p mimic. E. miR-377-3p suppressed the protein expression of E2F3. Assays were performed in triplicate. * P
    Figure Legend Snippet: E2F3 proto-oncogene is a target of miR-377-3p at specific 3′-UTR sites A. miR-377-3p is lowly expressed in primary human lung cancer tissues (n=96) and NSCLC cell lines. B. Scatter plots showing the inverse association between miR-377-3p level and E2F3 mRNA expression in primary human lung cancer tissues (n=96). C. E2F3 is up-regulated in primary human lung cancer tissues (n=96). D. Left pmiR-RB-REPORT™ dual-luciferase reporter vector. Middle The 3′-UTR of E2F3 harbors two miR-377-3p cognate sites. Right Relative luciferase activity of reporter plasmids carrying wild-type or mutant E2F3 3′-UTR in A549 and H1299 cells co-transfected with negative control (NC) or miR-377-3p mimic. E. miR-377-3p suppressed the protein expression of E2F3. Assays were performed in triplicate. * P

    Techniques Used: Expressing, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis, Transfection, Negative Control

    NEAT1 promotes NSCLC cell growth in vivo by inhibiting miR-377-3p/E2F3 axis A-C. Tumor size, volume, and weight of subcutaneous implantation models of A549 cell are shown. D. NEAT1 expression in tumors isolated from NC, NEAT1, sh-NEAT1, miR-377-3p, and NEAT1+miR-377-3p groups. E. The protein expression of E2F3 in tumors isolated from NC, NEAT1, sh-NEAT1, miR-377-3p, and NEAT1+miR-377-3p groups. F. Immunohistochemistry of Ki67 in tumors isolated from NC, NEAT1, sh-NEAT1, miR-377-3p, and NEAT1+miR-377-3p groups. G. Statistics of Ki 67 IHC. Assays were performed in triplicate. * P
    Figure Legend Snippet: NEAT1 promotes NSCLC cell growth in vivo by inhibiting miR-377-3p/E2F3 axis A-C. Tumor size, volume, and weight of subcutaneous implantation models of A549 cell are shown. D. NEAT1 expression in tumors isolated from NC, NEAT1, sh-NEAT1, miR-377-3p, and NEAT1+miR-377-3p groups. E. The protein expression of E2F3 in tumors isolated from NC, NEAT1, sh-NEAT1, miR-377-3p, and NEAT1+miR-377-3p groups. F. Immunohistochemistry of Ki67 in tumors isolated from NC, NEAT1, sh-NEAT1, miR-377-3p, and NEAT1+miR-377-3p groups. G. Statistics of Ki 67 IHC. Assays were performed in triplicate. * P

    Techniques Used: In Vivo, Expressing, Isolation, Immunohistochemistry

    NEAT1's oncogenic activity is in part through negative regulation of miRNA-377-3p in NSCLC cells Up-regulated miR-377-3p in A549 and H1299 cells, which stably overexpressed NEAT1, largely reversed the favorable effects of NEAT1 on cell proliferation A-B. migration C. and invasion D. Moreover, overexpression of miR-377-3p largely increased the cell apoptosis inhibited by NEAT1 E-F. Assays were performed in triplicate. * P
    Figure Legend Snippet: NEAT1's oncogenic activity is in part through negative regulation of miRNA-377-3p in NSCLC cells Up-regulated miR-377-3p in A549 and H1299 cells, which stably overexpressed NEAT1, largely reversed the favorable effects of NEAT1 on cell proliferation A-B. migration C. and invasion D. Moreover, overexpression of miR-377-3p largely increased the cell apoptosis inhibited by NEAT1 E-F. Assays were performed in triplicate. * P

    Techniques Used: Activity Assay, Stable Transfection, Migration, Over Expression

    12) Product Images from "Expression and Localization of the Cell Adhesion Molecule SgIGSF during Regeneration of the Olfactory Epithelium in Mice"

    Article Title: Expression and Localization of the Cell Adhesion Molecule SgIGSF during Regeneration of the Olfactory Epithelium in Mice

    Journal: Acta Histochemica et Cytochemica

    doi: 10.1267/ahc.06027

    Conventional ( A ) and real-time quantitative ( B ) RT-PCR analyses for SgIGSF mRNA expression in olfactory mucosa after olfactory nerve transection. ( A ) Amplified products for total RNA from olfactory mucosa 0, 4, 7, 11, 15 and 35 days after transection Y (lanes 1–6) were electrophoresed and stained with ethidium bromide. A representative result is shown. ( B ) The relative level of SgIGSF mRNA against GAPDH mRNA at 0 day, arbitrarily set as 1, is plotted against the corresponding values after transection. Each point represents mean±SD of 6 samples. * Significantly different from 0 day value (P
    Figure Legend Snippet: Conventional ( A ) and real-time quantitative ( B ) RT-PCR analyses for SgIGSF mRNA expression in olfactory mucosa after olfactory nerve transection. ( A ) Amplified products for total RNA from olfactory mucosa 0, 4, 7, 11, 15 and 35 days after transection Y (lanes 1–6) were electrophoresed and stained with ethidium bromide. A representative result is shown. ( B ) The relative level of SgIGSF mRNA against GAPDH mRNA at 0 day, arbitrarily set as 1, is plotted against the corresponding values after transection. Each point represents mean±SD of 6 samples. * Significantly different from 0 day value (P

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

    13) Product Images from "Transcription factor EGR1 directs tendon differentiation and promotes tendon repair"

    Article Title: Transcription factor EGR1 directs tendon differentiation and promotes tendon repair

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI67521

    EGR1 promotes the formation of tendon-like tissues in a rat model for tendon injury. ( A ) Achilles tendons of adult nude rats were separated from the plantaris and soleus tendons. A total transverse section of the Achilles tendon was created, and both ends were immediately sutured back together with surgical sutures. C3H10T1/2 or C3H10T1/2-EGR1 cells were then implanted in the injured/sutured tendon. C3H10T1/2- and C3H10T1/2-EGR1–grafted tendons were morphologically and histologically analyzed by H E staining of sections along the axis of the tendon, 2 ( B – E ) and 3 ( F – I ) weeks after manipulation. ( F and G ) Holes left by the suture points are indicated by ellipses. All the longitudinal sections ( B – I ) are orientated bone to the left and muscle to the right. ( J ) qRT-PCR analyses of tendon gene expression in nonmanipulated tendons, sham tendons (with no cell application), C3H10T1/2-grafted tendons, and C3H10T1/2-EGR1–grafted tendons 2 weeks after the operation. mRNA levels of nonmanipulated tendons were normalized to 1. ( K ) Collagen quantity was assessed using a hydroxyproline assay in nonmanipulated tendons, sham tendons, C3H10T1/2-grafted tendons, and C3H10T1/2-EGR1–grafted tendons 2 and 3 weeks postoperation. Error bars represent SD ( J and K ). * P
    Figure Legend Snippet: EGR1 promotes the formation of tendon-like tissues in a rat model for tendon injury. ( A ) Achilles tendons of adult nude rats were separated from the plantaris and soleus tendons. A total transverse section of the Achilles tendon was created, and both ends were immediately sutured back together with surgical sutures. C3H10T1/2 or C3H10T1/2-EGR1 cells were then implanted in the injured/sutured tendon. C3H10T1/2- and C3H10T1/2-EGR1–grafted tendons were morphologically and histologically analyzed by H E staining of sections along the axis of the tendon, 2 ( B – E ) and 3 ( F – I ) weeks after manipulation. ( F and G ) Holes left by the suture points are indicated by ellipses. All the longitudinal sections ( B – I ) are orientated bone to the left and muscle to the right. ( J ) qRT-PCR analyses of tendon gene expression in nonmanipulated tendons, sham tendons (with no cell application), C3H10T1/2-grafted tendons, and C3H10T1/2-EGR1–grafted tendons 2 weeks after the operation. mRNA levels of nonmanipulated tendons were normalized to 1. ( K ) Collagen quantity was assessed using a hydroxyproline assay in nonmanipulated tendons, sham tendons, C3H10T1/2-grafted tendons, and C3H10T1/2-EGR1–grafted tendons 2 and 3 weeks postoperation. Error bars represent SD ( J and K ). * P

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

    Link between Egr1 and TGF-β signaling pathway components during tendon cell differentiation in postnatal tendons and injured tendons. ( A ) EGR1-producing C3H10T1/2 cells displayed increased mRNA expression levels of the TGF-β signaling pathway components, Tgfb2 , Tgfbr2 , and Smad7 , compared with control C3H10T1/2 cells. mRNA levels for each gene in the control C3H10T1/2 cells were established at 1. ( B ) Tgfb2 mRNA levels and TGF-β2 quantity were determined in manipulated rat tendons grafted with C3H10T1/2-EGR1 versus control C3H10T1/2 cells 2 weeks postoperation. ( C ) Application of human recombinant TGF-β2 in C3H10T1/2 cells led to an increase in Scx and Col1a1 expression, while that of Egr1 and Tnmd was not induced 1 or 24 hours after TGF-β2 exposure. ( D ) Application of a specific TGF-β inhibitor SB43 on C3H10T1/2-EGR1 cells diminished the mRNA expression levels of Scx and Col1a1 genes. mRNA levels of C3H10T1/2-EGR1 cells treated with DMSO were normalized to 1. ( E ) ChIP assays were performed on tendons from postnatal mice with antibodies against EGR1. ChIP products were analyzed by PCR. Primers targeting a 293-bp fragment of the Tgfb2 promoter identified DNA regions immunoprecipitated by EGR1. ( F ) qRT-PCR analyses of TGF-β pathway components in Egr1 –/– injured tendons versus WT injured tendons 1 week after injury. mRNA levels of injured tendons from Egr1 –/– and WT mice were normalized to those of Gapdh in each experiment. For qRT-PCR analyses, the error bars represent SEM. * P
    Figure Legend Snippet: Link between Egr1 and TGF-β signaling pathway components during tendon cell differentiation in postnatal tendons and injured tendons. ( A ) EGR1-producing C3H10T1/2 cells displayed increased mRNA expression levels of the TGF-β signaling pathway components, Tgfb2 , Tgfbr2 , and Smad7 , compared with control C3H10T1/2 cells. mRNA levels for each gene in the control C3H10T1/2 cells were established at 1. ( B ) Tgfb2 mRNA levels and TGF-β2 quantity were determined in manipulated rat tendons grafted with C3H10T1/2-EGR1 versus control C3H10T1/2 cells 2 weeks postoperation. ( C ) Application of human recombinant TGF-β2 in C3H10T1/2 cells led to an increase in Scx and Col1a1 expression, while that of Egr1 and Tnmd was not induced 1 or 24 hours after TGF-β2 exposure. ( D ) Application of a specific TGF-β inhibitor SB43 on C3H10T1/2-EGR1 cells diminished the mRNA expression levels of Scx and Col1a1 genes. mRNA levels of C3H10T1/2-EGR1 cells treated with DMSO were normalized to 1. ( E ) ChIP assays were performed on tendons from postnatal mice with antibodies against EGR1. ChIP products were analyzed by PCR. Primers targeting a 293-bp fragment of the Tgfb2 promoter identified DNA regions immunoprecipitated by EGR1. ( F ) qRT-PCR analyses of TGF-β pathway components in Egr1 –/– injured tendons versus WT injured tendons 1 week after injury. mRNA levels of injured tendons from Egr1 –/– and WT mice were normalized to those of Gapdh in each experiment. For qRT-PCR analyses, the error bars represent SEM. * P

    Techniques Used: Cell Differentiation, Expressing, Recombinant, Chromatin Immunoprecipitation, Mouse Assay, Polymerase Chain Reaction, Immunoprecipitation, Quantitative RT-PCR

    EGR1-producing C3H10T1/2 cells increase the expression of tendon markers and lose their capacity to differentiate into bone and fat lineages. ( A ) EGR1-producing C3H10T1/2 cells showed a fibroblastic phenotype compared with C3H10T1/2 cells. EGR1-producing C3H10T1/2 cells displayed increased mRNA expression levels of Scx ( B ), of the all the tendon-associated collagens, Col1a1 , Col1a2 , Col3a1 , Col5a1 , Col6a1 , Col12a1 , and Col14a1 ( C ), and of the tendon-associated molecules, Tnmd , Tnc , Fn1 , Dcn , Bgn , and Fbn1 ( D ), but no change in Mkx ( B ) or Eln ( D ) expression levels compared with C3H10T1/2 cells. Relative expression levels of mRNAs for the markers associated with chondrogenic ( E ), osteogenic ( F ), and adipogenic ( G ) differentiation were not increased in EGR1-producing C3H10T1/2 cells compared with C3H10T1/2 cells. Relative mRNA expression levels for Sox6 (chondrogenesis) ( E ), Bglap , and Osx (osteogenesis) ( F ) were downregulated in the presence of EGR1 compared with control C3H10T1/2 cells. Error bars represent SEM. * P
    Figure Legend Snippet: EGR1-producing C3H10T1/2 cells increase the expression of tendon markers and lose their capacity to differentiate into bone and fat lineages. ( A ) EGR1-producing C3H10T1/2 cells showed a fibroblastic phenotype compared with C3H10T1/2 cells. EGR1-producing C3H10T1/2 cells displayed increased mRNA expression levels of Scx ( B ), of the all the tendon-associated collagens, Col1a1 , Col1a2 , Col3a1 , Col5a1 , Col6a1 , Col12a1 , and Col14a1 ( C ), and of the tendon-associated molecules, Tnmd , Tnc , Fn1 , Dcn , Bgn , and Fbn1 ( D ), but no change in Mkx ( B ) or Eln ( D ) expression levels compared with C3H10T1/2 cells. Relative expression levels of mRNAs for the markers associated with chondrogenic ( E ), osteogenic ( F ), and adipogenic ( G ) differentiation were not increased in EGR1-producing C3H10T1/2 cells compared with C3H10T1/2 cells. Relative mRNA expression levels for Sox6 (chondrogenesis) ( E ), Bglap , and Osx (osteogenesis) ( F ) were downregulated in the presence of EGR1 compared with control C3H10T1/2 cells. Error bars represent SEM. * P

    Techniques Used: Expressing

    EGR1 promotes the formation of tendon-like structures from C3H10T1/2 cells. ( A and B ) C3H10T1/2 ( A ) or C3H10T1/2-EGR1 ( B ) fibrin gel constructs. The 3-week-old tendon-like constructs made with C3H10T1/2-EGR1 cells were larger than those made with C3H10T1/2 cells. ( C ) Diameters were significantly larger in the presence of Egr1 compared with those of controls. ( D ) There was no significant increase in cell proliferation based on Ki67 + cell numbers in C3H10T1/2-EGR1 tendon constructs compared with C3H10T1/2 tendon constructs. ( E ) Longitudinal sections of C3H10T1/2 or C3H10T1/2-EGR1 fibrin gel constructs were immunostained with type I collagen antibody. ( F ) An increase in the expression levels of Scx , Col1a1 , and Col1a2 transcripts was observed in tendon-like constructs from C3H10T1/2-EGR1 cells compared with control constructs from C3H10T1/2 cells. * P
    Figure Legend Snippet: EGR1 promotes the formation of tendon-like structures from C3H10T1/2 cells. ( A and B ) C3H10T1/2 ( A ) or C3H10T1/2-EGR1 ( B ) fibrin gel constructs. The 3-week-old tendon-like constructs made with C3H10T1/2-EGR1 cells were larger than those made with C3H10T1/2 cells. ( C ) Diameters were significantly larger in the presence of Egr1 compared with those of controls. ( D ) There was no significant increase in cell proliferation based on Ki67 + cell numbers in C3H10T1/2-EGR1 tendon constructs compared with C3H10T1/2 tendon constructs. ( E ) Longitudinal sections of C3H10T1/2 or C3H10T1/2-EGR1 fibrin gel constructs were immunostained with type I collagen antibody. ( F ) An increase in the expression levels of Scx , Col1a1 , and Col1a2 transcripts was observed in tendon-like constructs from C3H10T1/2-EGR1 cells compared with control constructs from C3H10T1/2 cells. * P

    Techniques Used: Construct, Expressing

    14) Product Images from "Targeting BRAFV600E with PLX4720 Displays Potent Antimigratory and Anti-invasive Activity in Preclinical Models of Human Thyroid Cancer"

    Article Title: Targeting BRAFV600E with PLX4720 Displays Potent Antimigratory and Anti-invasive Activity in Preclinical Models of Human Thyroid Cancer

    Journal: The Oncologist

    doi: 10.1634/theoncologist.2010-0317

    p-ERK-1/ERK-2 expression in thyroid cancer cells. (A): B-Raf V600E mutation in 8505c cells; TPC-1 cells have wt B-Raf and harbor the PIK3CA H1047R mutation and RET/PTC-1 translocation. (B): One hour of 1 μM PLX4720 treatment resulted in lower p-ERK-1/ERK-2 levels (by Western blotting) in 8505c cells, but higher levels in TPC-1 cells. p-ERK-1/ERK-2 levels were lower in 8505c cells and TPC-1 cells with 10 μM PLX4720. (C): 72 hours of PLX4720 treatment resulted in lower p-ERK-1/ERK-2 levels in 8505c cells and higher levels in TPC-1 cells. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular signal–related kinase; p, phosphorylated; wt, wild-type.
    Figure Legend Snippet: p-ERK-1/ERK-2 expression in thyroid cancer cells. (A): B-Raf V600E mutation in 8505c cells; TPC-1 cells have wt B-Raf and harbor the PIK3CA H1047R mutation and RET/PTC-1 translocation. (B): One hour of 1 μM PLX4720 treatment resulted in lower p-ERK-1/ERK-2 levels (by Western blotting) in 8505c cells, but higher levels in TPC-1 cells. p-ERK-1/ERK-2 levels were lower in 8505c cells and TPC-1 cells with 10 μM PLX4720. (C): 72 hours of PLX4720 treatment resulted in lower p-ERK-1/ERK-2 levels in 8505c cells and higher levels in TPC-1 cells. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular signal–related kinase; p, phosphorylated; wt, wild-type.

    Techniques Used: Expressing, Mutagenesis, Translocation Assay, Western Blot

    Tumor growth and lung metastasis in an orthotopic mouse model of human anaplastic thyroid cancer. (A): Three weeks of PLX4720 treatment resulted in lower orthotopic tumor growth than in controls (*** p
    Figure Legend Snippet: Tumor growth and lung metastasis in an orthotopic mouse model of human anaplastic thyroid cancer. (A): Three weeks of PLX4720 treatment resulted in lower orthotopic tumor growth than in controls (*** p

    Techniques Used:

    PLX4720 inhibits B-Raf V600E -positive thyroid tumor aggressiveness in vivo. (A): Control mice with B-Raf V600E 8505c orthotopic tumors (H E stain) were large (A1) and showed extrathyroidal extension into the trachea (A1, A2) and skeletal muscle (A3) with atypical mitoses ( A4 , arrow); PLX4720-treated tumors (H E stain) were small and discrete (A5–A7) (arrows) and largely confined to the thyroid bed, with atypical pyknotic nuclei (A8) . (B): Control SCID mice (H E stain) had metastatic foci of carcinoma to the lung interstitium (arrows, B1, B2 ) as well as a paratracheal lymph node (asterisk, (B3) ; higher power, arrow (B4) ); PLX4720-treated mice had no evidence of metastasis to the lung ( B5, B6 ) or lymph nodes ( B7, B8 ). (C): PLX4720-treated tumors had greater nuclear staining for TTF-1 and PAX-8 (right) than untreated control mice (left). Abbreviations: H E, hematoxylin and eosin; SCID, severe combined immunodeficient; TTF-1, thyroid transcription factor.
    Figure Legend Snippet: PLX4720 inhibits B-Raf V600E -positive thyroid tumor aggressiveness in vivo. (A): Control mice with B-Raf V600E 8505c orthotopic tumors (H E stain) were large (A1) and showed extrathyroidal extension into the trachea (A1, A2) and skeletal muscle (A3) with atypical mitoses ( A4 , arrow); PLX4720-treated tumors (H E stain) were small and discrete (A5–A7) (arrows) and largely confined to the thyroid bed, with atypical pyknotic nuclei (A8) . (B): Control SCID mice (H E stain) had metastatic foci of carcinoma to the lung interstitium (arrows, B1, B2 ) as well as a paratracheal lymph node (asterisk, (B3) ; higher power, arrow (B4) ); PLX4720-treated mice had no evidence of metastasis to the lung ( B5, B6 ) or lymph nodes ( B7, B8 ). (C): PLX4720-treated tumors had greater nuclear staining for TTF-1 and PAX-8 (right) than untreated control mice (left). Abbreviations: H E, hematoxylin and eosin; SCID, severe combined immunodeficient; TTF-1, thyroid transcription factor.

    Techniques Used: In Vivo, Mouse Assay, H&E Stain, Staining

    Migration and invasion assays with PLX4720 treatment in thyroid cancer cell models. Treatment with 1 μM (A) or 10 μM (B) PLX4720 reduced migration and invasion in 8505c cells (*** p
    Figure Legend Snippet: Migration and invasion assays with PLX4720 treatment in thyroid cancer cell models. Treatment with 1 μM (A) or 10 μM (B) PLX4720 reduced migration and invasion in 8505c cells (*** p

    Techniques Used: Migration

    PLX4720 inhibits proliferation in thyroid cancer cells. (A–C): 1 μM PLX4720 reduced BrdU incorporation in TPC-1 cells (* p = .01); 10 μM PLX4720 reduced BrdU uptake in 8505c and TPC-1 cells (*** p
    Figure Legend Snippet: PLX4720 inhibits proliferation in thyroid cancer cells. (A–C): 1 μM PLX4720 reduced BrdU incorporation in TPC-1 cells (* p = .01); 10 μM PLX4720 reduced BrdU uptake in 8505c and TPC-1 cells (*** p

    Techniques Used: BrdU Incorporation Assay

    Gene expression with PLX4720 treatment. Quantitative real-time RT-PCR shows mRNA copy number of CCND1 (A) , TSP-1 (B) , ITGA6 (C) , and TTF-1 (D) mRNA in 8505c and TPC-1 cells after 24 hours or 72 hours of treatment with PLX4720 (either 1 μM or 10 μM). Abbreviations: CCND1, cyclin D1; ITGA6, integrin α6; RT-PCR, reverse transcriptase polymerase chain reaction; TSP-1, thrombospondin-1; TTF-1, thyroid transcription factor; wt, wild-type.
    Figure Legend Snippet: Gene expression with PLX4720 treatment. Quantitative real-time RT-PCR shows mRNA copy number of CCND1 (A) , TSP-1 (B) , ITGA6 (C) , and TTF-1 (D) mRNA in 8505c and TPC-1 cells after 24 hours or 72 hours of treatment with PLX4720 (either 1 μM or 10 μM). Abbreviations: CCND1, cyclin D1; ITGA6, integrin α6; RT-PCR, reverse transcriptase polymerase chain reaction; TSP-1, thrombospondin-1; TTF-1, thyroid transcription factor; wt, wild-type.

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

    15) Product Images from "αMSH inhibits adipose inflammation via reducing FoxOs transcription and blocking Akt/JNK pathway in mice"

    Article Title: αMSH inhibits adipose inflammation via reducing FoxOs transcription and blocking Akt/JNK pathway in mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17465

    Foxo4 negatively regulate MC5R transcription in αMSH inhibited inflammation in mice adipocytes (A) Fragments of MC5R promoter fused to a luciferase reporter plasmid or PGL3-basic (control) were co-transfected into cells together with Renlilla plasmid and pAd-Foxo4 (n=3). Luciferase activity was corrected for Renilla luciferase activity and normalized to control activity (n=3). (B) Chromatin immunoprecipitation (ChIP) analysis of Foxo4 and MC5R interaction. (C, D) After pAd-Foxo4 together with αMSH or pc-MC5R in LPS/saline treatment, MC5R mRNA level was determined in adipocytes (n=3). (E) When adipocytes were treated with pAd-Foxo4 and αMSH in LPS/saline treatment, mRNA levels of Foxo4 , IL-6 , TNFα , MCP-1 and Leptin were analyzed (n=3). Values are means ± SD. vs. control group, * P
    Figure Legend Snippet: Foxo4 negatively regulate MC5R transcription in αMSH inhibited inflammation in mice adipocytes (A) Fragments of MC5R promoter fused to a luciferase reporter plasmid or PGL3-basic (control) were co-transfected into cells together with Renlilla plasmid and pAd-Foxo4 (n=3). Luciferase activity was corrected for Renilla luciferase activity and normalized to control activity (n=3). (B) Chromatin immunoprecipitation (ChIP) analysis of Foxo4 and MC5R interaction. (C, D) After pAd-Foxo4 together with αMSH or pc-MC5R in LPS/saline treatment, MC5R mRNA level was determined in adipocytes (n=3). (E) When adipocytes were treated with pAd-Foxo4 and αMSH in LPS/saline treatment, mRNA levels of Foxo4 , IL-6 , TNFα , MCP-1 and Leptin were analyzed (n=3). Values are means ± SD. vs. control group, * P

    Techniques Used: Mouse Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Chromatin Immunoprecipitation

    Proposed mechanism of the inhibition of αMSH on adipose inflammation αMSH decreases adipose inflammation via blunting the phosphorylation of Akt/JNK. Akt phosphorylation activates Foxo1 and Foxo3a, while JNK phosphorylation activates Foxo4. What is more, Foxo4 acts via binding the promoter of MC5R, which is the receptor of αMSH.
    Figure Legend Snippet: Proposed mechanism of the inhibition of αMSH on adipose inflammation αMSH decreases adipose inflammation via blunting the phosphorylation of Akt/JNK. Akt phosphorylation activates Foxo1 and Foxo3a, while JNK phosphorylation activates Foxo4. What is more, Foxo4 acts via binding the promoter of MC5R, which is the receptor of αMSH.

    Techniques Used: Inhibition, Binding Assay

    FoxOs reverse the inhibition of αMSH on adipose inflammation in mice Mice white adipose tissue was isolated after intraperitoneal injection with pAd-Foxo1 and with another 500 nM αMSH, then mRNA levels of Foxo1 , MC5R (A) , M1, M2 markers (C) , IL-6 , MCP-1 , Leptin (D) were measured (n=6). After intraperitoneal injection with pAd-Foxo1 and with another 500 nM αMSH, serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (B) were detected (n=6). After intraperitoneal injection with pAd-Foxo3a and with another 500 nM αMSH to mice, mRNA levels of Foxo3a , MC5R (E) , M1, M2 markers (G) , IL-6 , MCP-1 , Leptin (H) were measured in white adipose tissue (n=6). Serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (F) were also detected (n=6). Mice were intraperitoneal injected with pAd-Foxo4 and another 500 nM αMSH, serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (J) and mRNA levels of Foxo4 , MC5R (I) , M1, M2 markers (K) , IL-6 , MCP-1 , Leptin (L) were measured (n=6). Values are means ± SD. vs. control group, * P
    Figure Legend Snippet: FoxOs reverse the inhibition of αMSH on adipose inflammation in mice Mice white adipose tissue was isolated after intraperitoneal injection with pAd-Foxo1 and with another 500 nM αMSH, then mRNA levels of Foxo1 , MC5R (A) , M1, M2 markers (C) , IL-6 , MCP-1 , Leptin (D) were measured (n=6). After intraperitoneal injection with pAd-Foxo1 and with another 500 nM αMSH, serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (B) were detected (n=6). After intraperitoneal injection with pAd-Foxo3a and with another 500 nM αMSH to mice, mRNA levels of Foxo3a , MC5R (E) , M1, M2 markers (G) , IL-6 , MCP-1 , Leptin (H) were measured in white adipose tissue (n=6). Serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (F) were also detected (n=6). Mice were intraperitoneal injected with pAd-Foxo4 and another 500 nM αMSH, serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (J) and mRNA levels of Foxo4 , MC5R (I) , M1, M2 markers (K) , IL-6 , MCP-1 , Leptin (L) were measured (n=6). Values are means ± SD. vs. control group, * P

    Techniques Used: Inhibition, Mouse Assay, Isolation, Injection

    FoxOs abolish the suppression of αMSH on inflammation in mice adipocytes (A) Oil Red O staining for differentiated primary adipocytes isolated from epididymal white adipose tissue after administration of αMSH for 1 h (left). Relative concentration of TG in adipocytes (middle) and FFA in cell culture medium (right) were detected (n=3). With αMSH treatment, mRNA levels of MC5R (B), IL-6 , TNFα , MCP-1 and Leptin (C) , Foxo1 , Foxo3a , Foxo4 and Foxo6 (D) , protein levels of p-AKT and total Akt (E) were detected in adipocytes (n=3). (F) Adipocytes treated with pAd-Foxo1 and αMSH, protein levels of IL-6, TNFα, MCP-1 and Leptin were measured (n=3). (G) Expression levels for Foxo3a, p-Akt, Akt and IL-6 protein after cells treated with pAd-Foxo3a and αMSH (n=3). (H) Normalized mRNA levels of Foxo4 , MC5R , Leptin and IL-6 with pAd-Foxo4 and αMSH treatments (n=3). Values are means ± SD. vs. control group, * P
    Figure Legend Snippet: FoxOs abolish the suppression of αMSH on inflammation in mice adipocytes (A) Oil Red O staining for differentiated primary adipocytes isolated from epididymal white adipose tissue after administration of αMSH for 1 h (left). Relative concentration of TG in adipocytes (middle) and FFA in cell culture medium (right) were detected (n=3). With αMSH treatment, mRNA levels of MC5R (B), IL-6 , TNFα , MCP-1 and Leptin (C) , Foxo1 , Foxo3a , Foxo4 and Foxo6 (D) , protein levels of p-AKT and total Akt (E) were detected in adipocytes (n=3). (F) Adipocytes treated with pAd-Foxo1 and αMSH, protein levels of IL-6, TNFα, MCP-1 and Leptin were measured (n=3). (G) Expression levels for Foxo3a, p-Akt, Akt and IL-6 protein after cells treated with pAd-Foxo3a and αMSH (n=3). (H) Normalized mRNA levels of Foxo4 , MC5R , Leptin and IL-6 with pAd-Foxo4 and αMSH treatments (n=3). Values are means ± SD. vs. control group, * P

    Techniques Used: Mouse Assay, Staining, Isolation, Concentration Assay, Cell Culture, Expressing

    Foxo1 and Foxo3a reverse the inhibition of αMSH on LPS-induced inflammation in mice adipocytes (A) Primary adipocytes were cultured and incubated for 0 h, 12 h, 24 h and 48 h in the presence of 1 μg/mL LPS. Cell viability was detected by CCK-8 (n=3). (B) Relative mRNA expressions of IL-6 , Leptin , MC5R , Foxo1 , Foxo3a , Foxo4 , Foxo6 and Caspase3 with αMSH treatment were analyzed (n=3). (C) After adipocytes treated with pAd-Foxo1 and incubated for LPS/saline and αMSH, mRNA levels of Foxo1 , IL-6 , MCP-1 and Leptin were detected (n=3). (D) Adipocytes were treated with pAd-Foxo3a and incubated for LPS/saline and αMSH, then measured mRNA levels of Foxo3a , IL-6 , MCP-1 and Leptin (n=3). Values are means ± SD. vs. control group, * P
    Figure Legend Snippet: Foxo1 and Foxo3a reverse the inhibition of αMSH on LPS-induced inflammation in mice adipocytes (A) Primary adipocytes were cultured and incubated for 0 h, 12 h, 24 h and 48 h in the presence of 1 μg/mL LPS. Cell viability was detected by CCK-8 (n=3). (B) Relative mRNA expressions of IL-6 , Leptin , MC5R , Foxo1 , Foxo3a , Foxo4 , Foxo6 and Caspase3 with αMSH treatment were analyzed (n=3). (C) After adipocytes treated with pAd-Foxo1 and incubated for LPS/saline and αMSH, mRNA levels of Foxo1 , IL-6 , MCP-1 and Leptin were detected (n=3). (D) Adipocytes were treated with pAd-Foxo3a and incubated for LPS/saline and αMSH, then measured mRNA levels of Foxo3a , IL-6 , MCP-1 and Leptin (n=3). Values are means ± SD. vs. control group, * P

    Techniques Used: Inhibition, Mouse Assay, Cell Culture, Incubation, CCK-8 Assay

    Akt/JNK signal pathway is impaired in the inhibition of αMSH on adipocyte inflammation and FoxOs expressions Mouse adipocytes were pretreated with αMSH and MK-2206 or SP600125, respectively. Relative protein levels of Akt, p-Akt ser473 , JNK, p-JNK Thr183 (A) , Foxo1, Foxo3a, Foxo4, IL-6 and Leptin (B) with or without MK-2206 (n=3). Representative immunoblots and densitometric quantification for Akt, p-Akt ser473 , JNK, p-JNK Thr183 (C) , Foxo1, Foxo3a, Foxo4, IL-6 and Leptin protein (D) with or without SP600125 (n=3). The level of total GAPDH was used as the loading control. Values are mean ± SD. * P
    Figure Legend Snippet: Akt/JNK signal pathway is impaired in the inhibition of αMSH on adipocyte inflammation and FoxOs expressions Mouse adipocytes were pretreated with αMSH and MK-2206 or SP600125, respectively. Relative protein levels of Akt, p-Akt ser473 , JNK, p-JNK Thr183 (A) , Foxo1, Foxo3a, Foxo4, IL-6 and Leptin (B) with or without MK-2206 (n=3). Representative immunoblots and densitometric quantification for Akt, p-Akt ser473 , JNK, p-JNK Thr183 (C) , Foxo1, Foxo3a, Foxo4, IL-6 and Leptin protein (D) with or without SP600125 (n=3). The level of total GAPDH was used as the loading control. Values are mean ± SD. * P

    Techniques Used: Inhibition, Western Blot

    αMSH weakens LPS-induced adipose inflammation by inhibiting FoxOs expressions in mice (A) Serum protein levels of IL-1β, IL-6, IL-4 and IL-10 after mice with LPS or saline intraperitoneal injection (n=6). (B) Measurement of serum αMSH level before and after LPS or saline intraperitoneal injection (n=6). Epididymis white adipose tissue was isolated after intraperitoneal injection with LPS or saline, then mRNA levels of MC5R (C) , Foxo1 , Foxo3a , Foxo4 and Foxo6 (D) were detected (n=6). Base on the LPS/saline injection, mice were injected with another 500 nM αMSH, then further detected for relative serum protein levels of IL-1β, IL-6, IL-4 (E) and mRNA levels of M1, M2 markers (F) , MC5R (G) , IL-6 , MCP-1 , Leptin (H) , Foxo1 , Foxo3a , Foxo4 and Foxo6 (I) in white adipose tissue (n=6). Values are means ± SD. vs. control group, * P
    Figure Legend Snippet: αMSH weakens LPS-induced adipose inflammation by inhibiting FoxOs expressions in mice (A) Serum protein levels of IL-1β, IL-6, IL-4 and IL-10 after mice with LPS or saline intraperitoneal injection (n=6). (B) Measurement of serum αMSH level before and after LPS or saline intraperitoneal injection (n=6). Epididymis white adipose tissue was isolated after intraperitoneal injection with LPS or saline, then mRNA levels of MC5R (C) , Foxo1 , Foxo3a , Foxo4 and Foxo6 (D) were detected (n=6). Base on the LPS/saline injection, mice were injected with another 500 nM αMSH, then further detected for relative serum protein levels of IL-1β, IL-6, IL-4 (E) and mRNA levels of M1, M2 markers (F) , MC5R (G) , IL-6 , MCP-1 , Leptin (H) , Foxo1 , Foxo3a , Foxo4 and Foxo6 (I) in white adipose tissue (n=6). Values are means ± SD. vs. control group, * P

    Techniques Used: Mouse Assay, Injection, Isolation

    16) Product Images from "A Novel Histone Deacetylase Inhibitor Exhibits Antitumor Activity via Apoptosis Induction, F-Actin Disruption and Gene Acetylation in Lung Cancer"

    Article Title: A Novel Histone Deacetylase Inhibitor Exhibits Antitumor Activity via Apoptosis Induction, F-Actin Disruption and Gene Acetylation in Lung Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012417

    The antitumor activity of OSU-HDAC-44 via cytokinese defect, F-actin disruption, apoptosis induction, and gene acetylation. OSU-HDAC-44 is a novel pan-HDAC inhibitor that exhibits a broad spectrum of antitumor activities in NSCLC cell and xenograft models, which involves histone acetylation-dependent activation of gene transcription in nucleus. For example, re-expression of NR4A1 and FOXO4 along with caspase activation induces intrinsic apoptosis. In addition, RhoA/F-actin motility control is inhibited by srGAP1 resulting from activation by OSU-HDAC-44. OSU-HDAC-44 also induces post-translational down-regulation of mitotic regulators, Aurora B and survivin leading to cytokinese defect and apoptosis.
    Figure Legend Snippet: The antitumor activity of OSU-HDAC-44 via cytokinese defect, F-actin disruption, apoptosis induction, and gene acetylation. OSU-HDAC-44 is a novel pan-HDAC inhibitor that exhibits a broad spectrum of antitumor activities in NSCLC cell and xenograft models, which involves histone acetylation-dependent activation of gene transcription in nucleus. For example, re-expression of NR4A1 and FOXO4 along with caspase activation induces intrinsic apoptosis. In addition, RhoA/F-actin motility control is inhibited by srGAP1 resulting from activation by OSU-HDAC-44. OSU-HDAC-44 also induces post-translational down-regulation of mitotic regulators, Aurora B and survivin leading to cytokinese defect and apoptosis.

    Techniques Used: Activity Assay, Activation Assay, Expressing

    Chemical structure, molecular docking analysis, and the effect of OSU-HDAC-44 on cell viability. ( A ) Chemical structure of OSU-HDAC-44 and SAHA. ( B ) Molecular docking analysis of OSU-HDAC-44 and SAHA. The structures of OSU-HDAC-44 and SAHA were calculated and the docking mode on catalytic domain of HDAC8 was predicted using the docking program GOLD 4.0.1. ( C ) Dose-dependent effects of OSU-HDAC-44 ( left ) and SAHA ( right ) on cell viability in IMR90, H1299, A549 and CL1-1 cells. Cells were treated with 0.5–10 µM of OSU-HDAC-44 or SAHA for 48 h, and cell viability was assessed by trypan blue exclusion assay. ( D ) OSU-HDAC-44 synergized with cisplatin to suppress cell proliferation. Cells were exposed to cisplatin (Cis) alone for 4 h, OSU-HDAC-44 (HDAC-44) alone for 48 h, or pretreated with OSU-HDAC-44 for 48 h before cisplatin treatment for 4 h, and then drug were withdrew and cells were incubated with drug-free media for additional 48 h. Cell viability was assessed by trypan blue exclusion assay. CL1-1 cells were treated with 4.4 µM cisplatin or 0.3 µM OSU-HDAC-44. A549 cells were treated with 1.6 µM cisplatin or 0.2 µM OSU-HDAC-44. Data represent mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: Chemical structure, molecular docking analysis, and the effect of OSU-HDAC-44 on cell viability. ( A ) Chemical structure of OSU-HDAC-44 and SAHA. ( B ) Molecular docking analysis of OSU-HDAC-44 and SAHA. The structures of OSU-HDAC-44 and SAHA were calculated and the docking mode on catalytic domain of HDAC8 was predicted using the docking program GOLD 4.0.1. ( C ) Dose-dependent effects of OSU-HDAC-44 ( left ) and SAHA ( right ) on cell viability in IMR90, H1299, A549 and CL1-1 cells. Cells were treated with 0.5–10 µM of OSU-HDAC-44 or SAHA for 48 h, and cell viability was assessed by trypan blue exclusion assay. ( D ) OSU-HDAC-44 synergized with cisplatin to suppress cell proliferation. Cells were exposed to cisplatin (Cis) alone for 4 h, OSU-HDAC-44 (HDAC-44) alone for 48 h, or pretreated with OSU-HDAC-44 for 48 h before cisplatin treatment for 4 h, and then drug were withdrew and cells were incubated with drug-free media for additional 48 h. Cell viability was assessed by trypan blue exclusion assay. CL1-1 cells were treated with 4.4 µM cisplatin or 0.3 µM OSU-HDAC-44. A549 cells were treated with 1.6 µM cisplatin or 0.2 µM OSU-HDAC-44. Data represent mean ± SEM from three independent experiments. * P

    Techniques Used: Trypan Blue Exclusion Assay, Incubation

    The body weight, H E staining of major organs, and hematological biochemistry examinations of tested animals. ( A ) OSU-HDAC-44 treatments did not cause significant body weight loss of tested animals. ( B ) H E staining of paraffin-embedded, 5 µm thick sections of the heart, liver, lung and kidney from OSU-HDAC-44-treated and untreated mice with A549 xenografts. There were no apparent histopathologic differences between these tissues sections (original magnification ×200). ( C ) Hematological biochemistry tests including GOT, GPT, albumin, BUN, creatinine, and WBC were examined and the results showed no significant differences between DMSO and OSU-HDAC-44 treatment.
    Figure Legend Snippet: The body weight, H E staining of major organs, and hematological biochemistry examinations of tested animals. ( A ) OSU-HDAC-44 treatments did not cause significant body weight loss of tested animals. ( B ) H E staining of paraffin-embedded, 5 µm thick sections of the heart, liver, lung and kidney from OSU-HDAC-44-treated and untreated mice with A549 xenografts. There were no apparent histopathologic differences between these tissues sections (original magnification ×200). ( C ) Hematological biochemistry tests including GOT, GPT, albumin, BUN, creatinine, and WBC were examined and the results showed no significant differences between DMSO and OSU-HDAC-44 treatment.

    Techniques Used: Staining, Mouse Assay

    Effect of OSU-HDAC-44 on the biomarkers associated with broad inhibition on numerous HDACs. Dose-dependent effects ( A ) and time-dependent effects ( B ) of OSU-HDAC-44 on the histone and non-histone proteins. Ac-H3, acetylated histone H3; Ac-H4, acetylated histone H4; Ac-p53, acetylated p53; p53, total p53. ( C ) OSU-HDAC-44 suppressed activities of class I (HDAC1 and HDAC8), class II (HDAC4 and HDAC6), and class IV (HDAC11) HDACs. Different HDAC isotypes were immunoprecipitated from H1299 nuclear extract by specific antibodies, and then subjected to in vitro HDAC inhibition assay as described in Materials and Methods section. Data represent mean ± SEM from three independent experiments. ** P
    Figure Legend Snippet: Effect of OSU-HDAC-44 on the biomarkers associated with broad inhibition on numerous HDACs. Dose-dependent effects ( A ) and time-dependent effects ( B ) of OSU-HDAC-44 on the histone and non-histone proteins. Ac-H3, acetylated histone H3; Ac-H4, acetylated histone H4; Ac-p53, acetylated p53; p53, total p53. ( C ) OSU-HDAC-44 suppressed activities of class I (HDAC1 and HDAC8), class II (HDAC4 and HDAC6), and class IV (HDAC11) HDACs. Different HDAC isotypes were immunoprecipitated from H1299 nuclear extract by specific antibodies, and then subjected to in vitro HDAC inhibition assay as described in Materials and Methods section. Data represent mean ± SEM from three independent experiments. ** P

    Techniques Used: Inhibition, Immunoprecipitation, In Vitro

    OSU-HDAC-44 effectively induced apoptosis and inhibited A549 xenograft growth. ( A ) Mice bearing the established A549 tumors (∼50 mm 3 ) were injected intraperitoneally with 7.5, 15 or 30 mg/kg of OSU-HDAC-44 or 1.5 mg/kg of TSA 3 days/week for three weeks. Eight mice per group were used in the xenograft experiment. The tumor volumes of mice were measured. Points, mean; error bars, 95% confidence intervals. P values were for comparisons with vehicle control (* P
    Figure Legend Snippet: OSU-HDAC-44 effectively induced apoptosis and inhibited A549 xenograft growth. ( A ) Mice bearing the established A549 tumors (∼50 mm 3 ) were injected intraperitoneally with 7.5, 15 or 30 mg/kg of OSU-HDAC-44 or 1.5 mg/kg of TSA 3 days/week for three weeks. Eight mice per group were used in the xenograft experiment. The tumor volumes of mice were measured. Points, mean; error bars, 95% confidence intervals. P values were for comparisons with vehicle control (* P

    Techniques Used: Mouse Assay, Injection

    OSU-HDAC-44 induces cytokinesis inhibition and subsequently leads to intrinsic apoptosis. ( A ) The effects of OSU-HDAC-44 on cell cycle distribution in A549 and H1299 cells. Cells were treated with 2.5 µM OSU-HDAC-44 or 5 µM SAHA for indicated times and assessed by flow cytometry. Left , results from one representative experiment are shown. Right , the mean percentage of G2/M and sub-G1 fraction population is plotted in the histogram. ( B ) The bi-nucleated cells and dysregulation of F-actin induced by OSU-HSAC-44. Cells were treated with 2.5 µM OSU-HDAC-44 for 48 h, and then fixed and stained with DAPI (DNA) and phalloidin (F-actin). Asterisk pointed to the bi-nucleus. Scale bars: 30 µm. ( C ) OSU-HDAC-44 induced degradation of Aurora B and survivin via 26S proteasome pathway. Upper , time-dependent decreases in Aurora B and survivin protein levels after 2.5 µM OSU-HDAC-44 treatment. Middle , A549 cells were treated with 2.5 µM OSU-HDAC-44 in the presence or absence of MG132 for 24 h. Lower , A549 cells were treated with 2.5 µM OSU-HDAC-44 for 24 h and cell lysate was subjected to IP assay using anti-Aurora B or anti-survivin specific antibodies and blotted with anti-ubiquitination antibody (Anti-Ub). ( D ) Caspase activity assay ( left) and Western blot analyses ( Right) confirmed that OSU-HDAC-44 induced intrinsic apoptosis pathway. Cells were treated with 2.5 µM OSU-HDAC-44 for indicated times and the subjected to caspase activity assay and Western blot analyses. Data represent mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: OSU-HDAC-44 induces cytokinesis inhibition and subsequently leads to intrinsic apoptosis. ( A ) The effects of OSU-HDAC-44 on cell cycle distribution in A549 and H1299 cells. Cells were treated with 2.5 µM OSU-HDAC-44 or 5 µM SAHA for indicated times and assessed by flow cytometry. Left , results from one representative experiment are shown. Right , the mean percentage of G2/M and sub-G1 fraction population is plotted in the histogram. ( B ) The bi-nucleated cells and dysregulation of F-actin induced by OSU-HSAC-44. Cells were treated with 2.5 µM OSU-HDAC-44 for 48 h, and then fixed and stained with DAPI (DNA) and phalloidin (F-actin). Asterisk pointed to the bi-nucleus. Scale bars: 30 µm. ( C ) OSU-HDAC-44 induced degradation of Aurora B and survivin via 26S proteasome pathway. Upper , time-dependent decreases in Aurora B and survivin protein levels after 2.5 µM OSU-HDAC-44 treatment. Middle , A549 cells were treated with 2.5 µM OSU-HDAC-44 in the presence or absence of MG132 for 24 h. Lower , A549 cells were treated with 2.5 µM OSU-HDAC-44 for 24 h and cell lysate was subjected to IP assay using anti-Aurora B or anti-survivin specific antibodies and blotted with anti-ubiquitination antibody (Anti-Ub). ( D ) Caspase activity assay ( left) and Western blot analyses ( Right) confirmed that OSU-HDAC-44 induced intrinsic apoptosis pathway. Cells were treated with 2.5 µM OSU-HDAC-44 for indicated times and the subjected to caspase activity assay and Western blot analyses. Data represent mean ± SEM from three independent experiments. * P

    Techniques Used: Inhibition, Flow Cytometry, Cytometry, Staining, Caspase Activity Assay, Western Blot

    OSU-HDAC-44 decreased RhoA activity via srGAP1 induction, leading to F-actin dysregulation. ( A ) Chromatin-immunoprecipitation-PCR analyses confirmed that treatment with 2.5 µM OSU-HDAC-44 for 2 h induced acetylation of histone H3 (H3K9K14Ac) in the promoter region of srGAP1 , NR4A1 and FOXO4 genes. ( B ) OSU-HDAC-44 increased the mRNA levels of srGAP1 , NR4A1 and FOXO4 genes using real-time RT-PCR analyses. Cells were treated with 2.5 µM OSU-HDAC-44 for 24 h and total RNA was extracted for the real-time RT-PCR analyses. Data represent mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: OSU-HDAC-44 decreased RhoA activity via srGAP1 induction, leading to F-actin dysregulation. ( A ) Chromatin-immunoprecipitation-PCR analyses confirmed that treatment with 2.5 µM OSU-HDAC-44 for 2 h induced acetylation of histone H3 (H3K9K14Ac) in the promoter region of srGAP1 , NR4A1 and FOXO4 genes. ( B ) OSU-HDAC-44 increased the mRNA levels of srGAP1 , NR4A1 and FOXO4 genes using real-time RT-PCR analyses. Cells were treated with 2.5 µM OSU-HDAC-44 for 24 h and total RNA was extracted for the real-time RT-PCR analyses. Data represent mean ± SEM from three independent experiments. * P

    Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Quantitative RT-PCR

    17) Product Images from "Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development"

    Article Title: Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development

    Journal: BMC Biology

    doi: 10.1186/1741-7007-12-5

    EmIR1 electron microscopic analyses. The anti-EmIR1 antiserum and gold-coupled anti-rabbit antibodies were used to detect EmIR1 in the metacestode germinal layer. A) Overview showing the location of the laminated layer, the germinal layer, undifferentiated (stem) cells and glycogen storage cells. B) Glycogen storage cell showing massive anti-EmIR1 staining (arrows). Scale bar in larger image represents 0.6 μm, scale bar in insert represents 3.6 μm.
    Figure Legend Snippet: EmIR1 electron microscopic analyses. The anti-EmIR1 antiserum and gold-coupled anti-rabbit antibodies were used to detect EmIR1 in the metacestode germinal layer. A) Overview showing the location of the laminated layer, the germinal layer, undifferentiated (stem) cells and glycogen storage cells. B) Glycogen storage cell showing massive anti-EmIR1 staining (arrows). Scale bar in larger image represents 0.6 μm, scale bar in insert represents 3.6 μm.

    Techniques Used: Staining

    Effects of insulin on metacestode glucose uptake. Uptake of C 14 -D-glucose by metacestode vesicles in the presence of 10 μM insulin alone,and insulin together with Na 3 VO 4 . Control was set to 1 and results were normalised against the control. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P
    Figure Legend Snippet: Effects of insulin on metacestode glucose uptake. Uptake of C 14 -D-glucose by metacestode vesicles in the presence of 10 μM insulin alone,and insulin together with Na 3 VO 4 . Control was set to 1 and results were normalised against the control. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P

    Techniques Used:

    Effects of insulin on the phosphorylation of metacestode vesicle proteins. A) Detection of EmIR1 in vesicle membrane fractions (MF). Vesicles from in vitro culture were homogenized and the MF was isolated. Western blot detection was carried out on MF and whole vesicle preparations (Ves) using the anti-EmIR1 antiserum. ‘Pro’ and ‘β’ mark the receptor pro-form and β-subunits. B) Phosphorylation of EmIR1 in response to insulin. Metacestode MF was stimulated for 10 minutes with either 100 nM insulin or IGF-I, followed by 30 minutes incubation with 100 μM HNMPA(AM) 3 or control DMSO. Phosphorylation of membrane proteins was carried out for 40 minutes in the presence of [ 32 P] γ-ATP. Proteins of the MF were precipitated using the anti-EmIR1 antiserum. Proteins were then separated by 8% SDS-PAGE and phosphorylation was detected by autoradiography. Bands are visible at the size of the EmIR1 β-subunit and below. C) Phosphorylation of EmIR1 after insulin stimulation of metacestode vesicles. Vesicles were stimulated (+) or not (−) with 100 nM insulin for 10 minutes. Following solubilisation of membrane proteins, the EmIR1 β-subunit was precipitated using the anti-EmIR1 antiserum and separated by SDS-PAGE. Western blot detection was carried out using the anti-EmIR1 antiserum (lower panel) or an anti-phospho-tyrosine antibody (upper panel). D) Phosphorylation of Echinococcus PI3K/Akt pathway components in response to insulin. Vesicles were stimulated with 10 nM insulin for the times indicated above. Vesicle lysates were subsequently separated by SDS-PAGE and probed using antibodies against the phosphorylated Akt substrate motif or phospho 4E-BP as indicated. β-Actin was used as loading control. E) Inhibition of 4E-BP phosphorylation through HNMPA(AM) 3 . Vesicles were incubated for two hours with 100 μM HNMPA(AM) 3 (HNM+) or the PI3K inhibitor LY294002 (LY+) before stimulation with 10 nM insulin. Crude lysates were probed with the anti-phospho 4E-BP antibody. β-Actin was used as loading control. DMSO, dimethyl sulphoxide; HNMPA, 2-hydroxynaphthalen-1-yl-methylphosphonic acid.
    Figure Legend Snippet: Effects of insulin on the phosphorylation of metacestode vesicle proteins. A) Detection of EmIR1 in vesicle membrane fractions (MF). Vesicles from in vitro culture were homogenized and the MF was isolated. Western blot detection was carried out on MF and whole vesicle preparations (Ves) using the anti-EmIR1 antiserum. ‘Pro’ and ‘β’ mark the receptor pro-form and β-subunits. B) Phosphorylation of EmIR1 in response to insulin. Metacestode MF was stimulated for 10 minutes with either 100 nM insulin or IGF-I, followed by 30 minutes incubation with 100 μM HNMPA(AM) 3 or control DMSO. Phosphorylation of membrane proteins was carried out for 40 minutes in the presence of [ 32 P] γ-ATP. Proteins of the MF were precipitated using the anti-EmIR1 antiserum. Proteins were then separated by 8% SDS-PAGE and phosphorylation was detected by autoradiography. Bands are visible at the size of the EmIR1 β-subunit and below. C) Phosphorylation of EmIR1 after insulin stimulation of metacestode vesicles. Vesicles were stimulated (+) or not (−) with 100 nM insulin for 10 minutes. Following solubilisation of membrane proteins, the EmIR1 β-subunit was precipitated using the anti-EmIR1 antiserum and separated by SDS-PAGE. Western blot detection was carried out using the anti-EmIR1 antiserum (lower panel) or an anti-phospho-tyrosine antibody (upper panel). D) Phosphorylation of Echinococcus PI3K/Akt pathway components in response to insulin. Vesicles were stimulated with 10 nM insulin for the times indicated above. Vesicle lysates were subsequently separated by SDS-PAGE and probed using antibodies against the phosphorylated Akt substrate motif or phospho 4E-BP as indicated. β-Actin was used as loading control. E) Inhibition of 4E-BP phosphorylation through HNMPA(AM) 3 . Vesicles were incubated for two hours with 100 μM HNMPA(AM) 3 (HNM+) or the PI3K inhibitor LY294002 (LY+) before stimulation with 10 nM insulin. Crude lysates were probed with the anti-phospho 4E-BP antibody. β-Actin was used as loading control. DMSO, dimethyl sulphoxide; HNMPA, 2-hydroxynaphthalen-1-yl-methylphosphonic acid.

    Techniques Used: In Vitro, Isolation, Western Blot, Incubation, SDS Page, Autoradiography, Inhibition

    Expression of EmIR1 and EmIR2 in E. multilocularis larval stages. A) Semi-quantitative RT-PCR of emir1 and emir2 expression. Serial 1/10 dilutions of cDNA from metacestode vesicles (MC), primary cells (PC) as well as dormant (PS-) and low pH/pepsin-activated protoscoleces (PS+) were subjected to gene-specific PCR using intron-flanking primers. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. The constitutively expressed gene elp was used as control. B) Western blot and immunoprecipitation employing the EmIR1 anti-serum. EmIR1 was immunoprecipitated from metacestode vesicles and treated with β-mercaptoethanol (beta-MeOH) at concentrations of 0%, 1% and 10%. Probes were then separated on a 12.5% polyacrylamide gel and developed using the anti-EmIR1 antiserum. ‘pro’ and ‘beta’ indicate the pro-form and the β-subunit of EmIR, respectively. C) Immunoprecipitation and Western blot using the anti-EmIR2 serum. EmIR2 was immunoprecipitated from protoscolex preparations, samples were then supplemented with 1% or 10% of β-mercaptoethanol (β-ME) and separated on a 10% SDS gel. Western blot was carried out using the anti-EmIR2 antiserum. D) Immunodetection of EmIR1 and EmIR2 in different larval stages using immune sera. Parasite larvae were lysed, protein preparations were then separated by SDS-PAGE, blotted onto a membrane and detected by the antisera. The purified anti-EmIR2 immune serum recognized the EmIR2 β-subunit at 87 kDa and a second band at 60 kDa. The EmIR1 β-subunit was detected at 150 kDa using the anti-EmIR1 immune serum. Actin was used as a loading control. Mc, metacestode vesicles; Pc, primary cells; Ps-, dormant protoscoleces; Ps+, activated protoscoleces.
    Figure Legend Snippet: Expression of EmIR1 and EmIR2 in E. multilocularis larval stages. A) Semi-quantitative RT-PCR of emir1 and emir2 expression. Serial 1/10 dilutions of cDNA from metacestode vesicles (MC), primary cells (PC) as well as dormant (PS-) and low pH/pepsin-activated protoscoleces (PS+) were subjected to gene-specific PCR using intron-flanking primers. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. The constitutively expressed gene elp was used as control. B) Western blot and immunoprecipitation employing the EmIR1 anti-serum. EmIR1 was immunoprecipitated from metacestode vesicles and treated with β-mercaptoethanol (beta-MeOH) at concentrations of 0%, 1% and 10%. Probes were then separated on a 12.5% polyacrylamide gel and developed using the anti-EmIR1 antiserum. ‘pro’ and ‘beta’ indicate the pro-form and the β-subunit of EmIR, respectively. C) Immunoprecipitation and Western blot using the anti-EmIR2 serum. EmIR2 was immunoprecipitated from protoscolex preparations, samples were then supplemented with 1% or 10% of β-mercaptoethanol (β-ME) and separated on a 10% SDS gel. Western blot was carried out using the anti-EmIR2 antiserum. D) Immunodetection of EmIR1 and EmIR2 in different larval stages using immune sera. Parasite larvae were lysed, protein preparations were then separated by SDS-PAGE, blotted onto a membrane and detected by the antisera. The purified anti-EmIR2 immune serum recognized the EmIR2 β-subunit at 87 kDa and a second band at 60 kDa. The EmIR1 β-subunit was detected at 150 kDa using the anti-EmIR1 immune serum. Actin was used as a loading control. Mc, metacestode vesicles; Pc, primary cells; Ps-, dormant protoscoleces; Ps+, activated protoscoleces.

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Western Blot, Immunoprecipitation, SDS-Gel, Immunodetection, SDS Page, Purification

    EmIR1 immunohistochemistry on metacestode vesicles. Cryosections of in vitro cultivated metacestode vesicles were probed with the anti-EmIR1 antiserum (anti-EmIR1) and detected with a FITC-coupled anti-rabbit-antibody. Nuclei were visualized by Hoechst-staining (Hoechst). Parasite surface structures were visualized using a general anti- Echinococcus metacestode antibody (Anti-Echi; Ingold et al ., 2001 [ 50 ]). LL, laminated layer; GSC, glycogen storing cells. FITC, fluorescein isothiocyanate.
    Figure Legend Snippet: EmIR1 immunohistochemistry on metacestode vesicles. Cryosections of in vitro cultivated metacestode vesicles were probed with the anti-EmIR1 antiserum (anti-EmIR1) and detected with a FITC-coupled anti-rabbit-antibody. Nuclei were visualized by Hoechst-staining (Hoechst). Parasite surface structures were visualized using a general anti- Echinococcus metacestode antibody (Anti-Echi; Ingold et al ., 2001 [ 50 ]). LL, laminated layer; GSC, glycogen storing cells. FITC, fluorescein isothiocyanate.

    Techniques Used: Immunohistochemistry, In Vitro, Staining

    Effects of insulin on E. multilocularis larval development. A) Morphology of primary cell aggregates. Primary cells were isolated from axenic metacestode vesicles and cultivated in 2% FCS/(D)MEM supplemented with or without 10 nM human insulin for one week. The aggregates were fixed and embedded in Technovit 8100. Sections (4 μm) were stained with haematoxylin/eosin. B) Formation of metacestode vesicles. Primary cells were cultivated in conditioned medium supplemented with human insulin for three weeks and mature metacestode vesicles were counted. Control was set to 1 and results were normalised against the control. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P
    Figure Legend Snippet: Effects of insulin on E. multilocularis larval development. A) Morphology of primary cell aggregates. Primary cells were isolated from axenic metacestode vesicles and cultivated in 2% FCS/(D)MEM supplemented with or without 10 nM human insulin for one week. The aggregates were fixed and embedded in Technovit 8100. Sections (4 μm) were stained with haematoxylin/eosin. B) Formation of metacestode vesicles. Primary cells were cultivated in conditioned medium supplemented with human insulin for three weeks and mature metacestode vesicles were counted. Control was set to 1 and results were normalised against the control. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P

    Techniques Used: Isolation, Staining

    Effects of HNMPA(AM) 3 on parasite larvae. A) Primary cells were isolated from axenic metacestode vesicles and cultivated in 2% FCS/(D)MEM supplemented with 10 nM human insulin, with or without HNMPA(AM) 3 . After three weeks of incubation, mature vesicles were counted. Insulin was added to the cultures in order to obtain vesicle formation within three weeks. B) Formation of primary cell aggregates in the presence of HNMPA(AM) 3. Primary cells were incubated as in (A) for seven days before aggregates were fixed, embedded in Technovit 8100 and 4 μm sections stained with haematoxylin/eosin. Note the profound effect of HNMPA(AM) 3 on parasite aggregates already after seven days. Ctrl, DMSO control. C) Protoscoleces were treated with HNMPA(AM) 3 for two weeks under axenic conditions. Protoscolex viability was analysed by counter-staining with methylene blue. D) Metacestode vesicles were treated for one week with 100 μM HNMPA(AM) 3 under axenic conditions. Survival was assessed by counting physically damaged vesicles. Vesicles were incubated in the presence of conditioned medium for optimal maintenance and survival conditions. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P
    Figure Legend Snippet: Effects of HNMPA(AM) 3 on parasite larvae. A) Primary cells were isolated from axenic metacestode vesicles and cultivated in 2% FCS/(D)MEM supplemented with 10 nM human insulin, with or without HNMPA(AM) 3 . After three weeks of incubation, mature vesicles were counted. Insulin was added to the cultures in order to obtain vesicle formation within three weeks. B) Formation of primary cell aggregates in the presence of HNMPA(AM) 3. Primary cells were incubated as in (A) for seven days before aggregates were fixed, embedded in Technovit 8100 and 4 μm sections stained with haematoxylin/eosin. Note the profound effect of HNMPA(AM) 3 on parasite aggregates already after seven days. Ctrl, DMSO control. C) Protoscoleces were treated with HNMPA(AM) 3 for two weeks under axenic conditions. Protoscolex viability was analysed by counter-staining with methylene blue. D) Metacestode vesicles were treated for one week with 100 μM HNMPA(AM) 3 under axenic conditions. Survival was assessed by counting physically damaged vesicles. Vesicles were incubated in the presence of conditioned medium for optimal maintenance and survival conditions. (*) P values below 0.05, (**) very significant for P between 0.001 and 0.01, (***) extremely significant for P

    Techniques Used: Isolation, Incubation, Staining

    18) Product Images from "Expression of interleukin (IL)-19 and IL-24 in inflammatory bowel disease patients: a cross-sectional study"

    Article Title: Expression of interleukin (IL)-19 and IL-24 in inflammatory bowel disease patients: a cross-sectional study

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12285

    Interleukin (IL)-19- and IL-24-expressing peripheral blood cells in patients with ulcerative colitis or Crohn's disease. Bar graphs show percentage of (a) CD4 + /CD14 − /IL-19 + - and CD4 + /CD14 − /IL-24 + -expressing T cells, (b) CD8 + /CD14 −
    Figure Legend Snippet: Interleukin (IL)-19- and IL-24-expressing peripheral blood cells in patients with ulcerative colitis or Crohn's disease. Bar graphs show percentage of (a) CD4 + /CD14 − /IL-19 + - and CD4 + /CD14 − /IL-24 + -expressing T cells, (b) CD8 + /CD14 −

    Techniques Used: Expressing

    Interleukin (IL)-19 and IL-24 mRNA levels in colonic mucosa from patients with inflammatory bowel disease and controls. (a) IL-19 gene expression. (b) IL-24 gene expression. Reverse transcription–quantitative polymerase chain reaction (RT-qPCR)
    Figure Legend Snippet: Interleukin (IL)-19 and IL-24 mRNA levels in colonic mucosa from patients with inflammatory bowel disease and controls. (a) IL-19 gene expression. (b) IL-24 gene expression. Reverse transcription–quantitative polymerase chain reaction (RT-qPCR)

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

    19) Product Images from "A Human Immunoglobulin ? Locus Is Similarly Well Expressed in Mice and Humans "

    Article Title: A Human Immunoglobulin ? Locus Is Similarly Well Expressed in Mice and Humans

    Journal: The Journal of Experimental Medicine

    doi:

    Hypermutated human Vλ sequences from sorted B220 + and PNA + PP B cells from HuIgλ + YAC/κ +/− mice. The sequences are a representative selection of the functional Vλ-Jλ rearrangements (indicated by the triangles in Fig. 1 ) isolated from RT-PCR.
    Figure Legend Snippet: Hypermutated human Vλ sequences from sorted B220 + and PNA + PP B cells from HuIgλ + YAC/κ +/− mice. The sequences are a representative selection of the functional Vλ-Jλ rearrangements (indicated by the triangles in Fig. 1 ) isolated from RT-PCR.

    Techniques Used: Mouse Assay, Selection, Functional Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    The HuIgλYAC accommodates a 380-kb region of the human λ L chain locus in authentic configuration with all Vλ genes of cluster A ( 21 , 22 , 40 ), the Jλ-Cλ segments, and the 3′ enhancer ( 17 ). Black boxes represent functional Vλ genes (3-27, 3-25, 2-23, 3-22, 3-21, 3-19, 2-18, 3-16, 2-14, 2-11, 3-10, 3-9, 2-8, 4-3, and 3-1) and white boxes show Vλ genes with open reading frames (2-33, 3-32, and 3-12) that have not been identified in productive rearrangements of human lymphocytes ( 40 ). Pseudogenes are not shown. Black triangles (▴) indicate V gene use in functionally Igλ rearrangements (mutated [see Fig. 5 ] and unmutated) found by RT-PCR in spleen and sorted PP cells from HuIgλ mice. Rearrangement to Jλ1 was found in 5 sequences, Jλ2 in 18, and Jλ3 in 8. The unique NotI restriction site is indicated. Probes to assess the integrity of the HuIgλYAC, LA (left arm) and Cλ2+3 are indicated.
    Figure Legend Snippet: The HuIgλYAC accommodates a 380-kb region of the human λ L chain locus in authentic configuration with all Vλ genes of cluster A ( 21 , 22 , 40 ), the Jλ-Cλ segments, and the 3′ enhancer ( 17 ). Black boxes represent functional Vλ genes (3-27, 3-25, 2-23, 3-22, 3-21, 3-19, 2-18, 3-16, 2-14, 2-11, 3-10, 3-9, 2-8, 4-3, and 3-1) and white boxes show Vλ genes with open reading frames (2-33, 3-32, and 3-12) that have not been identified in productive rearrangements of human lymphocytes ( 40 ). Pseudogenes are not shown. Black triangles (▴) indicate V gene use in functionally Igλ rearrangements (mutated [see Fig. 5 ] and unmutated) found by RT-PCR in spleen and sorted PP cells from HuIgλ mice. Rearrangement to Jλ1 was found in 5 sequences, Jλ2 in 18, and Jλ3 in 8. The unique NotI restriction site is indicated. Probes to assess the integrity of the HuIgλYAC, LA (left arm) and Cλ2+3 are indicated.

    Techniques Used: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    20) Product Images from "β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice"

    Article Title: β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice

    Journal: Cardiovascular Diabetology

    doi: 10.1186/s12933-018-0747-3

    Deletion of GC-A in β-cells from GC - A fl/fl ; RipCre tg (β GC-A KO) mice. a PCR analysis. Genomic DNA from different tissues was assayed for the appearance of the ~ 700-bp amplicon which results from complete recombination of the floxed GC-A gene segment. Genomic DNA was from white adipose tissue (WAT), skeletal muscle (Sk M), isolated pancreatic islets and hearts. b Quantitative RT-PCR analysis. GC-A mRNA expression levels in pancreatic islets from control and β GC-A KO mice. Values are the ratio of GC-A mRNA level relative to β2 microglobulin, expressed as x-fold vs control islets (20 samples per genotype). c Cyclic GMP determinations. Concentration-dependent effects of ANP on intracellular cGMP contents of pancreatic islets prepared and cultured from control mice (15 min incubation in the presence of the phosphodiesterase inhibitor IBMX; n = 4 per condition). d Comparison of the cGMP responses of β GC-A KO and control islets to 100 nM ANP (15 min incubation in the presence of IBMX; n = 4 per genotype and condition). e Insulin release. Effects of ANP on glucose-dependent insulin secretion by pancreatic islets prepared from β GC-A KO and control littermates (1 h incubation; n = 4). f Systolic, mean and diastolic arterial blood pressure levels of β GC-A KO and control littermates (n = 16 per genotype). *P
    Figure Legend Snippet: Deletion of GC-A in β-cells from GC - A fl/fl ; RipCre tg (β GC-A KO) mice. a PCR analysis. Genomic DNA from different tissues was assayed for the appearance of the ~ 700-bp amplicon which results from complete recombination of the floxed GC-A gene segment. Genomic DNA was from white adipose tissue (WAT), skeletal muscle (Sk M), isolated pancreatic islets and hearts. b Quantitative RT-PCR analysis. GC-A mRNA expression levels in pancreatic islets from control and β GC-A KO mice. Values are the ratio of GC-A mRNA level relative to β2 microglobulin, expressed as x-fold vs control islets (20 samples per genotype). c Cyclic GMP determinations. Concentration-dependent effects of ANP on intracellular cGMP contents of pancreatic islets prepared and cultured from control mice (15 min incubation in the presence of the phosphodiesterase inhibitor IBMX; n = 4 per condition). d Comparison of the cGMP responses of β GC-A KO and control islets to 100 nM ANP (15 min incubation in the presence of IBMX; n = 4 per genotype and condition). e Insulin release. Effects of ANP on glucose-dependent insulin secretion by pancreatic islets prepared from β GC-A KO and control littermates (1 h incubation; n = 4). f Systolic, mean and diastolic arterial blood pressure levels of β GC-A KO and control littermates (n = 16 per genotype). *P

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Amplification, Isolation, Quantitative RT-PCR, Expressing, Concentration Assay, Aqueous Normal-phase Chromatography, Cell Culture, Incubation

    21) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.02177-17

    Validation of K8 RNA binding property and identification of K8-associated RNAs using a CLIP-seq approach. (A) TPA-induced BCBL-1 cells were UV irradiated and subjected to immunoprecipitation with K8 antibody. Different amounts of RNase A (++, 2 μg/ml; +, 1 μg/ml) were added to the washing buffer during immunoprecipitation. Coprecipitated RNA was labeled with biotin at the 3′ end and spotted onto a nylon membrane. The biotinylated RNA was then analyzed with a chemiluminescent nucleic acid detection module kit. (B) Schematic illustration of the CLIP-seq procedure. TPA-induced BCBL-1 cells were UV irradiated and immunoprecipitated with K8 antibody. Different amounts of RNase A were added to the washing buffer. The coprecipitated RNAs were phosphorylated at the 5′ end with T4 polynucleotide kinase (PNK) and dephosphorylated with alkaline phosphatase at the 3′ end. The RNAs were ligated to the 3′ linker and then to the 5′ linker labeled with γ- 32 P. The RNA-protein complex was resolved in SDS-PAGE, and RNAs were isolated and amplified by RT-PCR. The PCR products were analyzed by high-throughput sequencing. (C) Autoradiogram of γ- 32 P-labeled RNA cross-linked to K8. Immunoprecipitation was performed with anti-K8 or anti-ORF45 antibody. (Bottom) Input of K8 and β-actin. (D) RT-PCR products from K8-CLIP. (E) Experimental design of CLIP-seq. For each set of CLIP experiments (IgG, KSHV-negative [BJAB], KSHV1 [BCBL-1], and KSHV2 [BCBL-1]), two biological repeats were carried out. Each biological repeat contained three technical repeats. (F) Library statistics of CLIP-seq. Two biological repeats from K8-CLIP-seq (KSHV1 and KSHV2) contained 67,953,806 and 81,632,254 clean reads, respectively. In contrast, there were only 402,896 and 3,472,238 clean reads in the IgG control and KSHV negative (BJAB) data sets, respectively. (G) Libraries from two biological repeats were highly similar. The correlation coefficient between two biological repeats (KSHV1 and KSHV2) was above 0.97.
    Figure Legend Snippet: Validation of K8 RNA binding property and identification of K8-associated RNAs using a CLIP-seq approach. (A) TPA-induced BCBL-1 cells were UV irradiated and subjected to immunoprecipitation with K8 antibody. Different amounts of RNase A (++, 2 μg/ml; +, 1 μg/ml) were added to the washing buffer during immunoprecipitation. Coprecipitated RNA was labeled with biotin at the 3′ end and spotted onto a nylon membrane. The biotinylated RNA was then analyzed with a chemiluminescent nucleic acid detection module kit. (B) Schematic illustration of the CLIP-seq procedure. TPA-induced BCBL-1 cells were UV irradiated and immunoprecipitated with K8 antibody. Different amounts of RNase A were added to the washing buffer. The coprecipitated RNAs were phosphorylated at the 5′ end with T4 polynucleotide kinase (PNK) and dephosphorylated with alkaline phosphatase at the 3′ end. The RNAs were ligated to the 3′ linker and then to the 5′ linker labeled with γ- 32 P. The RNA-protein complex was resolved in SDS-PAGE, and RNAs were isolated and amplified by RT-PCR. The PCR products were analyzed by high-throughput sequencing. (C) Autoradiogram of γ- 32 P-labeled RNA cross-linked to K8. Immunoprecipitation was performed with anti-K8 or anti-ORF45 antibody. (Bottom) Input of K8 and β-actin. (D) RT-PCR products from K8-CLIP. (E) Experimental design of CLIP-seq. For each set of CLIP experiments (IgG, KSHV-negative [BJAB], KSHV1 [BCBL-1], and KSHV2 [BCBL-1]), two biological repeats were carried out. Each biological repeat contained three technical repeats. (F) Library statistics of CLIP-seq. Two biological repeats from K8-CLIP-seq (KSHV1 and KSHV2) contained 67,953,806 and 81,632,254 clean reads, respectively. In contrast, there were only 402,896 and 3,472,238 clean reads in the IgG control and KSHV negative (BJAB) data sets, respectively. (G) Libraries from two biological repeats were highly similar. The correlation coefficient between two biological repeats (KSHV1 and KSHV2) was above 0.97.

    Techniques Used: RNA Binding Assay, Cross-linking Immunoprecipitation, Irradiation, Immunoprecipitation, Labeling, SDS Page, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Next-Generation Sequencing

    Association of K8 with ori-Lyt DNA is mediated by RNA. (A) Schematic illustration of the KSHV ori-Lyt core domain and DNA fragments that were used in the DNA affinity assay. (B) Three biotinylated ori-Lyt DNA fragments and an irrelevant DNA fragment from the ORF45 coding region as a control were prepared by PCR, conjugated on magnetic beads, and incubated with TPA-induced BCBL-1 nuclear extract with and without treatment with RNase A. After washing, samples were assayed by Western blotting with antibodies as indicated. (C) Binding of K8 to ori-Lyt DNA was determined in BAC16 (BAC WT) and BAC-K8GDDGR by ChIP assay with anti-K8 antibody. The positions of the amplicons (3.1F and 12F) are shown in panel A. The error bars indicate SD.
    Figure Legend Snippet: Association of K8 with ori-Lyt DNA is mediated by RNA. (A) Schematic illustration of the KSHV ori-Lyt core domain and DNA fragments that were used in the DNA affinity assay. (B) Three biotinylated ori-Lyt DNA fragments and an irrelevant DNA fragment from the ORF45 coding region as a control were prepared by PCR, conjugated on magnetic beads, and incubated with TPA-induced BCBL-1 nuclear extract with and without treatment with RNase A. After washing, samples were assayed by Western blotting with antibodies as indicated. (C) Binding of K8 to ori-Lyt DNA was determined in BAC16 (BAC WT) and BAC-K8GDDGR by ChIP assay with anti-K8 antibody. The positions of the amplicons (3.1F and 12F) are shown in panel A. The error bars indicate SD.

    Techniques Used: Polymerase Chain Reaction, Magnetic Beads, Incubation, Western Blot, Binding Assay, BAC Assay, Chromatin Immunoprecipitation

    22) Product Images from "Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR"

    Article Title: Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2011.04.002

    JAK2V617F Real-Time PCR Assay with PNA and LNA Blocking
    Figure Legend Snippet: JAK2V617F Real-Time PCR Assay with PNA and LNA Blocking

    Techniques Used: Real-time Polymerase Chain Reaction, Blocking Assay

    Standard curves (using twofold dilution steps) generated by JAK2V617F real-time PCR using PNA blocking ( squares ) and LNA blocking ( triangles ). Data are given as mean Ct ± SD.
    Figure Legend Snippet: Standard curves (using twofold dilution steps) generated by JAK2V617F real-time PCR using PNA blocking ( squares ) and LNA blocking ( triangles ). Data are given as mean Ct ± SD.

    Techniques Used: Generated, Real-time Polymerase Chain Reaction, Blocking Assay

    23) Product Images from "Glycogen Synthase Kinase (GSK) 3? Phosphorylates and Protects Nuclear Myosin 1c from Proteasome-Mediated Degradation to Activate rDNA Transcription in Early G1 Cells"

    Article Title: Glycogen Synthase Kinase (GSK) 3? Phosphorylates and Protects Nuclear Myosin 1c from Proteasome-Mediated Degradation to Activate rDNA Transcription in Early G1 Cells

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004390

    At G1, NM1 is ubiquitinated in a GSK3β-dependent manner by the E3 ligase UBR5. ( A ) Lysates prepared from GSK3β +/+ MEFs and GSK3β −/− MEFs at G1 transiently expressing HA-tagged ubiquitin, treated with MG132 where indicated, were subjected to immunoprecipitations with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin. ( B ) Lysates from HeLa cells synchronized in G1 co-transfected with GSK3β RNAi oligonucleotides or scrambled scrRNAi oligonucleotides and transiently expressing HA-tagged ubiquitin. Where indicated lysates were obtained from HeLa cells treated with MG132. Immunoprecipitations were performed from all lysates with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin. ( C ) Lysates from G1-blocked GSK3β −/− MEFs were subjected to RNAi-mediated gene silencing of the E3 ligases UBR5 and Fbxw8 or to scrRNAi oligonucleotides transiently expressing HA-tagged ubiquitin. The lysates were subjected to immunoprecipitations with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin.
    Figure Legend Snippet: At G1, NM1 is ubiquitinated in a GSK3β-dependent manner by the E3 ligase UBR5. ( A ) Lysates prepared from GSK3β +/+ MEFs and GSK3β −/− MEFs at G1 transiently expressing HA-tagged ubiquitin, treated with MG132 where indicated, were subjected to immunoprecipitations with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin. ( B ) Lysates from HeLa cells synchronized in G1 co-transfected with GSK3β RNAi oligonucleotides or scrambled scrRNAi oligonucleotides and transiently expressing HA-tagged ubiquitin. Where indicated lysates were obtained from HeLa cells treated with MG132. Immunoprecipitations were performed from all lysates with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin. ( C ) Lysates from G1-blocked GSK3β −/− MEFs were subjected to RNAi-mediated gene silencing of the E3 ligases UBR5 and Fbxw8 or to scrRNAi oligonucleotides transiently expressing HA-tagged ubiquitin. The lysates were subjected to immunoprecipitations with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection

    GSK3β phosphorylates NM1. ( A ) GSK3β, NM1 and actin are co-precipitated from nuclear protein extracts prepared from growing GSK3β +/+ MEFs. Bound proteins were detected on immunoblots with antibodies against WSTF, SNF2h, NM1, PCAF, GSK3β (CGR11) and actin. 10% of the input is shown in Lane 1. IP, immunoprecipitation. ( B ) Schematic representation of V5-tagged wt and mutated NM1 constructs stably expressed in HEK293T cell lines. ( C ) Co-precipitations of GSK3β from total lysates obtained from HEK293T cells stably expressing wt and mutated V5-tagged NM1 constructs as indicated. 10% of the input is shown. IP, immunoprecipitation. ( D ) Lysates were prepared from growing GSK3β +/+ MEFs and GSK3β −/− MEFs or from GSK3β +/+ MEFs and GSK3β −/− MEFs arrested in G1 by serum starvation. Where indicated extracts were subjected to alkaline phosphatase (AP) treatment. Lysates were analyzed on immunoblots for NM1 and actin. ( E ) Kinase assays were performed on lysates from G1-arrested GSK3β −/− MEFs untreated or treated with the proteasome inhibitor MG132, supplemented with γ- 33 P-ATP. Where indicated the lysates were incubated with recombinant GSK3β. To monitor NM1 phosphorylation, the lysates were subjected to immunoprecipitations with anti-NM1 antibodies. Phosphorylated NM1 was detected by phosphorimaging against the levels of unphosphorylated NM1 detected on immunoblots. ( F ) Kinase assays were performed on endogenous NM1 or actin immunoprecipitated from lysates of G1-arrested GSK3β −/− MEFs treated with MG132; after immunoprecipitations the beads were washed and incubated with γ- 33 P-ATP and recombinant GSK3β. Phosphorylated NM1 (NM1*) and autophosphorylated GSK3β (GSK3β*) were detected by phosphorimaging. The immunoprecipitated endogenous NM1 and actin were detected on immunoblots. Non-specific IgGs were used as negative control for the immunoprecipitations. ( G–H ) Tandem MS spectra of phosphoprylated and non-phosphorylated peptide DGIIDFTSGSELLITK identified within the primary NM1 sequence immunoprecipitated from G1-arrested lysates of GSK3β +/+ MEFs and GSK3β −/− MEFs, respectively.
    Figure Legend Snippet: GSK3β phosphorylates NM1. ( A ) GSK3β, NM1 and actin are co-precipitated from nuclear protein extracts prepared from growing GSK3β +/+ MEFs. Bound proteins were detected on immunoblots with antibodies against WSTF, SNF2h, NM1, PCAF, GSK3β (CGR11) and actin. 10% of the input is shown in Lane 1. IP, immunoprecipitation. ( B ) Schematic representation of V5-tagged wt and mutated NM1 constructs stably expressed in HEK293T cell lines. ( C ) Co-precipitations of GSK3β from total lysates obtained from HEK293T cells stably expressing wt and mutated V5-tagged NM1 constructs as indicated. 10% of the input is shown. IP, immunoprecipitation. ( D ) Lysates were prepared from growing GSK3β +/+ MEFs and GSK3β −/− MEFs or from GSK3β +/+ MEFs and GSK3β −/− MEFs arrested in G1 by serum starvation. Where indicated extracts were subjected to alkaline phosphatase (AP) treatment. Lysates were analyzed on immunoblots for NM1 and actin. ( E ) Kinase assays were performed on lysates from G1-arrested GSK3β −/− MEFs untreated or treated with the proteasome inhibitor MG132, supplemented with γ- 33 P-ATP. Where indicated the lysates were incubated with recombinant GSK3β. To monitor NM1 phosphorylation, the lysates were subjected to immunoprecipitations with anti-NM1 antibodies. Phosphorylated NM1 was detected by phosphorimaging against the levels of unphosphorylated NM1 detected on immunoblots. ( F ) Kinase assays were performed on endogenous NM1 or actin immunoprecipitated from lysates of G1-arrested GSK3β −/− MEFs treated with MG132; after immunoprecipitations the beads were washed and incubated with γ- 33 P-ATP and recombinant GSK3β. Phosphorylated NM1 (NM1*) and autophosphorylated GSK3β (GSK3β*) were detected by phosphorimaging. The immunoprecipitated endogenous NM1 and actin were detected on immunoblots. Non-specific IgGs were used as negative control for the immunoprecipitations. ( G–H ) Tandem MS spectra of phosphoprylated and non-phosphorylated peptide DGIIDFTSGSELLITK identified within the primary NM1 sequence immunoprecipitated from G1-arrested lysates of GSK3β +/+ MEFs and GSK3β −/− MEFs, respectively.

    Techniques Used: Western Blot, Immunoprecipitation, Construct, Stable Transfection, Expressing, Incubation, Recombinant, Negative Control, Mass Spectrometry, Sequencing

    GSK3β-dependent NM1 phosphorylation suppresses proteasome mediated degradation and mediates association with chromatin. ( A ) Cell cycle profile analyzed at the indicated time points, after release from a G1 arrest by serum starvation, on immunoblots of the corresponding lysates for NM1, cyclin A, cyclin E, p27 and β-actin. ( B ) Relative NM1 mRNA levels in GSK3β +/+ MEFs and GSK3β −/− MEFs monitored by RT-qPCR using β-tubulin mRNA as internal control. ( C ) rRNA synthesis in GSK3β +/+ MEFs and GSK3β −/− MEFs arrested in G1 by serum starvation. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT-qPCR using tubulin mRNA as internal control [p = 3.2e-09 (***)]. ( D ) Lysates from GSK3β −/− MEFs untreated or treated with the proteasome inhibitor MG132, released from a G1 block were collected at the indicated time points and analyzed on immunoblots for NM1 and β-actin. ( E ) ChIP and qPCR analysis on chromatin isolated from GSK3β +/+ MEFs and GSK3β −/− MEFs synchronized in G1, untreated or treated with MG132, at the rRNA gene promoter with antibodies against NM1 and GSK3β (CGR11). Significances p(−MG132) = 2.2e-05 (***) and p(+MG132) = 3.0e-05 (***) were respectively calculated against the NM1 values obtained in GSK3β +/+ MEFs not treated with MG132. ( F ) Immunoblots of total lysates from GSK3β +/+ MEFs untreated or treated with the kinase inhibitor BIO. Analysis was performed with antibodies to NM1, actin, and the GSK3β antibodies 27C10 and CGR11 as indicated. ( G ) ChIP and qPCR analysis on chromatin isolated from GSK3β +/+ MEFs at G1, untreated or treated with BIO, at the rRNA gene promoter with antibodies against NM1 and GSK3β (CGR11). The significance p = 0.009 (**) was calculated against the NM1 values obtained in GSK3β +/+ MEFs not treated with BIO.
    Figure Legend Snippet: GSK3β-dependent NM1 phosphorylation suppresses proteasome mediated degradation and mediates association with chromatin. ( A ) Cell cycle profile analyzed at the indicated time points, after release from a G1 arrest by serum starvation, on immunoblots of the corresponding lysates for NM1, cyclin A, cyclin E, p27 and β-actin. ( B ) Relative NM1 mRNA levels in GSK3β +/+ MEFs and GSK3β −/− MEFs monitored by RT-qPCR using β-tubulin mRNA as internal control. ( C ) rRNA synthesis in GSK3β +/+ MEFs and GSK3β −/− MEFs arrested in G1 by serum starvation. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT-qPCR using tubulin mRNA as internal control [p = 3.2e-09 (***)]. ( D ) Lysates from GSK3β −/− MEFs untreated or treated with the proteasome inhibitor MG132, released from a G1 block were collected at the indicated time points and analyzed on immunoblots for NM1 and β-actin. ( E ) ChIP and qPCR analysis on chromatin isolated from GSK3β +/+ MEFs and GSK3β −/− MEFs synchronized in G1, untreated or treated with MG132, at the rRNA gene promoter with antibodies against NM1 and GSK3β (CGR11). Significances p(−MG132) = 2.2e-05 (***) and p(+MG132) = 3.0e-05 (***) were respectively calculated against the NM1 values obtained in GSK3β +/+ MEFs not treated with MG132. ( F ) Immunoblots of total lysates from GSK3β +/+ MEFs untreated or treated with the kinase inhibitor BIO. Analysis was performed with antibodies to NM1, actin, and the GSK3β antibodies 27C10 and CGR11 as indicated. ( G ) ChIP and qPCR analysis on chromatin isolated from GSK3β +/+ MEFs at G1, untreated or treated with BIO, at the rRNA gene promoter with antibodies against NM1 and GSK3β (CGR11). The significance p = 0.009 (**) was calculated against the NM1 values obtained in GSK3β +/+ MEFs not treated with BIO.

    Techniques Used: Western Blot, Quantitative RT-PCR, Blocking Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation

    A speculative model in which GSK3β phosphorylates the NM1 C-terminal tail at G1. ( I ) In the presence of GSK3β, NM1 is phosphorylated and binds to rDNA chromatin. This phosphorylation event triggers a domino effect that leads to stabilization of the actomyosin complex and B-WICH multi-protein assembly on the rDNA. This mechanism leads to recruitment of PCAF, maintains the levels of H3K9 acetylation and activates transcription. ( II ) When GSK3β does not phosphorylate NM1, NM1 becomes polyubiquitinated by UBR5 and degraded by the proteasome. Consequently, the WICH complex is not assembled on the chromatin. At G1 NM1 degradation leads to suppression of pol I transcription and alterations in cell cycle progression.
    Figure Legend Snippet: A speculative model in which GSK3β phosphorylates the NM1 C-terminal tail at G1. ( I ) In the presence of GSK3β, NM1 is phosphorylated and binds to rDNA chromatin. This phosphorylation event triggers a domino effect that leads to stabilization of the actomyosin complex and B-WICH multi-protein assembly on the rDNA. This mechanism leads to recruitment of PCAF, maintains the levels of H3K9 acetylation and activates transcription. ( II ) When GSK3β does not phosphorylate NM1, NM1 becomes polyubiquitinated by UBR5 and degraded by the proteasome. Consequently, the WICH complex is not assembled on the chromatin. At G1 NM1 degradation leads to suppression of pol I transcription and alterations in cell cycle progression.

    Techniques Used:

    GSK3β distributes through the entire rDNA transcription unit, occupying the rRNA gene promoter and transcribed sequences. ( A ) Schematic representation of the primary structure of human GSK3β, including the N-terminal stretch of amino acids used as epitope for the GSK3β antibody CGR11. ( B ) Immunoblots of total lysates obtained from GSK3β +/+ MEFs, GSK3β −/− MEFs and HeLa cells analyzed with the anti-GSK3β antibodies CGR11 and 27C10 and with an anti-actin antibody. ( C ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter, 18S, 5.8S, 28S rDNA and IGS with the anti- GSK3β antibodies CGR11 and 27C10. Positions of all primers are indicated in bracket. The structure of individual mouse ribosomal rDNA repeat is shown to show the location of the different rDNA fragments analyzed. ( D ) ChIP-Seq performed on GSK3β +/+ MEFs. The previously sequenced mouse rDNA repeat BK000964 was utilized in our analysis procedure. The frequency of hits by sequences matching the region spanning the rDNA repeat sequence and IGS is shown by the resulting graph.
    Figure Legend Snippet: GSK3β distributes through the entire rDNA transcription unit, occupying the rRNA gene promoter and transcribed sequences. ( A ) Schematic representation of the primary structure of human GSK3β, including the N-terminal stretch of amino acids used as epitope for the GSK3β antibody CGR11. ( B ) Immunoblots of total lysates obtained from GSK3β +/+ MEFs, GSK3β −/− MEFs and HeLa cells analyzed with the anti-GSK3β antibodies CGR11 and 27C10 and with an anti-actin antibody. ( C ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter, 18S, 5.8S, 28S rDNA and IGS with the anti- GSK3β antibodies CGR11 and 27C10. Positions of all primers are indicated in bracket. The structure of individual mouse ribosomal rDNA repeat is shown to show the location of the different rDNA fragments analyzed. ( D ) ChIP-Seq performed on GSK3β +/+ MEFs. The previously sequenced mouse rDNA repeat BK000964 was utilized in our analysis procedure. The frequency of hits by sequences matching the region spanning the rDNA repeat sequence and IGS is shown by the resulting graph.

    Techniques Used: Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing

    GSK3β regulates pol I transcription activation. ( A ) rRNA synthesis in GSK3β +/+ MEFs and GSK3β −/− MEFs. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT–qPCR using actin mRNA as internal control. Error bars represent the standard deviation of three independent experiments [p = 3.39e-05 (***)]. ( B ) FUrD incorporation assays on living GSK3β −/− and GSK3β +/+ MEFs subjected to DRB treatment. Transcription was monitored by a short FUrd pulse to monitor incorporation into nascent nucleolar transcripts. After fixation, cells were co-stained with a fluorochrome conjugated anti-BrdU antibody to detect the incorporated FUrd and with a human auto-immune serum against pol I (S57299). Detection was by confocal microscopy. Scale bar, 5 ìm. ( C ) MeDIP and qPCR analysis on growing GSK3β +/+ MEFs and GSK3β −/− MEFs performed with an antibody for 5-methylcytidine. qPCR analysis on the precipitated DNA was performed with primers amplifying rRNA gene promoter and reference genes TSH2B and GAPDH. ( D ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter and 18S with the pol I specific autoimmune serum S57299 and an anti-UBF antibody. ( E ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter, 18S and IGS with the anti-GSK3β antibody CGR11 and antibodies against WSTF, SNF2h, NM1, actin and non-specific rabbit IgGs. ( F ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter with antibodies against H3K9Ac, H3K4me3 and PCAF.
    Figure Legend Snippet: GSK3β regulates pol I transcription activation. ( A ) rRNA synthesis in GSK3β +/+ MEFs and GSK3β −/− MEFs. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT–qPCR using actin mRNA as internal control. Error bars represent the standard deviation of three independent experiments [p = 3.39e-05 (***)]. ( B ) FUrD incorporation assays on living GSK3β −/− and GSK3β +/+ MEFs subjected to DRB treatment. Transcription was monitored by a short FUrd pulse to monitor incorporation into nascent nucleolar transcripts. After fixation, cells were co-stained with a fluorochrome conjugated anti-BrdU antibody to detect the incorporated FUrd and with a human auto-immune serum against pol I (S57299). Detection was by confocal microscopy. Scale bar, 5 ìm. ( C ) MeDIP and qPCR analysis on growing GSK3β +/+ MEFs and GSK3β −/− MEFs performed with an antibody for 5-methylcytidine. qPCR analysis on the precipitated DNA was performed with primers amplifying rRNA gene promoter and reference genes TSH2B and GAPDH. ( D ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter and 18S with the pol I specific autoimmune serum S57299 and an anti-UBF antibody. ( E ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter, 18S and IGS with the anti-GSK3β antibody CGR11 and antibodies against WSTF, SNF2h, NM1, actin and non-specific rabbit IgGs. ( F ) ChIP and qPCR on growing GSK3β +/+ MEFs and GSK3β −/− MEFs at the rRNA gene promoter with antibodies against H3K9Ac, H3K4me3 and PCAF.

    Techniques Used: Activation Assay, Quantitative RT-PCR, Standard Deviation, Staining, Confocal Microscopy, Methylated DNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    The effects of GSK3β knockout on the morphology of the nucleolus. ( A ) The size and number of nucleoli in GSK3β +/+ and GSK3β −/− MEFs were analyzed in double-stained preparations using anti-UBF (red) and anti-nucleolin (green) antibodies as nucleolar markers. ( B ) Quantitative evaluation of the number of nucleoli per cell. The histogram shows the average number of nucleoli per cell based on the analysis of 212 GSK3β +/+ MEFs and 211 GSK3β −/− MEFs, from two independent experiments. ( C ) The ultrastructure of the nucleolus in GSK3β +/+ and GSK3β −/− MEFs analyzed by transmission electron microcopy. GSK3β +/+ nucleolus. DFC: dense fibrillar component; FC: fibrillar center; GC: granular component; chrom: dense chromatin. The magnification bars represent 0.5 µm. ( D ) GSK3β +/+ and GSK3β −/− MEFs stained with an antibody against nucleolin (green) and counterstained with DAPI (blue) to visualize patterns of chromatin condensation. Dense chromatin of GSK3β −/− MEFs is found in small patches that are often located at the nuclear periphery, whereas in GSK3β +/+ cells they are often larger and more centrally located. ( E ) Transmission electron microscopy images showing the accumulation of dense chromatin near the nuclear envelope in GSK3β −/− MEFs. Nuc: nucleus; Cyt: cytoplasm. The bar represents 200 nm.
    Figure Legend Snippet: The effects of GSK3β knockout on the morphology of the nucleolus. ( A ) The size and number of nucleoli in GSK3β +/+ and GSK3β −/− MEFs were analyzed in double-stained preparations using anti-UBF (red) and anti-nucleolin (green) antibodies as nucleolar markers. ( B ) Quantitative evaluation of the number of nucleoli per cell. The histogram shows the average number of nucleoli per cell based on the analysis of 212 GSK3β +/+ MEFs and 211 GSK3β −/− MEFs, from two independent experiments. ( C ) The ultrastructure of the nucleolus in GSK3β +/+ and GSK3β −/− MEFs analyzed by transmission electron microcopy. GSK3β +/+ nucleolus. DFC: dense fibrillar component; FC: fibrillar center; GC: granular component; chrom: dense chromatin. The magnification bars represent 0.5 µm. ( D ) GSK3β +/+ and GSK3β −/− MEFs stained with an antibody against nucleolin (green) and counterstained with DAPI (blue) to visualize patterns of chromatin condensation. Dense chromatin of GSK3β −/− MEFs is found in small patches that are often located at the nuclear periphery, whereas in GSK3β +/+ cells they are often larger and more centrally located. ( E ) Transmission electron microscopy images showing the accumulation of dense chromatin near the nuclear envelope in GSK3β −/− MEFs. Nuc: nucleus; Cyt: cytoplasm. The bar represents 200 nm.

    Techniques Used: Knock-Out, Staining, Transmission Assay, Electron Microscopy

    24) Product Images from "Examining protein-protein interactions using endogenously tagged yeast arrays: The Cross-and-Capture system"

    Article Title: Examining protein-protein interactions using endogenously tagged yeast arrays: The Cross-and-Capture system

    Journal: Genome Research

    doi: 10.1101/gr.6667007

    Generation and verification of tagged protein arrays. ( A ) To tag ORFX as bait (V5–6×HIS) and prey (V5–3×VSV), a set of primers is used that anneal to identical binding sites within the template plasmids and have flanking sequence homologous to ORFX . PCR products generated from the bait and prey templates are transformed into a - and α-cells, respectively. Homologous recombination occurs between the variable portion of the 5′ primer (light blue) and the 3′ terminus of the ORF, and between the variable portion of the 3′ primer (red) and the 3′ UTR) of ORFX . Transformants are selected on G418 plates, and colony PCR is performed to verify integration of the Kan r downstream of the desired ORF. Abbreviations: TEF, translational elongation factor; TEFp, TEF promoter; TEFt, TEF terminator: Kan r , kanamycin resistance; loxp, site for CRE specific homologous recombination. ( B . The asterisk (*) denotes possible misloading or protein degradation. Note in the RAD51 lane the multiple protein products. Expected protein sizes are listed in Supplemental Table 1. ( C ) Analysis of effects on cell growth by tagging essential genes. A total of 24 strains with essential genes tagged as baits (6×HIS) and preys (3×VSV) were grown to saturation and spotted in 10-fold dilutions on YPD. Pictures were taken after 2 d at 30°C.
    Figure Legend Snippet: Generation and verification of tagged protein arrays. ( A ) To tag ORFX as bait (V5–6×HIS) and prey (V5–3×VSV), a set of primers is used that anneal to identical binding sites within the template plasmids and have flanking sequence homologous to ORFX . PCR products generated from the bait and prey templates are transformed into a - and α-cells, respectively. Homologous recombination occurs between the variable portion of the 5′ primer (light blue) and the 3′ terminus of the ORF, and between the variable portion of the 3′ primer (red) and the 3′ UTR) of ORFX . Transformants are selected on G418 plates, and colony PCR is performed to verify integration of the Kan r downstream of the desired ORF. Abbreviations: TEF, translational elongation factor; TEFp, TEF promoter; TEFt, TEF terminator: Kan r , kanamycin resistance; loxp, site for CRE specific homologous recombination. ( B . The asterisk (*) denotes possible misloading or protein degradation. Note in the RAD51 lane the multiple protein products. Expected protein sizes are listed in Supplemental Table 1. ( C ) Analysis of effects on cell growth by tagging essential genes. A total of 24 strains with essential genes tagged as baits (6×HIS) and preys (3×VSV) were grown to saturation and spotted in 10-fold dilutions on YPD. Pictures were taken after 2 d at 30°C.

    Techniques Used: Binding Assay, Sequencing, Polymerase Chain Reaction, Generated, Transformation Assay, Homologous Recombination

    25) Product Images from "Association of a Cac8I polymorphism in the IGF1 gene with growth traits in Indian goats"

    Article Title: Association of a Cac8I polymorphism in the IGF1 gene with growth traits in Indian goats

    Journal: Journal of Genetic Engineering & Biotechnology

    doi: 10.1016/j.jgeb.2017.04.002

    Pattern of Cac8I digestion of A224G of 294 bp fragment of IGF1 with AG and GG genotypes. Lane 1: 294 bp PCR product, Lanes 2, 4 5: AG genotype, Lane 3: GG genotype, Lane 6: 50 bp marker.
    Figure Legend Snippet: Pattern of Cac8I digestion of A224G of 294 bp fragment of IGF1 with AG and GG genotypes. Lane 1: 294 bp PCR product, Lanes 2, 4 5: AG genotype, Lane 3: GG genotype, Lane 6: 50 bp marker.

    Techniques Used: Polymerase Chain Reaction, Marker

    26) Product Images from "DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes"

    Article Title: DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw141

    DUX4 interacts with p300/CBP. ( A ) Schematic diagram of the lentiviral constructs carrying doxycycline-inducible DUX4 and rtTA. sgTRE: second generation tet-response element for doxycycline-induced expression. ( B ) Immunoblot showing doxycycline dose-dependent inducible expression of DUX4-flag in LHCN-M2 cells. ( C ) Time course of induction of DUX4 downstream target genes, FRG2 and ZSCAN4 . ( D ) Cell death of immortalized human LHCN-M2 myoblasts 48 hours after DUX4 induction with 250 ng/ml doxycycline. ( E ) Silver stained SDS-PAGE gel of DUX4-flag associated proteins, 6 h after 250 ng/ml doxycycline treatment. ( F ) Immunoblots of DUX4-associated factors, p300 and CBP, in immunoprecipitates with Flag-DUX4. ( G ) Direct interaction of DUX4 with p300. Left panel: Coomassie staining of GST and GST-DUX4 proteins purified from E. coli . Right panel: immunoblot for recombinant p300 after GST-pull down, showing interaction with GST-DUX4 but not GST alone.
    Figure Legend Snippet: DUX4 interacts with p300/CBP. ( A ) Schematic diagram of the lentiviral constructs carrying doxycycline-inducible DUX4 and rtTA. sgTRE: second generation tet-response element for doxycycline-induced expression. ( B ) Immunoblot showing doxycycline dose-dependent inducible expression of DUX4-flag in LHCN-M2 cells. ( C ) Time course of induction of DUX4 downstream target genes, FRG2 and ZSCAN4 . ( D ) Cell death of immortalized human LHCN-M2 myoblasts 48 hours after DUX4 induction with 250 ng/ml doxycycline. ( E ) Silver stained SDS-PAGE gel of DUX4-flag associated proteins, 6 h after 250 ng/ml doxycycline treatment. ( F ) Immunoblots of DUX4-associated factors, p300 and CBP, in immunoprecipitates with Flag-DUX4. ( G ) Direct interaction of DUX4 with p300. Left panel: Coomassie staining of GST and GST-DUX4 proteins purified from E. coli . Right panel: immunoblot for recombinant p300 after GST-pull down, showing interaction with GST-DUX4 but not GST alone.

    Techniques Used: Construct, Expressing, Staining, SDS Page, Western Blot, Purification, Recombinant

    p300 is recruited to the DUX4 binding site in ZSCAN4 . ( A ) Location of PCR amplicons in the ZSCAN4 gene used for ChIP analyses. The previously-identified DUX4-binding site is at +1430, in exon 2. ( B–G ) ChIP assays with various antibodies at the indicated ZSCAN4 loci (ZSCAN4-P: promoter region, ZSCAN4-E: exon 2 region). LHCN-M2 cells were treated with 250 ng/ml dox for 6 h to express DUX4 or DUX4-ΔC and subjected to ChIP analyses.% input of H3K18Ac, H3K27Ac, and H3K4me3 were normalized with% input of H3 ( n = 3, error bars represent SEM). Note that both DUX4 and DUX4ΔC are recruited to the DUX4 site in exon 2, but p300 is not recruited by DUX4ΔC, and acetylation of H3K18 is not increased by DUX4ΔC.
    Figure Legend Snippet: p300 is recruited to the DUX4 binding site in ZSCAN4 . ( A ) Location of PCR amplicons in the ZSCAN4 gene used for ChIP analyses. The previously-identified DUX4-binding site is at +1430, in exon 2. ( B–G ) ChIP assays with various antibodies at the indicated ZSCAN4 loci (ZSCAN4-P: promoter region, ZSCAN4-E: exon 2 region). LHCN-M2 cells were treated with 250 ng/ml dox for 6 h to express DUX4 or DUX4-ΔC and subjected to ChIP analyses.% input of H3K18Ac, H3K27Ac, and H3K4me3 were normalized with% input of H3 ( n = 3, error bars represent SEM). Note that both DUX4 and DUX4ΔC are recruited to the DUX4 site in exon 2, but p300 is not recruited by DUX4ΔC, and acetylation of H3K18 is not increased by DUX4ΔC.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    The C-terminal domain of DUX4 is required for p300 interaction and DUX4-mediated transcriptional activation, but not for repression of target genes. ( A ) Schematic diagram of DUX4-ΔC and full length DUX4. The C-terminal 98 amino acids were deleted in DUX4-ΔC. ( B ) Western blots indicating the level of DUX4-ΔC and DUX4 expression in three independent LHCN-M2 clonal cell lines. Whole cell lysates were prepared after 6 h of dox treatment. Note that the C-terminal deletion is expressed much more abundantly. ( C ) p300/CBP binds to the C-terminal domain of DUX4. DUX4-ΔC-flag and DUX4-flag were immunoprecipitated with flag antibody 6 h after 250 ng dox treatment. Endogenous p300 and CBP were coprecipitated with DUX4-flag, but not with DUX4-ΔC-flag. ( D ) RT-qPCR analysis of two upregulated genes (ZSCAN4 and CCNA1), and two downregulated genes (MYOD1 and MYF5) in DUX4-ΔC or DUX4 inducible LHCN-M2 cells 6 h after 250 ng/ml dox treatment. Expression is normalized to GAPDH ( n = 3, three independent clones, error bars represent SEM, t-test: *** P
    Figure Legend Snippet: The C-terminal domain of DUX4 is required for p300 interaction and DUX4-mediated transcriptional activation, but not for repression of target genes. ( A ) Schematic diagram of DUX4-ΔC and full length DUX4. The C-terminal 98 amino acids were deleted in DUX4-ΔC. ( B ) Western blots indicating the level of DUX4-ΔC and DUX4 expression in three independent LHCN-M2 clonal cell lines. Whole cell lysates were prepared after 6 h of dox treatment. Note that the C-terminal deletion is expressed much more abundantly. ( C ) p300/CBP binds to the C-terminal domain of DUX4. DUX4-ΔC-flag and DUX4-flag were immunoprecipitated with flag antibody 6 h after 250 ng dox treatment. Endogenous p300 and CBP were coprecipitated with DUX4-flag, but not with DUX4-ΔC-flag. ( D ) RT-qPCR analysis of two upregulated genes (ZSCAN4 and CCNA1), and two downregulated genes (MYOD1 and MYF5) in DUX4-ΔC or DUX4 inducible LHCN-M2 cells 6 h after 250 ng/ml dox treatment. Expression is normalized to GAPDH ( n = 3, three independent clones, error bars represent SEM, t-test: *** P

    Techniques Used: Activation Assay, Western Blot, Expressing, Immunoprecipitation, Quantitative RT-PCR, Clone Assay

    Dominant negative activity of the p300 interacting C-terminal domain of DUX4. ( A ) Schematic of DUX4 expression constructs. 293T cells modified to express full-length DUX4 driven from the dox-inducible sgTRE promoter, shown above, were transfected with transient expression constructs, shown below. The number of C-terminal DUX4 amino acids is indicated. sgTRE: second generation tet-response element for dox-induced expression; CMV: CMV promoter for high level constitutive expression; NLS: SV40-nuclear localization signal; HD: homeodomain. GFP was added to stabilize DUX4 fragments. NLS was added to deliver DUX4 fragment in the nucleus. ( B ) DUX4 mediated ZSCAN4 mRNA expression. Cells were transfected, 24 h later 250 ng/ml dox was applied, and mRNA extracted 6 hours after that ( n = 3, error bars represent SEM). ( C ) Activity of a cotransfected DUX4-luciferase reporter. 293T cells were co-transfected with both full length DUX4 and empty vector (EV) or the tested fragments, together with DUX4 luciferase and Renilla control reporters ( n = 3, error bars represent SEM). ( D ) Western blots of DUX4 immunoprecipitations performed in the presence of GFP-NLS control or GFP-NLS-DUX4-C-terminal 98 amino acids. Interaction of full length DUX4 with p300 is inhibited by overexpression of the C-terminus of DUX4.
    Figure Legend Snippet: Dominant negative activity of the p300 interacting C-terminal domain of DUX4. ( A ) Schematic of DUX4 expression constructs. 293T cells modified to express full-length DUX4 driven from the dox-inducible sgTRE promoter, shown above, were transfected with transient expression constructs, shown below. The number of C-terminal DUX4 amino acids is indicated. sgTRE: second generation tet-response element for dox-induced expression; CMV: CMV promoter for high level constitutive expression; NLS: SV40-nuclear localization signal; HD: homeodomain. GFP was added to stabilize DUX4 fragments. NLS was added to deliver DUX4 fragment in the nucleus. ( B ) DUX4 mediated ZSCAN4 mRNA expression. Cells were transfected, 24 h later 250 ng/ml dox was applied, and mRNA extracted 6 hours after that ( n = 3, error bars represent SEM). ( C ) Activity of a cotransfected DUX4-luciferase reporter. 293T cells were co-transfected with both full length DUX4 and empty vector (EV) or the tested fragments, together with DUX4 luciferase and Renilla control reporters ( n = 3, error bars represent SEM). ( D ) Western blots of DUX4 immunoprecipitations performed in the presence of GFP-NLS control or GFP-NLS-DUX4-C-terminal 98 amino acids. Interaction of full length DUX4 with p300 is inhibited by overexpression of the C-terminus of DUX4.

    Techniques Used: Dominant Negative Mutation, Activity Assay, Expressing, Construct, Modification, Transfection, Luciferase, Plasmid Preparation, Western Blot, Over Expression

    27) Product Images from "TARBP2-Enhanced Resistance during Tamoxifen Treatment in Breast Cancer"

    Article Title: TARBP2-Enhanced Resistance during Tamoxifen Treatment in Breast Cancer

    Journal: Cancers

    doi: 10.3390/cancers11020210

    Tamoxifen induces SOX2 to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
    Figure Legend Snippet: Tamoxifen induces SOX2 to enhance tamoxifen resistance through TARBP2. ( A , B ) Expression of different stem cell markers after tamoxifen treatment. MCF-7 cells were treated with 2 μM tamoxifen for 48 h and then RNA was isolated to analyze the mRNA expression of stem cell markers by reverse-transcription PCR (qRT-PCR). The experiments were repeated at least 3 times, and ATP5E was used as a positive control for tamoxifen treatment ( A ). * p ≤ 0.05 by t -test. Cells as indicated in ( A ) were collected to analyze protein expression by western blotting ( B ). ( C , D ) Effect of SOX2 expression on tamoxifen sensitivity. MCF-7 cells were transfected with shRNA targeting SOX2 for 48 h and treated with different concentrations of tamoxifen (1, 2, 5, 10, 20 μM) for 72 h. The efficiency of SOX2 knock-down was examined by western blot ( C ), and the proliferation and colony formation were determined by MTT ( D ) and colony formation assays ( E ), respectively. MTT experimental results are given as the means ± SEM from at least three separate experiments that were performed in duplicate or triplicate and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01. ( F , G ) Tamoxifen downregulated the protein level of SOX2 through TARBP2. MCF-7 cells were transfected with shRNAs targeting TARBP2 for 48 h; 2 μM tamoxifen was then added to the culture medium for 48 h. The cells were harvested to determine the protein expressions by western blot. ( G – I ) TARBP2-regulated protein stability of SOX2 in tamoxifen-treated and resistant cells. Tamoxifen-treated (2 μM for 48 h) MCF-7 ( G ) and MCF-7/TR1 ( H ) cells were treated with 50 μg/mL cycloheximide to block protein synthesis and were then harvested at the indicated time point to analyze the expression of SOX2 by western blotting. ( I ) MCF-7 cells were transfected with the indicated shRNAs targeting TARBP2 for 48 h and treated with 2 μM tamoxifen for 48 h. Cells were add 50 μg/mL cycloheximide and harvested at the indicated time point to analyze the expression of SOX2 by western blotting. The degradation rates were plotted for the average ± SEM of at least three independent experiments and analyzed by two-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Techniques Used: Expressing, Isolation, Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control, Western Blot, Transfection, shRNA, MTT Assay, Blocking Assay

    Both SOX2 and TARBP2 expression are elevated in hormone therapy-resistant tumor cells. ( A ) The correlation of SOX2 expression with the overall survival of ER-positive breast cancer patients was analyzed and downloaded using Kaplan-Meier Plotter ( http://kmplot.com/ ). ( B , C ) Association of SOX2 expression and hormone therapy resistance in breast cancer tissues. Representative serial sections of Figure 1 B showed images of SOX2 IHC in primary tumors and tumors in lymph nodes in cases of cancer recurrence ( B ). Scale Bar: 100 uM. Statistics of SOX2 protein expression levels in primary tumors and metastatic tumor cells in cases of cancer recurrence ( C ). ( D ) Resistance mechanism for tamoxifen–induced TARBP2-SOX2 in breast cancer.
    Figure Legend Snippet: Both SOX2 and TARBP2 expression are elevated in hormone therapy-resistant tumor cells. ( A ) The correlation of SOX2 expression with the overall survival of ER-positive breast cancer patients was analyzed and downloaded using Kaplan-Meier Plotter ( http://kmplot.com/ ). ( B , C ) Association of SOX2 expression and hormone therapy resistance in breast cancer tissues. Representative serial sections of Figure 1 B showed images of SOX2 IHC in primary tumors and tumors in lymph nodes in cases of cancer recurrence ( B ). Scale Bar: 100 uM. Statistics of SOX2 protein expression levels in primary tumors and metastatic tumor cells in cases of cancer recurrence ( C ). ( D ) Resistance mechanism for tamoxifen–induced TARBP2-SOX2 in breast cancer.

    Techniques Used: Expressing, Immunohistochemistry

    28) Product Images from "MicroRNA-9-5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway"

    Article Title: MicroRNA-9-5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4062

    miR-9-5p enhances HCC cell migration by Klf4. (A) Wound healing assay of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic/inhibitor or siKlf4/Klf4 for 48 h. Images were taken in randomly selected fields at a higher magnification (×40). (B) Cell migration assay in SK-Hep-1 and HCC-LM3 cells transfected with 100 nM miR-9-5p mimic/inhibitor or 2 µ g Klf4/siKlf4. Images were acquired at a higher magnification (×40) at the fixed location of the panel. Numbers of migratory cells number were counted and are presented in the bar graph. Representative images are presented. * P
    Figure Legend Snippet: miR-9-5p enhances HCC cell migration by Klf4. (A) Wound healing assay of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic/inhibitor or siKlf4/Klf4 for 48 h. Images were taken in randomly selected fields at a higher magnification (×40). (B) Cell migration assay in SK-Hep-1 and HCC-LM3 cells transfected with 100 nM miR-9-5p mimic/inhibitor or 2 µ g Klf4/siKlf4. Images were acquired at a higher magnification (×40) at the fixed location of the panel. Numbers of migratory cells number were counted and are presented in the bar graph. Representative images are presented. * P

    Techniques Used: Migration, Wound Healing Assay, Cell Migration Assay, Transfection

    miRNAs that potentially downregulate Klf4 expression in HCC. (A) Crossed interaction of miRNAs, according to prediction databases to predict the miRNAs that bind to the 3′-untranslated region of Klf4. (B) The candidate miRNA mimics were transfected into Hep-3B cells, and the expression level of Klf4 was measured by using RT-qPCR. (C) The transfection efficiency of the candidate miRNAs' mimics was measured using RT-qPCR analysis in Hep-3B cells. * P
    Figure Legend Snippet: miRNAs that potentially downregulate Klf4 expression in HCC. (A) Crossed interaction of miRNAs, according to prediction databases to predict the miRNAs that bind to the 3′-untranslated region of Klf4. (B) The candidate miRNA mimics were transfected into Hep-3B cells, and the expression level of Klf4 was measured by using RT-qPCR. (C) The transfection efficiency of the candidate miRNAs' mimics was measured using RT-qPCR analysis in Hep-3B cells. * P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    miR-9-5p/Klf4 axis enhances HCC cell proliferation via an AKT/mTOR-associated pathway. (A) The transfection efficiency of miR-9-5p mimic or inhibitor, or siKlf4/Klf4 was measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis. (B) Cell proliferation viability was determined by cell counting kit-8 assay. A total number of 2,000 transfected cells dissolved in 100 µ l 10% fetal bovine serum were seeded into 96-well plates on days 1-5, and the absorbance was measured at 450 nm every 24 h to obtain a cell growth curve. (C) Flow cytometric analysis of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic or inhibitor, or siKlf4/Klf4 for 48 h. Representative data are featured, presenting the population of living cells (Annexin V-FITC-/PI-) in the left lower quadrant, early apoptotic cells (Annexin V-FITC+/PI-) in right lower quadrant, late apoptotic cells (Annexin V-FITC +/PI+) in the right upper quadrant and necrotic cells (Annexin V-FITC -/PI+) in the left upper quadrant. (D) Levels of AKT, p-AKT S473, mTOR, p-mTOR, Bcl-2 and Bax were examined by western blotting in SK-Hep-1 and HCC-LM3 cells transfected with miR-9-5p mimic/inhibitor or Klf4/siKlf4. (E) Flow cytometric analysis of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic/inhibitor together with AKTi (10 µ l, MK-2206, 2 HCl; dissolved in DMSO) for 48 h. (F) Cell proliferation analysis of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic/inhibitor with AKT inhibitor (10 µ l, MK-2206, 2 HCl; dissolved in DMSO) for 48 h. * P
    Figure Legend Snippet: miR-9-5p/Klf4 axis enhances HCC cell proliferation via an AKT/mTOR-associated pathway. (A) The transfection efficiency of miR-9-5p mimic or inhibitor, or siKlf4/Klf4 was measured using reverse transcription-quantitative polymerase chain reaction and western blot analysis. (B) Cell proliferation viability was determined by cell counting kit-8 assay. A total number of 2,000 transfected cells dissolved in 100 µ l 10% fetal bovine serum were seeded into 96-well plates on days 1-5, and the absorbance was measured at 450 nm every 24 h to obtain a cell growth curve. (C) Flow cytometric analysis of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic or inhibitor, or siKlf4/Klf4 for 48 h. Representative data are featured, presenting the population of living cells (Annexin V-FITC-/PI-) in the left lower quadrant, early apoptotic cells (Annexin V-FITC+/PI-) in right lower quadrant, late apoptotic cells (Annexin V-FITC +/PI+) in the right upper quadrant and necrotic cells (Annexin V-FITC -/PI+) in the left upper quadrant. (D) Levels of AKT, p-AKT S473, mTOR, p-mTOR, Bcl-2 and Bax were examined by western blotting in SK-Hep-1 and HCC-LM3 cells transfected with miR-9-5p mimic/inhibitor or Klf4/siKlf4. (E) Flow cytometric analysis of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic/inhibitor together with AKTi (10 µ l, MK-2206, 2 HCl; dissolved in DMSO) for 48 h. (F) Cell proliferation analysis of SK-Hep-1 and HCC-LM3 cells treated with miR-9-5p mimic/inhibitor with AKT inhibitor (10 µ l, MK-2206, 2 HCl; dissolved in DMSO) for 48 h. * P

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting, Flow Cytometry

    29) Product Images from "Involvement of COX-2/PGE2 Pathway in the Upregulation of MMP-9 Expression in Pancreatic Cancer"

    Article Title: Involvement of COX-2/PGE2 Pathway in the Upregulation of MMP-9 Expression in Pancreatic Cancer

    Journal: Gastroenterology Research and Practice

    doi: 10.1155/2011/214269

    Involvement of COX-2 in the upregulation of MMP-9 by TNF- α and LPS. (a) Western blotting revealed expression of COX-2 and MMP-9 was increased in BxPC-3 after treatment with exogenous TNF- α or LPS. (b) COX-2 mRNA expression was upregulated by TNF- α or LPS (* P
    Figure Legend Snippet: Involvement of COX-2 in the upregulation of MMP-9 by TNF- α and LPS. (a) Western blotting revealed expression of COX-2 and MMP-9 was increased in BxPC-3 after treatment with exogenous TNF- α or LPS. (b) COX-2 mRNA expression was upregulated by TNF- α or LPS (* P

    Techniques Used: Western Blot, Expressing

    Migration analysis. Migration of BxPC-3 cells was increased after treatment with TNF- α (* P
    Figure Legend Snippet: Migration analysis. Migration of BxPC-3 cells was increased after treatment with TNF- α (* P

    Techniques Used: Migration

    30) Product Images from "Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner"

    Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

    Journal: Autophagy

    doi: 10.1080/15548627.2015.1068489

    ( See previous page ). Bafilomycin A 1 promotes differentiation, elevates cell death and decreases chondrocyte proliferation in cultured metatarsal bones. Histological appearance of different zones of the growth plate of cultured bones showed increased hypertrophy upon Baf exposure ( A ). Baf caused an increase in phosphorylated RPS6 ( B ) and SQSTM1 accumulation ( C ) in chondrocytes when assessed by immunohistochemistry. Visualization of proteoglycan levels by Safranin O staining did not reveal any changes ( D ). Treatment with Baf elevated cell death as assessed by TUNEL labeling ( E ) and decreased cell proliferation as assessed by BrdU incorporation ( F ). Baf stimulated an increase in Col10a1 levels as assessed by in situ hybridization ( G ) and immunohistochemistry ( H ). Levels of mineralization were assessed by Von Kossa staining and showed no obvious differences ( I ). Osteoclasts were visualized by ACP5 staining and no changes were observed ( J ). All bones were exposed to Baf for 3 d. Scale bar: 100 μm. Statistical analysis is incorporated in the text.
    Figure Legend Snippet: ( See previous page ). Bafilomycin A 1 promotes differentiation, elevates cell death and decreases chondrocyte proliferation in cultured metatarsal bones. Histological appearance of different zones of the growth plate of cultured bones showed increased hypertrophy upon Baf exposure ( A ). Baf caused an increase in phosphorylated RPS6 ( B ) and SQSTM1 accumulation ( C ) in chondrocytes when assessed by immunohistochemistry. Visualization of proteoglycan levels by Safranin O staining did not reveal any changes ( D ). Treatment with Baf elevated cell death as assessed by TUNEL labeling ( E ) and decreased cell proliferation as assessed by BrdU incorporation ( F ). Baf stimulated an increase in Col10a1 levels as assessed by in situ hybridization ( G ) and immunohistochemistry ( H ). Levels of mineralization were assessed by Von Kossa staining and showed no obvious differences ( I ). Osteoclasts were visualized by ACP5 staining and no changes were observed ( J ). All bones were exposed to Baf for 3 d. Scale bar: 100 μm. Statistical analysis is incorporated in the text.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Immunohistochemistry, Staining, TUNEL Assay, Labeling, BrdU Incorporation Assay, In Situ Hybridization

    31) Product Images from "Bufalin suppresses hepatocarcinogenesis by targeting β-catenin/TCF signaling via cell cycle-related kinase"

    Article Title: Bufalin suppresses hepatocarcinogenesis by targeting β-catenin/TCF signaling via cell cycle-related kinase

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22113-2

    Bufalin reduces CCRK expression by inhibiting the transcription. ( A ) Bufalin decreased CCRK expression in the perinuclear region. Green immunofluorescence staining of CCRK was shown in representative images and nuclei were counterstained with DAPI. ( B ) Bufalin decreased CCRK mRNA level in a dose-dependent manner. Various concentrations of bufalin were incubated with PLC5 cells and quantitative RT-PCR was performed to detect CCRK transcript levels. ( C ) Bufalin inhibited CCRK expression and inactivated CCRK-induced β-catenin signaling in a dose-dependent manner. Western blot analysis was performed to detect the expression of CCRK, active and total β-catenin, CCND1 and EGFR. β-actin was used as a loading control. ( D ) Bufalin impeded the binding of transcriptional factor to CCRK promoter in PLC5 cells. Bufalin or vehicle was incubated with PLC5 cells and anti-AR antibody was used to pulldown antigen- CCRK promoter complex for quantitative PCR analysis. IgG antibody was used as a control in ChIP assay. ( E ) Bufalin impaired CCRK promoter activity using luciferase reporter assay. CCRK promoter constructor and Renilla were transfected into PLC5 cells followed by the treatment with bufalin or vehicle. Luciferase activities related to Renilla control were measured. *** P
    Figure Legend Snippet: Bufalin reduces CCRK expression by inhibiting the transcription. ( A ) Bufalin decreased CCRK expression in the perinuclear region. Green immunofluorescence staining of CCRK was shown in representative images and nuclei were counterstained with DAPI. ( B ) Bufalin decreased CCRK mRNA level in a dose-dependent manner. Various concentrations of bufalin were incubated with PLC5 cells and quantitative RT-PCR was performed to detect CCRK transcript levels. ( C ) Bufalin inhibited CCRK expression and inactivated CCRK-induced β-catenin signaling in a dose-dependent manner. Western blot analysis was performed to detect the expression of CCRK, active and total β-catenin, CCND1 and EGFR. β-actin was used as a loading control. ( D ) Bufalin impeded the binding of transcriptional factor to CCRK promoter in PLC5 cells. Bufalin or vehicle was incubated with PLC5 cells and anti-AR antibody was used to pulldown antigen- CCRK promoter complex for quantitative PCR analysis. IgG antibody was used as a control in ChIP assay. ( E ) Bufalin impaired CCRK promoter activity using luciferase reporter assay. CCRK promoter constructor and Renilla were transfected into PLC5 cells followed by the treatment with bufalin or vehicle. Luciferase activities related to Renilla control were measured. *** P

    Techniques Used: Expressing, Immunofluorescence, Staining, Incubation, Quantitative RT-PCR, Western Blot, Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Activity Assay, Luciferase, Reporter Assay, Transfection

    Bufalin blocks HCC cell proliferation and transformation through CCRK-mediated β-catenin/TCF signaling. ( A ) CCRK knockdown attenuated the effect of bufalin on suppressing cell proliferation. Stably CCRK-depriving PLC5 cells or parental cells were treated with indicated concentrations of bufalin or vehicle. Cell growths were detected using CCK-8 assay. Inhibitory rates were plotted as the percentage of viable cells treated with vehicle. ( B ) CCRK expression promoted bufalin-induced cell suppression. Stable CCRK-expressing LO2 cells or parental cells were treated with indicated concentrations of bufalin or vehicle followed by the detection of cell growths in CCK-8 assay. ( C and D ) Bufalin abrogated CCRK-induced anchorage-dependent ( C ) or -independent cell growth (D) detected by colony formation or soft agar assays, respectively. Representative images of colonies formed are shown. Original magnification ×100. ( E ) Bufalin impeded CCRK-induced G1/S cell cycle progression. ( F ) Bufalin suppressed CCRK-induced β-catenin/TCF signaling activity. Protein expressions of CCRK, active and total β-catenin, CCND1, EGFR and PCNA were detected and β-actin was used as a loading control. **P
    Figure Legend Snippet: Bufalin blocks HCC cell proliferation and transformation through CCRK-mediated β-catenin/TCF signaling. ( A ) CCRK knockdown attenuated the effect of bufalin on suppressing cell proliferation. Stably CCRK-depriving PLC5 cells or parental cells were treated with indicated concentrations of bufalin or vehicle. Cell growths were detected using CCK-8 assay. Inhibitory rates were plotted as the percentage of viable cells treated with vehicle. ( B ) CCRK expression promoted bufalin-induced cell suppression. Stable CCRK-expressing LO2 cells or parental cells were treated with indicated concentrations of bufalin or vehicle followed by the detection of cell growths in CCK-8 assay. ( C and D ) Bufalin abrogated CCRK-induced anchorage-dependent ( C ) or -independent cell growth (D) detected by colony formation or soft agar assays, respectively. Representative images of colonies formed are shown. Original magnification ×100. ( E ) Bufalin impeded CCRK-induced G1/S cell cycle progression. ( F ) Bufalin suppressed CCRK-induced β-catenin/TCF signaling activity. Protein expressions of CCRK, active and total β-catenin, CCND1, EGFR and PCNA were detected and β-actin was used as a loading control. **P

    Techniques Used: Transformation Assay, Stable Transfection, CCK-8 Assay, Expressing, Activity Assay

    Bufalin induces G1 phase arrest to inhibit HCC cell proliferation and transformation. ( A ) Bufalin inhibited the proliferation of PLC5 cells in a dose-dependent manner compared with LO2 cells. Indicated concentrations of bufalin were treated with PLC5 or LO2 cells for 48 hours. Cell viability was measured in CCK-8 assay and the inhibitory rate was calculated referring to vehicle treatment. ( B ) Bufalin inhibited the proliferation of PLC5 cells in a time-dependent manner compared with LO2 cells. 10 nmol/L of bufalin was incubated with PLC5 or LO2 cells for 5 consecutive days. Cell viability was measured every 24 hours and the inhibitory rate was calculated referring to vehicle treatment. ( C ) Bufalin inhibited focus formation of PLC5 cells in a dose-dependent manner. Representative images of colonies formed are shown. ( D ) Bufalin inhibited anchorage-independent growth of PLC5 cells in soft agar. Representative images of colonies formed are shown. Original magnification, ×100. ( E ) Bufalin impeded cell cycle transition by inducing G1 phase arrest in PLC5 cells. ( F ) The protein levels of CDC25A, CDK6 and CDKN1B were detected by western blot in bufalin-treated PLC5 cells. β-actin was used as an loading control. *P
    Figure Legend Snippet: Bufalin induces G1 phase arrest to inhibit HCC cell proliferation and transformation. ( A ) Bufalin inhibited the proliferation of PLC5 cells in a dose-dependent manner compared with LO2 cells. Indicated concentrations of bufalin were treated with PLC5 or LO2 cells for 48 hours. Cell viability was measured in CCK-8 assay and the inhibitory rate was calculated referring to vehicle treatment. ( B ) Bufalin inhibited the proliferation of PLC5 cells in a time-dependent manner compared with LO2 cells. 10 nmol/L of bufalin was incubated with PLC5 or LO2 cells for 5 consecutive days. Cell viability was measured every 24 hours and the inhibitory rate was calculated referring to vehicle treatment. ( C ) Bufalin inhibited focus formation of PLC5 cells in a dose-dependent manner. Representative images of colonies formed are shown. ( D ) Bufalin inhibited anchorage-independent growth of PLC5 cells in soft agar. Representative images of colonies formed are shown. Original magnification, ×100. ( E ) Bufalin impeded cell cycle transition by inducing G1 phase arrest in PLC5 cells. ( F ) The protein levels of CDC25A, CDK6 and CDKN1B were detected by western blot in bufalin-treated PLC5 cells. β-actin was used as an loading control. *P

    Techniques Used: Transformation Assay, CCK-8 Assay, Incubation, Western Blot

    Bufalin inhibits CCRK-driven β-catenin/TCF signaling-dependent tumorigenicity in nude mice. ( A ) Bufalin decreased CCRK-induced tumor growth in xenograft model compared with vehicle treatment. ( B ) Bufalin inhibited CCRK expression and then inactivated CCRK-induced β-catenin signaling. Expression levels of CCRK, active and total β-catenin, CCND1 and EGFR were detected in western blot. β-actin was used as a loading control. ( C ) Bufalin inhibited CCRK-induced intrahepatic neoplasm formation in orthotopic model compared with vehicle treatment. ( D ) Bufalin inhibited CCRK-induced tumor growth in orthotopic model. The tumor volume and weight were measured and compared between two groups treated with bufalin or vehicle. ( E ) Representative images of tumor and nontumor tissues in orthotopic HCC mouse models were shown in hematoxylin and eosin (H E) staining. Mouse models were treated with bufalin or vehicle. ( F ) Immunohistochemical staining of CCRK and β-catenin in the representative section from orthotopic mouse model. Positive cytoplasmic staining of CCRK and positive nuclear staining of β-catenin were observed in vehicle group, while bufalin treatment decreased the staining strength of CCRK in the cytoplasm and restricted β-catenin in the cytoplasm. **P
    Figure Legend Snippet: Bufalin inhibits CCRK-driven β-catenin/TCF signaling-dependent tumorigenicity in nude mice. ( A ) Bufalin decreased CCRK-induced tumor growth in xenograft model compared with vehicle treatment. ( B ) Bufalin inhibited CCRK expression and then inactivated CCRK-induced β-catenin signaling. Expression levels of CCRK, active and total β-catenin, CCND1 and EGFR were detected in western blot. β-actin was used as a loading control. ( C ) Bufalin inhibited CCRK-induced intrahepatic neoplasm formation in orthotopic model compared with vehicle treatment. ( D ) Bufalin inhibited CCRK-induced tumor growth in orthotopic model. The tumor volume and weight were measured and compared between two groups treated with bufalin or vehicle. ( E ) Representative images of tumor and nontumor tissues in orthotopic HCC mouse models were shown in hematoxylin and eosin (H E) staining. Mouse models were treated with bufalin or vehicle. ( F ) Immunohistochemical staining of CCRK and β-catenin in the representative section from orthotopic mouse model. Positive cytoplasmic staining of CCRK and positive nuclear staining of β-catenin were observed in vehicle group, while bufalin treatment decreased the staining strength of CCRK in the cytoplasm and restricted β-catenin in the cytoplasm. **P

    Techniques Used: Mouse Assay, Expressing, Western Blot, Staining, Immunohistochemistry

    Bufalin inhibits β-catenin/TCF signaling in HCC cells. ( A ) Bufalin induced the redistribution of β-catenin from nuclei to cytoplasm. Red immunofluorescence staining of β-catenin was shown in representative images and nuclei were counterstained with DAPI. ( B ) Bufalin inactivated β-catenin signaling in a dose-dependent manner. Western blot analysis was performed to detect the expression of active and total β-catenin, CCND1, EGFR and PCNA. β-actin was used as a loading control. ( C ) Bufalin decreased CCND1 and EGFR mRNA levels, but not CTNNB1 , in a dose-dependent manner. Various concentrations of bufalin were incubated with PLC5 cells and quantitative RT-PCR was performed to detect transcript levels. ***P
    Figure Legend Snippet: Bufalin inhibits β-catenin/TCF signaling in HCC cells. ( A ) Bufalin induced the redistribution of β-catenin from nuclei to cytoplasm. Red immunofluorescence staining of β-catenin was shown in representative images and nuclei were counterstained with DAPI. ( B ) Bufalin inactivated β-catenin signaling in a dose-dependent manner. Western blot analysis was performed to detect the expression of active and total β-catenin, CCND1, EGFR and PCNA. β-actin was used as a loading control. ( C ) Bufalin decreased CCND1 and EGFR mRNA levels, but not CTNNB1 , in a dose-dependent manner. Various concentrations of bufalin were incubated with PLC5 cells and quantitative RT-PCR was performed to detect transcript levels. ***P

    Techniques Used: Immunofluorescence, Staining, Western Blot, Expressing, Incubation, Quantitative RT-PCR

    32) Product Images from "SIRT1-dependent mitochondrial biogenesis supports therapeutic effects of resveratrol against neurodevelopment damage by fluoride"

    Article Title: SIRT1-dependent mitochondrial biogenesis supports therapeutic effects of resveratrol against neurodevelopment damage by fluoride

    Journal: Theranostics

    doi: 10.7150/thno.42387

    RSV protects cells from NaF-caused adverse neuronal effects via promoting SIRT1-depeendent PGC-1α-NRF1-TFAM signaling pathway. (A) SIRT1 deacetylase activity in SH-SY5Y cells using SIRT1 assay kit. (B) Immunoblot analysis of SIRT1 in SH-SY5Y cells and the corresponding quantification. (C, D) RT-qPCR (C) and immunoblot (D) analyses of PGC-1α, NRF1 and TFAM in SH-SY5Y cells. Quantification represents the levels of the indicated mRNA and protein normalized to GAPDH. (E, F) RT-qPCR (E) and immunoblot (F) analyses of representative subunits encoded by mtDNA in SH-SY5Y cells. Quantification represents the levels of the indicated mRNA and protein normalized to GAPDH. (G) RT-qPCR analysis of relative mtDNA contents in SH-SY5Y cells. (H, I) Representative flow plots of MMP levels (H) and mitoROS production (I) in SH-SY5Y cells using flow cytometry. (J) Levels of cell viability in SH-SY5Y cells determined by CCK-8 assay. SH-SY5Y cells were preincubated with 20 μM RSV for 2 h followed by co-culturing with 60 mg/L NaF and 3 mM NIC for 24 h. Data information: Data are presented as mean ± SD. Data were cumulative of at least three independent experiments. * P
    Figure Legend Snippet: RSV protects cells from NaF-caused adverse neuronal effects via promoting SIRT1-depeendent PGC-1α-NRF1-TFAM signaling pathway. (A) SIRT1 deacetylase activity in SH-SY5Y cells using SIRT1 assay kit. (B) Immunoblot analysis of SIRT1 in SH-SY5Y cells and the corresponding quantification. (C, D) RT-qPCR (C) and immunoblot (D) analyses of PGC-1α, NRF1 and TFAM in SH-SY5Y cells. Quantification represents the levels of the indicated mRNA and protein normalized to GAPDH. (E, F) RT-qPCR (E) and immunoblot (F) analyses of representative subunits encoded by mtDNA in SH-SY5Y cells. Quantification represents the levels of the indicated mRNA and protein normalized to GAPDH. (G) RT-qPCR analysis of relative mtDNA contents in SH-SY5Y cells. (H, I) Representative flow plots of MMP levels (H) and mitoROS production (I) in SH-SY5Y cells using flow cytometry. (J) Levels of cell viability in SH-SY5Y cells determined by CCK-8 assay. SH-SY5Y cells were preincubated with 20 μM RSV for 2 h followed by co-culturing with 60 mg/L NaF and 3 mM NIC for 24 h. Data information: Data are presented as mean ± SD. Data were cumulative of at least three independent experiments. * P

    Techniques Used: Pyrolysis Gas Chromatography, Histone Deacetylase Assay, Activity Assay, Quantitative RT-PCR, Flow Cytometry, CCK-8 Assay

    Disturbance of circulating mitochondrial biogenesis signaling molecules are associated with intelligence loss in children. (A) Fluoride concentration in drinking water and urine determined by a standardized ion selective electrode method. (B) Intelligence quotient (IQ) scores of children measured by CRT-RC2. (C-F) Levels of circulating SIRT1 (C), PGC-1α (D), NRF1 (E) and TFAM (F) in peripheral blood lymphocytes detected by ELISA assay. (G-J) Correlation between urinary fluoride concentration and IQ scores (G), as well as circulating SIRT1 (H), PGC-1α (I), TFAM (J) levels. (K-M) Correlation between IQ scores and circulating SIRT1 (K), PGC-1α (L), TFAM (M) levels. A total of 30 children in control areas and 30 children in high fluoride areas in Tianjin, China were recruited randomly. Data information: Data are presented as mean ± SD. Detailed statistical tests were shown as unpaired two-tailed Student's t test (A-F) and Pearson correlation coefficient analysis (G-M). * P
    Figure Legend Snippet: Disturbance of circulating mitochondrial biogenesis signaling molecules are associated with intelligence loss in children. (A) Fluoride concentration in drinking water and urine determined by a standardized ion selective electrode method. (B) Intelligence quotient (IQ) scores of children measured by CRT-RC2. (C-F) Levels of circulating SIRT1 (C), PGC-1α (D), NRF1 (E) and TFAM (F) in peripheral blood lymphocytes detected by ELISA assay. (G-J) Correlation between urinary fluoride concentration and IQ scores (G), as well as circulating SIRT1 (H), PGC-1α (I), TFAM (J) levels. (K-M) Correlation between IQ scores and circulating SIRT1 (K), PGC-1α (L), TFAM (M) levels. A total of 30 children in control areas and 30 children in high fluoride areas in Tianjin, China were recruited randomly. Data information: Data are presented as mean ± SD. Detailed statistical tests were shown as unpaired two-tailed Student's t test (A-F) and Pearson correlation coefficient analysis (G-M). * P

    Techniques Used: Concentration Assay, Pyrolysis Gas Chromatography, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    RSV improves SIRT1-relied mitochondrial biogenesis process in NaF-injured hippocampal tissues of offspring rats. (A) Immunoblot analyses of SIRT1, PGC-1 and NRF1 in hippocampal tissues ( n = 3 rats per group). GAPDH was used as the internal control. (B) Representative images of the IHC staining for SIRT1 + and NRF1 + neurons in hippocampal DG region. SIRT1 + and NRF1 + neuronal cells are demonstrated by black arrows and quantified. Scale bars represent 50 μm, n = 2 rats per group. (C) RT-qPCR analyses of relative mtDNA contents in hippocampal tissues ( n = 6 rats per group). (D) Immunoblot analyses of ATP6 in hippocampal tissues ( n = 3 rats per group). (E) Representative images of the IHC staining for ATP6 + neurons in hippocampal DG region. ATP6 + neuronal cells are demonstrated by black arrows and quantified. Scale bars represent 50 μm, n = 2 rats per group. Data information: Data are presented as mean ± SD. * P
    Figure Legend Snippet: RSV improves SIRT1-relied mitochondrial biogenesis process in NaF-injured hippocampal tissues of offspring rats. (A) Immunoblot analyses of SIRT1, PGC-1 and NRF1 in hippocampal tissues ( n = 3 rats per group). GAPDH was used as the internal control. (B) Representative images of the IHC staining for SIRT1 + and NRF1 + neurons in hippocampal DG region. SIRT1 + and NRF1 + neuronal cells are demonstrated by black arrows and quantified. Scale bars represent 50 μm, n = 2 rats per group. (C) RT-qPCR analyses of relative mtDNA contents in hippocampal tissues ( n = 6 rats per group). (D) Immunoblot analyses of ATP6 in hippocampal tissues ( n = 3 rats per group). (E) Representative images of the IHC staining for ATP6 + neurons in hippocampal DG region. ATP6 + neuronal cells are demonstrated by black arrows and quantified. Scale bars represent 50 μm, n = 2 rats per group. Data information: Data are presented as mean ± SD. * P

    Techniques Used: Pyrolysis Gas Chromatography, Immunohistochemistry, Staining, Quantitative RT-PCR

    A proposed model for the role of mitochondrial biogenesis process in developmental fluoride neurotoxicity and protective action of RSV. Mitochondrial biogenesis process plays a vital role in developmental fluoride neurotoxicity. Improvement in mitochondrial biogenesis by TFAM overexpression causes restoration of mitochondrial function, thus alleviating neurotoxic effects of fluoride. Importantly, RSV protects against developmental fluoride neurotoxicity by enhancing mitochondrial biogenesis and function activated by SIRT1-dependent PGC-1α/NRF1/TFAM signaling pathway, which is suppressed by SIRT1 antagonist NIC. Images of hippocampus, vacuum blood-collection tubes and developmental children were modified from http://p.ayxbk.com/image s/5/5b/Hippocampus_and_seahorse_cropped.JPG; https://www.hellorf.com/image/show/146119724?source=zcool term=tubes%20prepared , and http://www.jianbihua.cc/renwu/31334_2.html , respectively, with permission.
    Figure Legend Snippet: A proposed model for the role of mitochondrial biogenesis process in developmental fluoride neurotoxicity and protective action of RSV. Mitochondrial biogenesis process plays a vital role in developmental fluoride neurotoxicity. Improvement in mitochondrial biogenesis by TFAM overexpression causes restoration of mitochondrial function, thus alleviating neurotoxic effects of fluoride. Importantly, RSV protects against developmental fluoride neurotoxicity by enhancing mitochondrial biogenesis and function activated by SIRT1-dependent PGC-1α/NRF1/TFAM signaling pathway, which is suppressed by SIRT1 antagonist NIC. Images of hippocampus, vacuum blood-collection tubes and developmental children were modified from http://p.ayxbk.com/image s/5/5b/Hippocampus_and_seahorse_cropped.JPG; https://www.hellorf.com/image/show/146119724?source=zcool term=tubes%20prepared , and http://www.jianbihua.cc/renwu/31334_2.html , respectively, with permission.

    Techniques Used: Over Expression, Pyrolysis Gas Chromatography, Modification

    NaF exposure caused SIRT1 expression changes both in vitro and in vivo . (A) ChIP-PCR analyses for PGC-1α, NRF1 and TFAM binding to the SIRT1 promoter in SH-SY5Y cells. (B) SIRT1 deacetylase activity in SH-SY5Y cells measured by SIRT1 assay kits. (C, D) RT-qPCR (C) and immunoblot (D) analyses of SIRT1 in SH-SY5Y cells. Quantification represents the levels of the indicated mRNA and protein normalized to GAPDH. (E) Immunoblot analyses of SIRT1 in hippocampal tissues ( n = 3 rats per group). (F) Representative images of the IHC staining for SIRT1-expressing (SIRT1 + ) neurons in hippocampal CA1 region. SIRT1 + neuronal cells are demonstrated by black arrows and quantified. Scale bars represent 50 μm, n = 2 rats per group. Data information: Data are presented as mean ± SD. Data were cumulative of at least three independent experiments (A-D). * P
    Figure Legend Snippet: NaF exposure caused SIRT1 expression changes both in vitro and in vivo . (A) ChIP-PCR analyses for PGC-1α, NRF1 and TFAM binding to the SIRT1 promoter in SH-SY5Y cells. (B) SIRT1 deacetylase activity in SH-SY5Y cells measured by SIRT1 assay kits. (C, D) RT-qPCR (C) and immunoblot (D) analyses of SIRT1 in SH-SY5Y cells. Quantification represents the levels of the indicated mRNA and protein normalized to GAPDH. (E) Immunoblot analyses of SIRT1 in hippocampal tissues ( n = 3 rats per group). (F) Representative images of the IHC staining for SIRT1-expressing (SIRT1 + ) neurons in hippocampal CA1 region. SIRT1 + neuronal cells are demonstrated by black arrows and quantified. Scale bars represent 50 μm, n = 2 rats per group. Data information: Data are presented as mean ± SD. Data were cumulative of at least three independent experiments (A-D). * P

    Techniques Used: Expressing, In Vitro, In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Pyrolysis Gas Chromatography, Binding Assay, Histone Deacetylase Assay, Activity Assay, Quantitative RT-PCR, Immunohistochemistry, Staining

    33) Product Images from "Silibinin, Dexamethasone, and Doxycycline as Potential Therapeutic Agents for Treating Vesicant-Inflicted Ocular Injuries"

    Article Title: Silibinin, Dexamethasone, and Doxycycline as Potential Therapeutic Agents for Treating Vesicant-Inflicted Ocular Injuries

    Journal: Toxicology and applied pharmacology

    doi: 10.1016/j.taap.2012.07.014

    Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin formulation treatment on NM-induced VEGF expression in cultured rabbit eye corneal epithelium. The dissected rabbit corneas in culture were either untreated (UC), or
    Figure Legend Snippet: Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin formulation treatment on NM-induced VEGF expression in cultured rabbit eye corneal epithelium. The dissected rabbit corneas in culture were either untreated (UC), or

    Techniques Used: Expressing, Cell Culture

    Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin or silibinin formulation treatments on NM-induced epithelial thickness in cultured rabbit eye cornea. The dissected rabbit corneas in culture were either untreated
    Figure Legend Snippet: Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin or silibinin formulation treatments on NM-induced epithelial thickness in cultured rabbit eye cornea. The dissected rabbit corneas in culture were either untreated

    Techniques Used: Cell Culture

    Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin or silibinin formulation treatments on NM-induced microbullae formation (epithelial-stromal separation) in cultured rabbit eye cornea. The dissected rabbit corneas
    Figure Legend Snippet: Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin or silibinin formulation treatments on NM-induced microbullae formation (epithelial-stromal separation) in cultured rabbit eye cornea. The dissected rabbit corneas

    Techniques Used: Cell Culture

    Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin formulation treatment on NM-induced apoptotic cell death in cultured rabbit eye corneal epithelium. The dissected rabbit corneas in culture were either untreated (UC),
    Figure Legend Snippet: Effect of doxycycline, dexamethasone treatments alone or in combination, and silibinin formulation treatment on NM-induced apoptotic cell death in cultured rabbit eye corneal epithelium. The dissected rabbit corneas in culture were either untreated (UC),

    Techniques Used: Cell Culture

    Effect of doxycycline, dexamethasone treatments alone or in combination (A), and silibinin or silibinin formulation treatments (B) on NM-induced protein expression of COX-2 and MMP-9 in cultured rabbit eye cornea. The dissected rabbit corneas in culture
    Figure Legend Snippet: Effect of doxycycline, dexamethasone treatments alone or in combination (A), and silibinin or silibinin formulation treatments (B) on NM-induced protein expression of COX-2 and MMP-9 in cultured rabbit eye cornea. The dissected rabbit corneas in culture

    Techniques Used: Expressing, Cell Culture

    34) Product Images from "Silibinin inhibits prostate cancer invasion, motility and migration by suppressing vimentin and MMP-2 expression"

    Article Title: Silibinin inhibits prostate cancer invasion, motility and migration by suppressing vimentin and MMP-2 expression

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2009.94

    Dose- and time-dependent inhibitory effects of silibinin on migration of ARCaP M cells in vitro . The confluent ARCaP M monolayers were wounded by scraping and treated with silibinin (50, 100, and 200 μmol/L). Cell migration to the wound surface
    Figure Legend Snippet: Dose- and time-dependent inhibitory effects of silibinin on migration of ARCaP M cells in vitro . The confluent ARCaP M monolayers were wounded by scraping and treated with silibinin (50, 100, and 200 μmol/L). Cell migration to the wound surface

    Techniques Used: Migration, In Vitro

    Inhibitory effects of silibinin on the expression of vimentin and MMP-2 in ARCaP M cells. (A and B) Cells were treated with the indicated doses of silibinin (50, 100, and 200 μmol/L) for 24, 48, and 72 h, and then cell lysates were subjected to
    Figure Legend Snippet: Inhibitory effects of silibinin on the expression of vimentin and MMP-2 in ARCaP M cells. (A and B) Cells were treated with the indicated doses of silibinin (50, 100, and 200 μmol/L) for 24, 48, and 72 h, and then cell lysates were subjected to

    Techniques Used: Expressing

    Different sensitivities of prostate cancer cell lines to growth inhibition by silibinin. After cells were treated with the indicated doses of silibinin (50, 100, and 200 μmol/L) for 24, 48, 72, and 96 h, cell viabilities were determined by MTT
    Figure Legend Snippet: Different sensitivities of prostate cancer cell lines to growth inhibition by silibinin. After cells were treated with the indicated doses of silibinin (50, 100, and 200 μmol/L) for 24, 48, 72, and 96 h, cell viabilities were determined by MTT

    Techniques Used: Inhibition, MTT Assay

    Dose- and time-dependent inhibitory effects of silibinin on the invasion and motility of ARCaP M cells. ARCaP M cells were treated with different concentrations of silibinin (50, 100, and 200 μmol/L) for 24, 48, and 72 h and then harvested and seeded
    Figure Legend Snippet: Dose- and time-dependent inhibitory effects of silibinin on the invasion and motility of ARCaP M cells. ARCaP M cells were treated with different concentrations of silibinin (50, 100, and 200 μmol/L) for 24, 48, and 72 h and then harvested and seeded

    Techniques Used:

    35) Product Images from "Protective Effects of Silibinin and Its Possible Mechanism of Action in Mice Exposed to Chronic Unpredictable Mild Stress"

    Article Title: Protective Effects of Silibinin and Its Possible Mechanism of Action in Mice Exposed to Chronic Unpredictable Mild Stress

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2014.138

    Effects of silibinin on CUMS-induced depression-like behavior in the forced swimming test. All values are expressed as the mean ± SEM ( n =12 each), ** p
    Figure Legend Snippet: Effects of silibinin on CUMS-induced depression-like behavior in the forced swimming test. All values are expressed as the mean ± SEM ( n =12 each), ** p

    Techniques Used:

    Effects of silibinin on CUMS-induced depression-like behavior in the tail suspension test. All values are expressed as the mean ± SEM ( n =12 each), ** p
    Figure Legend Snippet: Effects of silibinin on CUMS-induced depression-like behavior in the tail suspension test. All values are expressed as the mean ± SEM ( n =12 each), ** p

    Techniques Used:

    Effects of silibinin on CUMS-induced depression-like behavior in the open field test. (A) The total number of squares crossed. (B) The frequency of rearing. All values are expressed as the mean ± SEM ( n =12 each), ** p
    Figure Legend Snippet: Effects of silibinin on CUMS-induced depression-like behavior in the open field test. (A) The total number of squares crossed. (B) The frequency of rearing. All values are expressed as the mean ± SEM ( n =12 each), ** p

    Techniques Used:

    36) Product Images from "Chemopreventive Effects of Silibinin on Colitis-Associated Tumorigenesis by Inhibiting IL-6/STAT3 Signaling Pathway"

    Article Title: Chemopreventive Effects of Silibinin on Colitis-Associated Tumorigenesis by Inhibiting IL-6/STAT3 Signaling Pathway

    Journal: Mediators of Inflammation

    doi: 10.1155/2018/1562010

    Silibinin supplementation inhibited proliferation and promoted apoptosis in intestinal tumor cells. (a, b) IMCE and HCT-116 cells were treated with silibinin at indicated concentrations for 72 h, respectively. Cell viability was then determined by MTT assay. Data are representative of three independent experiments and expressed as mean ± SD. (c, d) Colon sections from the AOM/DSS and AOM/DSS/SB groups were stained with Ki-67 (brown staining). (e, f) Colon sections from the two groups were stained with TUNEL. Green staining represented apoptotic cells. Scale bars, 50 μ m. Positive rate was determined by counting positively stained nuclei in tumor cells at 5 randomly selected fields from each group. SB: silibinin. n = 5~11 ( ∗ P
    Figure Legend Snippet: Silibinin supplementation inhibited proliferation and promoted apoptosis in intestinal tumor cells. (a, b) IMCE and HCT-116 cells were treated with silibinin at indicated concentrations for 72 h, respectively. Cell viability was then determined by MTT assay. Data are representative of three independent experiments and expressed as mean ± SD. (c, d) Colon sections from the AOM/DSS and AOM/DSS/SB groups were stained with Ki-67 (brown staining). (e, f) Colon sections from the two groups were stained with TUNEL. Green staining represented apoptotic cells. Scale bars, 50 μ m. Positive rate was determined by counting positively stained nuclei in tumor cells at 5 randomly selected fields from each group. SB: silibinin. n = 5~11 ( ∗ P

    Techniques Used: MTT Assay, Staining, TUNEL Assay

    Silibinin supplementation restored colonic barrier function and ameliorated the infiltration of macrophages. (a) Paraffin-embedded specimens of colonic tumor-adjacent tissue were subjected to immunofluorescent staining for ZO-1 distribution using an anti-ZO-1 antibody and a FITC-labeled secondary antibody (green staining). Nuclei were stained with DAPI (blue staining). (b) Real-time PCR analysis of ZO-1 expression in colonic epithelium was also shown. (c, d) PAS staining for goblet cells in tumor-adjacent tissue was shown, and diagrams presented average positive cells per crypt of each group. (e, f) Expression of F4/80 in tumor-adjacent tissue was assessed using immunohistochemical staining. The positive rate was determined by counting the absolute number of positive staining in at least 100 colonic crypts of each mouse. Scale bars, 50 μ m. SB: silibinin. n = 5~11 ( ∗∗ P
    Figure Legend Snippet: Silibinin supplementation restored colonic barrier function and ameliorated the infiltration of macrophages. (a) Paraffin-embedded specimens of colonic tumor-adjacent tissue were subjected to immunofluorescent staining for ZO-1 distribution using an anti-ZO-1 antibody and a FITC-labeled secondary antibody (green staining). Nuclei were stained with DAPI (blue staining). (b) Real-time PCR analysis of ZO-1 expression in colonic epithelium was also shown. (c, d) PAS staining for goblet cells in tumor-adjacent tissue was shown, and diagrams presented average positive cells per crypt of each group. (e, f) Expression of F4/80 in tumor-adjacent tissue was assessed using immunohistochemical staining. The positive rate was determined by counting the absolute number of positive staining in at least 100 colonic crypts of each mouse. Scale bars, 50 μ m. SB: silibinin. n = 5~11 ( ∗∗ P

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

    Silibinin treatment suppressed colitis-associated tumorigenesis. (a) Hematoxylin and eosin (H E) staining of colon tumor and colitis in the control, AOM/DSS, and AOM/DSS/SB groups. Scale bars, 50 μ m. (b, c) Tumor score and colitis score of each group. SB: silibinin. n = 5~11 ( ∗ P
    Figure Legend Snippet: Silibinin treatment suppressed colitis-associated tumorigenesis. (a) Hematoxylin and eosin (H E) staining of colon tumor and colitis in the control, AOM/DSS, and AOM/DSS/SB groups. Scale bars, 50 μ m. (b, c) Tumor score and colitis score of each group. SB: silibinin. n = 5~11 ( ∗ P

    Techniques Used: Staining

    Silibinin downregulated IL-6/STAT3 pathway in AOM/DSS mice. (a, b) The expression of IL-6 in colonic tissues from the control, AOM/DSS, and AOM/DSS/SB mice was visualized by immunohistochemical staining. The percentage of IL-6 positive staining in five randomly selected fields of each group was shown. (c, f) STAT3 (c) and p-STAT3 (e) in colonic tissues from the three groups were detected. Positive rate of STAT3 (d) and p-STAT3 (f) in five randomly selected fields of each group was also presented separately. Scale bars, 50 μ m. SB: silibinin; ns: not significant. n = 5~11 ( ∗∗∗ P
    Figure Legend Snippet: Silibinin downregulated IL-6/STAT3 pathway in AOM/DSS mice. (a, b) The expression of IL-6 in colonic tissues from the control, AOM/DSS, and AOM/DSS/SB mice was visualized by immunohistochemical staining. The percentage of IL-6 positive staining in five randomly selected fields of each group was shown. (c, f) STAT3 (c) and p-STAT3 (e) in colonic tissues from the three groups were detected. Positive rate of STAT3 (d) and p-STAT3 (f) in five randomly selected fields of each group was also presented separately. Scale bars, 50 μ m. SB: silibinin; ns: not significant. n = 5~11 ( ∗∗∗ P

    Techniques Used: Mouse Assay, Expressing, Immunohistochemistry, Staining

    Silibinin decreased inflammatory cytokine production in intestinal tumor cell lines and AOM/DSS mice. (a, b) Real-time PCR was performed to detect the inflammatory cytokines in intestinal tumor cells. Relative expressions of IL-6, IL-1 β , and TNF- α in IMCE (a) and HCT-116 cells (b) were presented. (c) Relative expression of IL-6, IL-1 β , and TNF- α in colonic tumor-adjacent tissue in the control, AOM/DSS, and AOM/DSS/SB groups. Data were representative of three independent experiments and expressed as mean ± SD. SB: silibinin; ns: not significant. n = 5~11 ( ∗ P
    Figure Legend Snippet: Silibinin decreased inflammatory cytokine production in intestinal tumor cell lines and AOM/DSS mice. (a, b) Real-time PCR was performed to detect the inflammatory cytokines in intestinal tumor cells. Relative expressions of IL-6, IL-1 β , and TNF- α in IMCE (a) and HCT-116 cells (b) were presented. (c) Relative expression of IL-6, IL-1 β , and TNF- α in colonic tumor-adjacent tissue in the control, AOM/DSS, and AOM/DSS/SB groups. Data were representative of three independent experiments and expressed as mean ± SD. SB: silibinin; ns: not significant. n = 5~11 ( ∗ P

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

    Silibinin treatment ameliorated colitis and tumor load in AOM/DSS mice. (a) The experimental course of AOM/DSS mouse model. (b) Body weight changes of all groups after treatment. (c) Macroscopic appearance of colon in the control, AOM/DSS, and AOM/DSS/SB groups. (d) Colon length after treatment at day 70. (e) Colon weight after AOM/DSS induction at day 70. (f) Colon was opened longitudinally after sacrifice and the amount of tumors were calculated. (g, h) Histogram showing the size distribution of tumors and average tumor load. SB: silibinin. n = 5~11 ( ∗ P
    Figure Legend Snippet: Silibinin treatment ameliorated colitis and tumor load in AOM/DSS mice. (a) The experimental course of AOM/DSS mouse model. (b) Body weight changes of all groups after treatment. (c) Macroscopic appearance of colon in the control, AOM/DSS, and AOM/DSS/SB groups. (d) Colon length after treatment at day 70. (e) Colon weight after AOM/DSS induction at day 70. (f) Colon was opened longitudinally after sacrifice and the amount of tumors were calculated. (g, h) Histogram showing the size distribution of tumors and average tumor load. SB: silibinin. n = 5~11 ( ∗ P

    Techniques Used: Mouse Assay

    Silibinin inhibited STAT3 phosphorylation in intestinal tumor cells. (a) Western blot showed the expression of p-STAT3 and STAT3 in IMCE cells after incubation with silibinin and/or LPS for 3 h, 6 h, 12 h, and 24 h, respectively. (b) ImageJ was applied to densitometric analyses to determine the expression of p-STAT3 and STAT3 in IMCE cells, and the data were normalized as relative ratio to β -actin. (c) The expression of p-STAT3 and STAT3 in HCT-116 cells after incubation with silibinin and/or LPS for 3 h, 6 h, 12 h, and 24 h, respectively. (d) Densitometric analyses to determine the expression of p-STAT3 and STAT3 in HCT-116 cells. (e) Western blot was also utilized to show the expression of p-STAT3 and STAT3 in the control, AOM/DSS, and AOM/DSS/SB groups. (f) Densitometric analyses were also involved in quantifying the expression of p-STAT3 and STAT3 in the control, AOM/DSS, and AOM/DSS/SB groups. SB: silibinin; ns: not significant. n = 5~11 ( ∗∗ P
    Figure Legend Snippet: Silibinin inhibited STAT3 phosphorylation in intestinal tumor cells. (a) Western blot showed the expression of p-STAT3 and STAT3 in IMCE cells after incubation with silibinin and/or LPS for 3 h, 6 h, 12 h, and 24 h, respectively. (b) ImageJ was applied to densitometric analyses to determine the expression of p-STAT3 and STAT3 in IMCE cells, and the data were normalized as relative ratio to β -actin. (c) The expression of p-STAT3 and STAT3 in HCT-116 cells after incubation with silibinin and/or LPS for 3 h, 6 h, 12 h, and 24 h, respectively. (d) Densitometric analyses to determine the expression of p-STAT3 and STAT3 in HCT-116 cells. (e) Western blot was also utilized to show the expression of p-STAT3 and STAT3 in the control, AOM/DSS, and AOM/DSS/SB groups. (f) Densitometric analyses were also involved in quantifying the expression of p-STAT3 and STAT3 in the control, AOM/DSS, and AOM/DSS/SB groups. SB: silibinin; ns: not significant. n = 5~11 ( ∗∗ P

    Techniques Used: Western Blot, Expressing, Incubation

    37) Product Images from "NFATc1 regulates the transcription of DNA damage-induced apoptosis suppressor"

    Article Title: NFATc1 regulates the transcription of DNA damage-induced apoptosis suppressor

    Journal: Data in Brief

    doi: 10.1016/j.dib.2015.11.011

    ChIP assay of RNA polymerase (ser5) in HEK293 cells. RNA polymerase II (phospho-ser5) binding to the DDIAS promoter in HEK293 cells. A ChIP assay was performed using an anti-RNA pol II antibody. An anti-IgG antibody was used as a negative control. PCR amplification was performed using the indicated DDIAS promoter-specific primers or control primers specific for GAPDH .
    Figure Legend Snippet: ChIP assay of RNA polymerase (ser5) in HEK293 cells. RNA polymerase II (phospho-ser5) binding to the DDIAS promoter in HEK293 cells. A ChIP assay was performed using an anti-RNA pol II antibody. An anti-IgG antibody was used as a negative control. PCR amplification was performed using the indicated DDIAS promoter-specific primers or control primers specific for GAPDH .

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Polymerase Chain Reaction, Amplification

    38) Product Images from "Curcuminoid-phospholipid complex induces apoptosis in mammary epithelial cells by STAT-3 signaling"

    Article Title: Curcuminoid-phospholipid complex induces apoptosis in mammary epithelial cells by STAT-3 signaling

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2008.40.6.647

    A representative experiment of cleaved caspase 3 assessed by Western blot: cells were incubated in medium RPMI 1640 3% FCS containing 10 ng/ml insulin, with EGF (lane 1), an association of EGF plus CURC (100 ng/ml, lane 2) and only CURC (100 ng/ml, lane
    Figure Legend Snippet: A representative experiment of cleaved caspase 3 assessed by Western blot: cells were incubated in medium RPMI 1640 3% FCS containing 10 ng/ml insulin, with EGF (lane 1), an association of EGF plus CURC (100 ng/ml, lane 2) and only CURC (100 ng/ml, lane

    Techniques Used: Western Blot, Incubation

    JSI-124 reduces CURC effect on cleaved caspase 3 assessed by Western blot: cells were incubated for 12 h in medium RPMI 1640, 3% FCS containing CURC (100 ng/ml) alone or with JSI-124 (10 and 100 nM).
    Figure Legend Snippet: JSI-124 reduces CURC effect on cleaved caspase 3 assessed by Western blot: cells were incubated for 12 h in medium RPMI 1640, 3% FCS containing CURC (100 ng/ml) alone or with JSI-124 (10 and 100 nM).

    Techniques Used: Western Blot, Incubation

    39) Product Images from "Killing of Kaposi's sarcoma-associated herpesvirus-infected fibroblasts during latent infection by activated natural killer cells"

    Article Title: Killing of Kaposi's sarcoma-associated herpesvirus-infected fibroblasts during latent infection by activated natural killer cells

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201040661

    Latently infected primary neonatal dermal KSHV-GFP fibroblast cells generated through co-culture with BCBL-1-GFP cells. (A) Micrograph of a single KSHV-GFP infected fibroblast 24 h after co-culture with BCBL-1-GFP cells (photographed at 200× magnification under phase-contrast and UV fluorescence). (B) Flow cytometric analysis of GFP expression by KSHV-GFP-infected fibroblasts (grey histogram) after G418 selection relative to uninfected fibroblasts (open histogram). (C) IFA detection of ORF 73 LANA protein in nuclei of KSHV-infected fibroblasts (photographed at 200× magnification under phase-contrast and UV fluorescence). (A–C) are representative of five independent experiments. (D) Total cellular KSHV viral load determined by quantitative real-time PCR analysis of K5 DNA by chronically KSHV-infected fibroblasts and BCBL-1 cells. Data are shown as mean copies/cell±SD ( n =3). (E) Expression of KSHV K3 and K5 lytic cycle associated mRNA production in resting and TPA-stimulated KSHV-infected fibroblasts and BCBL-1-GFP cells determined by quantitative real-time RT-PCR (qRT-PCR). KSHV-infected fibroblasts or BCBL-1-GFP cells were stimulated with TPA before harvesting for mRNA analysis by qRT-PCR. Data shown are the mean±SD of copies of mRNA/10 4 cells upon correction for cell input by qRT-PCR for GAPDH and are summarised from four experiments.
    Figure Legend Snippet: Latently infected primary neonatal dermal KSHV-GFP fibroblast cells generated through co-culture with BCBL-1-GFP cells. (A) Micrograph of a single KSHV-GFP infected fibroblast 24 h after co-culture with BCBL-1-GFP cells (photographed at 200× magnification under phase-contrast and UV fluorescence). (B) Flow cytometric analysis of GFP expression by KSHV-GFP-infected fibroblasts (grey histogram) after G418 selection relative to uninfected fibroblasts (open histogram). (C) IFA detection of ORF 73 LANA protein in nuclei of KSHV-infected fibroblasts (photographed at 200× magnification under phase-contrast and UV fluorescence). (A–C) are representative of five independent experiments. (D) Total cellular KSHV viral load determined by quantitative real-time PCR analysis of K5 DNA by chronically KSHV-infected fibroblasts and BCBL-1 cells. Data are shown as mean copies/cell±SD ( n =3). (E) Expression of KSHV K3 and K5 lytic cycle associated mRNA production in resting and TPA-stimulated KSHV-infected fibroblasts and BCBL-1-GFP cells determined by quantitative real-time RT-PCR (qRT-PCR). KSHV-infected fibroblasts or BCBL-1-GFP cells were stimulated with TPA before harvesting for mRNA analysis by qRT-PCR. Data shown are the mean±SD of copies of mRNA/10 4 cells upon correction for cell input by qRT-PCR for GAPDH and are summarised from four experiments.

    Techniques Used: Infection, Generated, Co-Culture Assay, Fluorescence, Flow Cytometry, Expressing, Selection, Immunofluorescence, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    40) Product Images from "Cyclin-dependent kinase inhibitor p21 controls adult neural stem cell expansion by regulating Sox2 gene expression"

    Article Title: Cyclin-dependent kinase inhibitor p21 controls adult neural stem cell expansion by regulating Sox2 gene expression

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2012.12.001

    DNA damage checkpoint activation in p21-null and Sox2 overexpressing cells ( A ) Crystal violet staining of murine p53 -null MEFs transduced with empty vector (EV) or constructs encoding the indicated factors. Sox2 overexpression does not induce cellular senescence in the absence of p53. Transduction with oncogenic Ras V12 has been included as a positive control. ( B ) Cellular lysates from NSCs transduced with the indicated constructs were subjected to immunoblotting using the specified antibodies. Sox2 overexpression induced an increase in p53 protein levels. ( C ) Wild-type or p53 -null NSCs were transduced with constructs encoding GFP or Sox2/GFP. Expression of Sox2 decreased the number of secondary neurospheres in the wild-type cells but did not affect the self-renewal of p53 -null NSCs. Treatment of Sox2/GFP-transduced cells with the ATM inhibitor KU55933 restored neurosphere formation to the levels found in GFP-transduced cells (n = 3). ( D ) Assessment in wild-type or p21 -null NSCs of p16 , and p19 Arf mRNA levels by qRT-PCR, showing an increase of endogenous p19 Arf expression in p21-deficient cells (n = 3). ( E ) Levels of immunostaining for p16, p19 Arf , and p53 in the nuclei of p21 -wild-type and null cells, as measured by high content fluorescence microscopy (n = 3). ( F ) p21-deficient neurospheres were transduced with control (-), p53 or p16/p19 Arf shRNAs. Graph showing the rescue of the growth arrest observed in p21 -deficient NSCs by knocking down p16/19 or p53 . The inset shows the knockdown efficiency of the indicated shRNAi constructs by qPCR analysis. Data are represented as the average ± s.e.m. of the indicated number of the experiments (n) (* p
    Figure Legend Snippet: DNA damage checkpoint activation in p21-null and Sox2 overexpressing cells ( A ) Crystal violet staining of murine p53 -null MEFs transduced with empty vector (EV) or constructs encoding the indicated factors. Sox2 overexpression does not induce cellular senescence in the absence of p53. Transduction with oncogenic Ras V12 has been included as a positive control. ( B ) Cellular lysates from NSCs transduced with the indicated constructs were subjected to immunoblotting using the specified antibodies. Sox2 overexpression induced an increase in p53 protein levels. ( C ) Wild-type or p53 -null NSCs were transduced with constructs encoding GFP or Sox2/GFP. Expression of Sox2 decreased the number of secondary neurospheres in the wild-type cells but did not affect the self-renewal of p53 -null NSCs. Treatment of Sox2/GFP-transduced cells with the ATM inhibitor KU55933 restored neurosphere formation to the levels found in GFP-transduced cells (n = 3). ( D ) Assessment in wild-type or p21 -null NSCs of p16 , and p19 Arf mRNA levels by qRT-PCR, showing an increase of endogenous p19 Arf expression in p21-deficient cells (n = 3). ( E ) Levels of immunostaining for p16, p19 Arf , and p53 in the nuclei of p21 -wild-type and null cells, as measured by high content fluorescence microscopy (n = 3). ( F ) p21-deficient neurospheres were transduced with control (-), p53 or p16/p19 Arf shRNAs. Graph showing the rescue of the growth arrest observed in p21 -deficient NSCs by knocking down p16/19 or p53 . The inset shows the knockdown efficiency of the indicated shRNAi constructs by qPCR analysis. Data are represented as the average ± s.e.m. of the indicated number of the experiments (n) (* p

    Techniques Used: Activation Assay, Staining, Transduction, Plasmid Preparation, Construct, Over Expression, Positive Control, Expressing, Quantitative RT-PCR, Immunostaining, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

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    Concentration Assay:

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    Article Snippet: .. Transcription assays To reveal active pol I transcription foci, living GSK3β+/+ MEFs and GSK3β−/− MEFs grown on cover slips were pre-incubated with DMEM supplemented with 75 µM DRB (Sigma Aldrich) for 1 h. The FURD (Sigma-Aldrich) was then added to a final concentration of 2 mM and cellular uptake was allowed for up to 10 min , . ..

    Incubation:

    Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor
    Article Snippet: .. For mouse peptides, 14 μg non-phosphorylated peptide was incubated with or without 2 μl (0.70 μg) of active recombinant GSK3α or 2 μl GSK3β (1000 units) in 18 μl GSK3 kinase buffer with 4 μCi γ-32 P-ATP at 30°C for 1 h. About 20 μl samples were added to the filter (Microcon-10kDa Centrifugal Unit, Millipore) and centrifuged at 8000 × g for 30 min. .. This centrifugation was needed to remove 32 P-labeled GSK3 due to auto-activation of the recombinant GSK3 during the kinase assay, as the molecular weight of GSK3 was larger than 10 kDa and hence could not pass through the 10 kDa-filter of Microcon-10kDa Centrifugal Units.

    Article Title: Differential Hyperphosphorylation of Tau-S199, -T231 and -S396 in Organotypic Brain Slices of Alzheimer Mice. A Model to Study Early Tau Hyperphosphorylation Using Okadaic Acid
    Article Snippet: .. RPB983Mu01) was incubated with 2 μl glycogensynthase-kinase-3β (GSK-3β) stock (170–200 nmol min/mg, Sigma G4296) in 25 μl tau kinase buffer (40 mM HEPES, 5 mM EGTA, 3 mM MgCl2, pH 7.6) including 2 mM ATP overnight at 37°C. ..

    Recombinant:

    Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor
    Article Snippet: .. For mouse peptides, 14 μg non-phosphorylated peptide was incubated with or without 2 μl (0.70 μg) of active recombinant GSK3α or 2 μl GSK3β (1000 units) in 18 μl GSK3 kinase buffer with 4 μCi γ-32 P-ATP at 30°C for 1 h. About 20 μl samples were added to the filter (Microcon-10kDa Centrifugal Unit, Millipore) and centrifuged at 8000 × g for 30 min. .. This centrifugation was needed to remove 32 P-labeled GSK3 due to auto-activation of the recombinant GSK3 during the kinase assay, as the molecular weight of GSK3 was larger than 10 kDa and hence could not pass through the 10 kDa-filter of Microcon-10kDa Centrifugal Units.

    Activity Assay:

    Article Title: Colorectal cancer cells require glycogen synthase kinase-3β for sustaining mitosis via translocated promoter region (TPR)-dynein interaction
    Article Snippet: .. To examine the effect of inhibition of GSK3β activity, cells were treated with 25 µM AR-A014418 [ ] or 25 µM SB-216763 [ ] (Sigma-Aldrich) and collected at the indicated periods. .. This study used antibodies against GSK3β (32391, Abcam) and TPR (sc-101294, Santa Cruz Biotechnology).

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    Inhibition:

    Article Title: Colorectal cancer cells require glycogen synthase kinase-3β for sustaining mitosis via translocated promoter region (TPR)-dynein interaction
    Article Snippet: .. To examine the effect of inhibition of GSK3β activity, cells were treated with 25 µM AR-A014418 [ ] or 25 µM SB-216763 [ ] (Sigma-Aldrich) and collected at the indicated periods. .. This study used antibodies against GSK3β (32391, Abcam) and TPR (sc-101294, Santa Cruz Biotechnology).

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  • 94
    Millipore sp1
    Pml silencing and RA treatment induce similar chromatin remodeling events on Oct4 PP (A) MNase mapping of the Oct4 regulatory region. Top panel shows the map of Oct4 gene promoter and enhancer region (DE, PE, PP) and four CRs. HindIII and ScaI sites and Southern blot probes (PI, PII and PIII for PP, PE and DE, respectively) are depicted. P19 stem, siPml- or RA-treated cells were subjected to MNase digestion for 5 minutes. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern hybridization. The panels directly under the map show EtBr-stained gels and bottom panels show Southern blots probed with PI, PII or PIII. (B) Restriction enzyme accessibility of Oct4 PP region in P19 stem, siPml-treated, or RA-treated cells. “*” signs mark diagnostic bands indicative of sensitive sites. The results are summarized on the map above these blots. Restriction sites are labeled under the top map. A small black box on each map depicts the <t>Sp1-binding</t> site and HRE cluster on CR1. Abbreviations: DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; TIS, transcription initiation site.
    Sp1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore g418 plates
    Generation and verification of tagged protein arrays. ( A ) To tag ORFX as bait (V5–6×HIS) and prey (V5–3×VSV), a set of primers is used that anneal to identical binding sites within the template plasmids and have flanking sequence homologous to ORFX . PCR products generated from the bait and prey templates are transformed into a - and α-cells, respectively. Homologous recombination occurs between the variable portion of the 5′ primer (light blue) and the 3′ terminus of the ORF, and between the variable portion of the 3′ primer (red) and the 3′ UTR) of ORFX . Transformants are selected on <t>G418</t> plates, and colony PCR is performed to verify integration of the Kan r downstream of the desired ORF. Abbreviations: TEF, translational elongation factor; TEFp, TEF promoter; TEFt, TEF terminator: Kan r , kanamycin resistance; loxp, site for CRE specific homologous recombination. ( B . The asterisk (*) denotes possible misloading or protein degradation. Note in the RAD51 lane the multiple protein products. Expected protein sizes are listed in Supplemental Table 1. ( C ) Analysis of effects on cell growth by tagging essential genes. A total of 24 strains with essential genes tagged as baits (6×HIS) and preys (3×VSV) were grown to saturation and spotted in 10-fold dilutions on YPD. Pictures were taken after 2 d at 30°C.
    G418 Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore l002
    <t>L002</t> reduces hypertension (HTN)-induced murine cardiac fibrosis and hypertrophy: (A) After two weeks of Ang II infusion and L002 treatment, cardiac sections from the four groups were stained with Masson's trichrome to identify collagen deposits. Upper panel shows perivascular deposition of collagen, while the lower panel identifies deposition of cardiac interstitial collagen. (B) Collagen deposition (perivascular + Interstitial) was quantified by ImagePro software program analysis, n = 7–12. (C) M-mode echocardiographic images showing thickness of left ventricular (LV) wall and LV diameters. (D) Quantification of the thickness of LV wall from echocardiography analysis, n = 4–12. (E) Post-mortem heart weight to body weight (HW/BW) were assessed in control and treatment groups. n = 7–12. (F) Shows systolic blood pressures (SBP) after two weeks of Ang II treatment as assessed by tail-cuff methods, n = 4–12. Data represented as mean ± SEM. *p
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    Pml silencing and RA treatment induce similar chromatin remodeling events on Oct4 PP (A) MNase mapping of the Oct4 regulatory region. Top panel shows the map of Oct4 gene promoter and enhancer region (DE, PE, PP) and four CRs. HindIII and ScaI sites and Southern blot probes (PI, PII and PIII for PP, PE and DE, respectively) are depicted. P19 stem, siPml- or RA-treated cells were subjected to MNase digestion for 5 minutes. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern hybridization. The panels directly under the map show EtBr-stained gels and bottom panels show Southern blots probed with PI, PII or PIII. (B) Restriction enzyme accessibility of Oct4 PP region in P19 stem, siPml-treated, or RA-treated cells. “*” signs mark diagnostic bands indicative of sensitive sites. The results are summarized on the map above these blots. Restriction sites are labeled under the top map. A small black box on each map depicts the Sp1-binding site and HRE cluster on CR1. Abbreviations: DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; TIS, transcription initiation site.

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter

    doi: 10.1002/stem.623

    Figure Lengend Snippet: Pml silencing and RA treatment induce similar chromatin remodeling events on Oct4 PP (A) MNase mapping of the Oct4 regulatory region. Top panel shows the map of Oct4 gene promoter and enhancer region (DE, PE, PP) and four CRs. HindIII and ScaI sites and Southern blot probes (PI, PII and PIII for PP, PE and DE, respectively) are depicted. P19 stem, siPml- or RA-treated cells were subjected to MNase digestion for 5 minutes. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern hybridization. The panels directly under the map show EtBr-stained gels and bottom panels show Southern blots probed with PI, PII or PIII. (B) Restriction enzyme accessibility of Oct4 PP region in P19 stem, siPml-treated, or RA-treated cells. “*” signs mark diagnostic bands indicative of sensitive sites. The results are summarized on the map above these blots. Restriction sites are labeled under the top map. A small black box on each map depicts the Sp1-binding site and HRE cluster on CR1. Abbreviations: DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; TIS, transcription initiation site.

    Article Snippet: ChIP assays were performed as described [ ], using the following antibodies: Pml (sc-18423, Santa Cruz), Daxx (07-471, Millipore), TR2 (sc-9087, Santa Cruz), Sp1 (07-645, Millipore), SF1 (sc-10976X, Santa Cruz), BAF155 (sc-9746X, Santa Cruz), Brg1 (sc-10768X, Santa Cruz), Brm (sc-28710X, Santa Cruz), G9a (3306, Cell Signaling), RIP140 (ab42126, Abcam), H3 (ab1791, Abcam), H3.3 (ab62642, Abcam), H3K9me3 ( , Millipore), H3K27me3 (ab6002-25, Abcam), AcH3 (06-599, Millipore), HP1γ (sc-10213, Santa Cruz) and RNAPII (05-623, Millipore).

    Techniques: Southern Blot, Hybridization, Staining, Diagnostic Assay, Labeling, Binding Assay

    Pml-NBs are involved in Oct4 activation through directly associating with the Oct4 PP region (A) The Oct4 -reporter activity in P19 cells co-transfected with a Pml expression vector at various doses. (B) The activities of Oct4 -reporters deleted in various CRs. Left panel depicts Oct4 -reporters each deleted in different CRs. Right panel shows Pml-activated reporter activities. The activity in Pml-transfected P19 was divided by that in control transfected P19, and plotted as fold induction. (C) Upper panel: Map of four CRs (gray boxes) on the Oct4 gene regulatory region and ChIP primer sets as indicated. The black box within CR1 depicts Sp1-binding site and hormone response elements (for TR2 and SF1 binding). Lower panels: The fold change of Pml-ChIP (left), Daxx-ChIP (middle), and CBP/P300-ChIP (right) determined by qPCR. Control cell: black bar. siPml-treated cell: white bar. RA-treated cell: grey bar. Abbreviations: CR, conserved region; DE, distal enhancer; PE, proximal promoter; PP, proximal promoter; TIS, transcription initiation site. SEM is each indicated by an error bar.

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter

    doi: 10.1002/stem.623

    Figure Lengend Snippet: Pml-NBs are involved in Oct4 activation through directly associating with the Oct4 PP region (A) The Oct4 -reporter activity in P19 cells co-transfected with a Pml expression vector at various doses. (B) The activities of Oct4 -reporters deleted in various CRs. Left panel depicts Oct4 -reporters each deleted in different CRs. Right panel shows Pml-activated reporter activities. The activity in Pml-transfected P19 was divided by that in control transfected P19, and plotted as fold induction. (C) Upper panel: Map of four CRs (gray boxes) on the Oct4 gene regulatory region and ChIP primer sets as indicated. The black box within CR1 depicts Sp1-binding site and hormone response elements (for TR2 and SF1 binding). Lower panels: The fold change of Pml-ChIP (left), Daxx-ChIP (middle), and CBP/P300-ChIP (right) determined by qPCR. Control cell: black bar. siPml-treated cell: white bar. RA-treated cell: grey bar. Abbreviations: CR, conserved region; DE, distal enhancer; PE, proximal promoter; PP, proximal promoter; TIS, transcription initiation site. SEM is each indicated by an error bar.

    Article Snippet: ChIP assays were performed as described [ ], using the following antibodies: Pml (sc-18423, Santa Cruz), Daxx (07-471, Millipore), TR2 (sc-9087, Santa Cruz), Sp1 (07-645, Millipore), SF1 (sc-10976X, Santa Cruz), BAF155 (sc-9746X, Santa Cruz), Brg1 (sc-10768X, Santa Cruz), Brm (sc-28710X, Santa Cruz), G9a (3306, Cell Signaling), RIP140 (ab42126, Abcam), H3 (ab1791, Abcam), H3.3 (ab62642, Abcam), H3K9me3 ( , Millipore), H3K27me3 (ab6002-25, Abcam), AcH3 (06-599, Millipore), HP1γ (sc-10213, Santa Cruz) and RNAPII (05-623, Millipore).

    Techniques: Activation Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

    Pml activates Oct4 gene along with transcription factors TR2, SF1, and Sp1 (A): Pml expression vector was transfected into P19 cells with or without TR2, SF1 and Sp1 expression vectors in the presence of the pCR1 reporter, and the normalized luciferase activity was determined. (B): P19 cells were subjected to RNA silencing individually with TR2, SF1 and Sp1 in the absence and presence of siPml. Protein levels were determined by western blot. Actin serves as the internal control.

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter

    doi: 10.1002/stem.623

    Figure Lengend Snippet: Pml activates Oct4 gene along with transcription factors TR2, SF1, and Sp1 (A): Pml expression vector was transfected into P19 cells with or without TR2, SF1 and Sp1 expression vectors in the presence of the pCR1 reporter, and the normalized luciferase activity was determined. (B): P19 cells were subjected to RNA silencing individually with TR2, SF1 and Sp1 in the absence and presence of siPml. Protein levels were determined by western blot. Actin serves as the internal control.

    Article Snippet: ChIP assays were performed as described [ ], using the following antibodies: Pml (sc-18423, Santa Cruz), Daxx (07-471, Millipore), TR2 (sc-9087, Santa Cruz), Sp1 (07-645, Millipore), SF1 (sc-10976X, Santa Cruz), BAF155 (sc-9746X, Santa Cruz), Brg1 (sc-10768X, Santa Cruz), Brm (sc-28710X, Santa Cruz), G9a (3306, Cell Signaling), RIP140 (ab42126, Abcam), H3 (ab1791, Abcam), H3.3 (ab62642, Abcam), H3K9me3 ( , Millipore), H3K27me3 (ab6002-25, Abcam), AcH3 (06-599, Millipore), HP1γ (sc-10213, Santa Cruz) and RNAPII (05-623, Millipore).

    Techniques: Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Western Blot

    Pml recruits transcription factors TR2, SF1, and Sp1, and Brg1-dependent chromatin remodeling complex on Oct4 proximal promoter )

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter

    doi: 10.1002/stem.623

    Figure Lengend Snippet: Pml recruits transcription factors TR2, SF1, and Sp1, and Brg1-dependent chromatin remodeling complex on Oct4 proximal promoter )

    Article Snippet: ChIP assays were performed as described [ ], using the following antibodies: Pml (sc-18423, Santa Cruz), Daxx (07-471, Millipore), TR2 (sc-9087, Santa Cruz), Sp1 (07-645, Millipore), SF1 (sc-10976X, Santa Cruz), BAF155 (sc-9746X, Santa Cruz), Brg1 (sc-10768X, Santa Cruz), Brm (sc-28710X, Santa Cruz), G9a (3306, Cell Signaling), RIP140 (ab42126, Abcam), H3 (ab1791, Abcam), H3.3 (ab62642, Abcam), H3K9me3 ( , Millipore), H3K27me3 (ab6002-25, Abcam), AcH3 (06-599, Millipore), HP1γ (sc-10213, Santa Cruz) and RNAPII (05-623, Millipore).

    Techniques:

    LM-PCR to detect nucleosome insertion and rearrangement on Oct4 PP (A) LM-PCR analysis of the Oct4 PP region in P19 stem, siPml-treated and RA-treated cells. Specific primers (F1, F2, F3, F4 and R1 depicted under the control map) were each used to determine the boundary of a specific nucleosome. Data show the PCR products resolved on sequencing gels with the size maker (base pair) loaded on the left. The deduced actual size (after subtracting 25 bp linker length) of the fragment indicative of a specific nuclesome boundary is shown on the right. (B) nucleosome positions on PP in the three groups of cells according to LM-PCR results shown in panel A. Primers are indicated with arrows. The black box on the map shows the Sp1-HRE cluster.

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Promyelocytic leukemia protein in retinoic acid-induced chromatin remodeling of Oct4 gene promoter

    doi: 10.1002/stem.623

    Figure Lengend Snippet: LM-PCR to detect nucleosome insertion and rearrangement on Oct4 PP (A) LM-PCR analysis of the Oct4 PP region in P19 stem, siPml-treated and RA-treated cells. Specific primers (F1, F2, F3, F4 and R1 depicted under the control map) were each used to determine the boundary of a specific nucleosome. Data show the PCR products resolved on sequencing gels with the size maker (base pair) loaded on the left. The deduced actual size (after subtracting 25 bp linker length) of the fragment indicative of a specific nuclesome boundary is shown on the right. (B) nucleosome positions on PP in the three groups of cells according to LM-PCR results shown in panel A. Primers are indicated with arrows. The black box on the map shows the Sp1-HRE cluster.

    Article Snippet: ChIP assays were performed as described [ ], using the following antibodies: Pml (sc-18423, Santa Cruz), Daxx (07-471, Millipore), TR2 (sc-9087, Santa Cruz), Sp1 (07-645, Millipore), SF1 (sc-10976X, Santa Cruz), BAF155 (sc-9746X, Santa Cruz), Brg1 (sc-10768X, Santa Cruz), Brm (sc-28710X, Santa Cruz), G9a (3306, Cell Signaling), RIP140 (ab42126, Abcam), H3 (ab1791, Abcam), H3.3 (ab62642, Abcam), H3K9me3 ( , Millipore), H3K27me3 (ab6002-25, Abcam), AcH3 (06-599, Millipore), HP1γ (sc-10213, Santa Cruz) and RNAPII (05-623, Millipore).

    Techniques: Polymerase Chain Reaction, Sequencing

    Acetylated Sp1 does not bind at the bak or p21 promoters . Panel A shows the organisation and sequence of the bak (Ai) and p21 (Aii) probes, with hypothetical and proven Sp1/3 binding sites underlined. Sequence numbers refer to distance from the transcriptional start site of each gene. Panel B shows that binding of Sp1 to both target sequences is decreased following butyrate treatment. Panel B: Western of mobility shift assay (WeMSA) analysis of binding to the Bak and p21 probes shows that Sp1 binding decreases following treatment with 10 mM sodium butyrate for 24 h compared to an untreated control (upper panel). Binding of acetyl-Sp1 could not be detected by WeMSA (lower panel). These data are representative of three independent repeats. Panel C: The binding of Sp1 to the bak (panel Ci) and p21 (panel Cii) promoter sequences was determined following treatment of HCT116 cells with a range of butyrate concentrations (0-20 mM). The upper panels show WeMSA gels immunoprobed for Sp1. As a loading control, the same extracts were also separated by SDS page, and immunoprobed with the same antibody (lower panels). Data shown in Ci and Cii are representative of at least two independent repeat experiments. Panel Ciii shows mean (+/- SD) of response at the Bak promoter. Whilst the levels of Sp1 are broadly constant, levels of Sp1 binding for both probes are reduced following treatment with butyrate.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Acetylated Sp1 does not bind at the bak or p21 promoters . Panel A shows the organisation and sequence of the bak (Ai) and p21 (Aii) probes, with hypothetical and proven Sp1/3 binding sites underlined. Sequence numbers refer to distance from the transcriptional start site of each gene. Panel B shows that binding of Sp1 to both target sequences is decreased following butyrate treatment. Panel B: Western of mobility shift assay (WeMSA) analysis of binding to the Bak and p21 probes shows that Sp1 binding decreases following treatment with 10 mM sodium butyrate for 24 h compared to an untreated control (upper panel). Binding of acetyl-Sp1 could not be detected by WeMSA (lower panel). These data are representative of three independent repeats. Panel C: The binding of Sp1 to the bak (panel Ci) and p21 (panel Cii) promoter sequences was determined following treatment of HCT116 cells with a range of butyrate concentrations (0-20 mM). The upper panels show WeMSA gels immunoprobed for Sp1. As a loading control, the same extracts were also separated by SDS page, and immunoprobed with the same antibody (lower panels). Data shown in Ci and Cii are representative of at least two independent repeat experiments. Panel Ciii shows mean (+/- SD) of response at the Bak promoter. Whilst the levels of Sp1 are broadly constant, levels of Sp1 binding for both probes are reduced following treatment with butyrate.

    Article Snippet: Antibodies used include: HRP conjugated mouse anti-Actin (ab20272, Abcam), rabbit anti-Sp1 (cat#07-645, Millipore), rabbit anti-Sp3 (D-20, Santa Cruz Biotechnology); rabbit anti-acetyl lysine (cat#ab3879, Chemicon); mouse anti-HDAC1 (cat#05-614, clone 2e10, Millipore); mouse anti-HDAC2 (cat#05-814, clone 3F3, Millpore); rabbit anti-HDAC3 (ab16047, Abcam).

    Techniques: Sequencing, Binding Assay, Western Blot, Mobility Shift, SDS Page

    Knock-down of Sp1 reveals multiple targets in the p53 signalling pathway are altered . Sp1 knockdown was achieved using Ambion siRNA. RNA was extracted 48 and 72 hours post transfection, labelled and hybridised to human U133 plus 2.0 arrays. Analysis of microarray data was carried out using GCOS, Array assist and the DAVID online analysis tool. Statistical significance was determined using an unpaired two-tailed t test, summarised in the workflow shown in Fig 5A. Knockdown was confirmed by QPCR using TaqMan ® gene expression assays (Fig 5B). Due to variability in knockdown produced by transient transfections, the biological replicates are shown as individual bars. The gene list identified using PLIER was sorted for statistically significant (p > 0.05) gene expression changes of greater than 1.2 fold. Subsequent analysis of this list using DAVID identified a number of changes occurring in the p53/P21 regulatory pathway (panel C; adapted from DAVID [[ 39 , 40 ], and http://www.genome.jp/kegg/ ). Stars denote genes which were significantly upregulated (red) or down-regulated (black). Validation of a subset of genes was carried out using Applied Biosystems TaqMan ® gene expression assays, according to manufacturer's instructions. QPCR expression data for Bid, p21, serpine, P53AIP and Sp1 are shown in Fig 5, panel B for HCT116 cells following Sp1 knockdown harvested 48 (panel Di) or 72 (panel Dii) hours post-transfection. The biological replicates are shown as individual bars; designated n1 and n2.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Knock-down of Sp1 reveals multiple targets in the p53 signalling pathway are altered . Sp1 knockdown was achieved using Ambion siRNA. RNA was extracted 48 and 72 hours post transfection, labelled and hybridised to human U133 plus 2.0 arrays. Analysis of microarray data was carried out using GCOS, Array assist and the DAVID online analysis tool. Statistical significance was determined using an unpaired two-tailed t test, summarised in the workflow shown in Fig 5A. Knockdown was confirmed by QPCR using TaqMan ® gene expression assays (Fig 5B). Due to variability in knockdown produced by transient transfections, the biological replicates are shown as individual bars. The gene list identified using PLIER was sorted for statistically significant (p > 0.05) gene expression changes of greater than 1.2 fold. Subsequent analysis of this list using DAVID identified a number of changes occurring in the p53/P21 regulatory pathway (panel C; adapted from DAVID [[ 39 , 40 ], and http://www.genome.jp/kegg/ ). Stars denote genes which were significantly upregulated (red) or down-regulated (black). Validation of a subset of genes was carried out using Applied Biosystems TaqMan ® gene expression assays, according to manufacturer's instructions. QPCR expression data for Bid, p21, serpine, P53AIP and Sp1 are shown in Fig 5, panel B for HCT116 cells following Sp1 knockdown harvested 48 (panel Di) or 72 (panel Dii) hours post-transfection. The biological replicates are shown as individual bars; designated n1 and n2.

    Article Snippet: Antibodies used include: HRP conjugated mouse anti-Actin (ab20272, Abcam), rabbit anti-Sp1 (cat#07-645, Millipore), rabbit anti-Sp3 (D-20, Santa Cruz Biotechnology); rabbit anti-acetyl lysine (cat#ab3879, Chemicon); mouse anti-HDAC1 (cat#05-614, clone 2e10, Millipore); mouse anti-HDAC2 (cat#05-814, clone 3F3, Millpore); rabbit anti-HDAC3 (ab16047, Abcam).

    Techniques: Transfection, Microarray, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Produced

    Sp1 acetylation increases in a co-linear manner with bak and p21 expression following sodium butyrate treatment . HCT116 cells were treated with increasing concentrations of butyrate (0-20 mM) for 24 hr, fixed and fluorescence immunostained either for Sp1 and Bak or Ace-Sp1 and p21. Cellular fluorescence was quantified by High-Content Analysis. Panel A shows protein expression levels of Sp1 (Panel Ai); acetyl-Sp1 (Panel Aii); Bak, (Panel Aiii) and p21 (Panel Aiv). Data are from a single pass experiment with three replicates, with 50 fields per replicate scored. The EC 50 value for each event in response to butyrate is shown in Panel Av. Panel B shows representative images of HCT116 cells following 24 h of 0 or 10 mM sodium butyrate treatment and fluorescence immunostaining for acetyl-Sp1 and p21. Panel Bi shows representative fields from the control (untreated) cells (upper panel) and the treated culture (lower panel). Staining patterns in the treated cells broadly fell into two main types, cell positive for acetyl-Sp1 alone (examples marked by arrows i) and cells positive staining for both acetyl Sp1 and p21 (marked by arrows ii). These subpopulations were distinguished by plotting acetyl-Sp1 vs. p21 fluorescence (see supplementary online data Fig 2 for gating strategy). Panel Bii shows quantitation of data from three independent experiments, showing numbers of cells in gated fractions that were dual stained, as indicated.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Sp1 acetylation increases in a co-linear manner with bak and p21 expression following sodium butyrate treatment . HCT116 cells were treated with increasing concentrations of butyrate (0-20 mM) for 24 hr, fixed and fluorescence immunostained either for Sp1 and Bak or Ace-Sp1 and p21. Cellular fluorescence was quantified by High-Content Analysis. Panel A shows protein expression levels of Sp1 (Panel Ai); acetyl-Sp1 (Panel Aii); Bak, (Panel Aiii) and p21 (Panel Aiv). Data are from a single pass experiment with three replicates, with 50 fields per replicate scored. The EC 50 value for each event in response to butyrate is shown in Panel Av. Panel B shows representative images of HCT116 cells following 24 h of 0 or 10 mM sodium butyrate treatment and fluorescence immunostaining for acetyl-Sp1 and p21. Panel Bi shows representative fields from the control (untreated) cells (upper panel) and the treated culture (lower panel). Staining patterns in the treated cells broadly fell into two main types, cell positive for acetyl-Sp1 alone (examples marked by arrows i) and cells positive staining for both acetyl Sp1 and p21 (marked by arrows ii). These subpopulations were distinguished by plotting acetyl-Sp1 vs. p21 fluorescence (see supplementary online data Fig 2 for gating strategy). Panel Bii shows quantitation of data from three independent experiments, showing numbers of cells in gated fractions that were dual stained, as indicated.

    Article Snippet: Antibodies used include: HRP conjugated mouse anti-Actin (ab20272, Abcam), rabbit anti-Sp1 (cat#07-645, Millipore), rabbit anti-Sp3 (D-20, Santa Cruz Biotechnology); rabbit anti-acetyl lysine (cat#ab3879, Chemicon); mouse anti-HDAC1 (cat#05-614, clone 2e10, Millipore); mouse anti-HDAC2 (cat#05-814, clone 3F3, Millpore); rabbit anti-HDAC3 (ab16047, Abcam).

    Techniques: Expressing, Fluorescence, High Content Screening, Immunostaining, Staining, Quantitation Assay

    Effect of HDACi on cell cycle, p21 expression, bak expression and Sp1 expression and acetylation . The extent of the concomitant response of Sp1 acetylation, cell cycle arrest and p21 up-regulation was determined using a high-content biology approach. HCT116 cells were treated with concentration ranges of 0-20 mM sodium butyrate, 0-20 mM valproic acid (VPA), 0-20 μM Oxamflatin, 0-20 μM Scriptaid, 0-20 μM APHA compound 8, 0-20 μM CHAHA. In all cases treatments were carried outfor 24 h. Cells were stained using immunocytochemistry for DNA content (Hoescht), p21, bak, Sp1 and acetyl-Sp1 as described in the methods section. Cells were analysed, on the basis of DNA content, for cell cycle phase and divided into G1 (filled circles), S (filled squares) or G2/M (filled triangles). Levels of protein were calculated from mean total fluorescence and are expressed in terms of fluorophore fluorescence relative to that observed in untreated cells. Sp1 (filled squares) and acetyl-Sp1 (open squares) are shown on the same graph.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Effect of HDACi on cell cycle, p21 expression, bak expression and Sp1 expression and acetylation . The extent of the concomitant response of Sp1 acetylation, cell cycle arrest and p21 up-regulation was determined using a high-content biology approach. HCT116 cells were treated with concentration ranges of 0-20 mM sodium butyrate, 0-20 mM valproic acid (VPA), 0-20 μM Oxamflatin, 0-20 μM Scriptaid, 0-20 μM APHA compound 8, 0-20 μM CHAHA. In all cases treatments were carried outfor 24 h. Cells were stained using immunocytochemistry for DNA content (Hoescht), p21, bak, Sp1 and acetyl-Sp1 as described in the methods section. Cells were analysed, on the basis of DNA content, for cell cycle phase and divided into G1 (filled circles), S (filled squares) or G2/M (filled triangles). Levels of protein were calculated from mean total fluorescence and are expressed in terms of fluorophore fluorescence relative to that observed in untreated cells. Sp1 (filled squares) and acetyl-Sp1 (open squares) are shown on the same graph.

    Article Snippet: Antibodies used include: HRP conjugated mouse anti-Actin (ab20272, Abcam), rabbit anti-Sp1 (cat#07-645, Millipore), rabbit anti-Sp3 (D-20, Santa Cruz Biotechnology); rabbit anti-acetyl lysine (cat#ab3879, Chemicon); mouse anti-HDAC1 (cat#05-614, clone 2e10, Millipore); mouse anti-HDAC2 (cat#05-814, clone 3F3, Millpore); rabbit anti-HDAC3 (ab16047, Abcam).

    Techniques: Expressing, Concentration Assay, Staining, Immunocytochemistry, Fluorescence

    Acetylation of Sp1 precedes p21 up-regulation in response to all HDACi . Panel A shows EC 50 values calculated from the concentration-response data shown in Fig 2, for each of Sp1 acetylation and p21 upregulation. There was generally close agreement between these values. The third column indicates the concentration used in subsequent timecourse experiments. Panel B shows timecourse experiments. HCT116 cells were treated with concentrations of HDACi as indicated in panel A, for times from 0 to 6 h. Timepoints less that 1 h are 10 min and 30 min. Cells were fixed and stained for acetyl-Sp1 and for p21 as before. Fluorescences were quantitated using high content approaches. Graphs are for acetyl-Sp1 (filled squares) p21 (filled triangles) and total Sp1 (filled diamonds). The results show the means of three repeat experiments with internal triplicates.

    Journal: Molecular Cancer

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    doi: 10.1186/1476-4598-9-275

    Figure Lengend Snippet: Acetylation of Sp1 precedes p21 up-regulation in response to all HDACi . Panel A shows EC 50 values calculated from the concentration-response data shown in Fig 2, for each of Sp1 acetylation and p21 upregulation. There was generally close agreement between these values. The third column indicates the concentration used in subsequent timecourse experiments. Panel B shows timecourse experiments. HCT116 cells were treated with concentrations of HDACi as indicated in panel A, for times from 0 to 6 h. Timepoints less that 1 h are 10 min and 30 min. Cells were fixed and stained for acetyl-Sp1 and for p21 as before. Fluorescences were quantitated using high content approaches. Graphs are for acetyl-Sp1 (filled squares) p21 (filled triangles) and total Sp1 (filled diamonds). The results show the means of three repeat experiments with internal triplicates.

    Article Snippet: Antibodies used include: HRP conjugated mouse anti-Actin (ab20272, Abcam), rabbit anti-Sp1 (cat#07-645, Millipore), rabbit anti-Sp3 (D-20, Santa Cruz Biotechnology); rabbit anti-acetyl lysine (cat#ab3879, Chemicon); mouse anti-HDAC1 (cat#05-614, clone 2e10, Millipore); mouse anti-HDAC2 (cat#05-814, clone 3F3, Millpore); rabbit anti-HDAC3 (ab16047, Abcam).

    Techniques: Concentration Assay, Staining

    Generation and verification of tagged protein arrays. ( A ) To tag ORFX as bait (V5–6×HIS) and prey (V5–3×VSV), a set of primers is used that anneal to identical binding sites within the template plasmids and have flanking sequence homologous to ORFX . PCR products generated from the bait and prey templates are transformed into a - and α-cells, respectively. Homologous recombination occurs between the variable portion of the 5′ primer (light blue) and the 3′ terminus of the ORF, and between the variable portion of the 3′ primer (red) and the 3′ UTR) of ORFX . Transformants are selected on G418 plates, and colony PCR is performed to verify integration of the Kan r downstream of the desired ORF. Abbreviations: TEF, translational elongation factor; TEFp, TEF promoter; TEFt, TEF terminator: Kan r , kanamycin resistance; loxp, site for CRE specific homologous recombination. ( B . The asterisk (*) denotes possible misloading or protein degradation. Note in the RAD51 lane the multiple protein products. Expected protein sizes are listed in Supplemental Table 1. ( C ) Analysis of effects on cell growth by tagging essential genes. A total of 24 strains with essential genes tagged as baits (6×HIS) and preys (3×VSV) were grown to saturation and spotted in 10-fold dilutions on YPD. Pictures were taken after 2 d at 30°C.

    Journal: Genome Research

    Article Title: Examining protein-protein interactions using endogenously tagged yeast arrays: The Cross-and-Capture system

    doi: 10.1101/gr.6667007

    Figure Lengend Snippet: Generation and verification of tagged protein arrays. ( A ) To tag ORFX as bait (V5–6×HIS) and prey (V5–3×VSV), a set of primers is used that anneal to identical binding sites within the template plasmids and have flanking sequence homologous to ORFX . PCR products generated from the bait and prey templates are transformed into a - and α-cells, respectively. Homologous recombination occurs between the variable portion of the 5′ primer (light blue) and the 3′ terminus of the ORF, and between the variable portion of the 3′ primer (red) and the 3′ UTR) of ORFX . Transformants are selected on G418 plates, and colony PCR is performed to verify integration of the Kan r downstream of the desired ORF. Abbreviations: TEF, translational elongation factor; TEFp, TEF promoter; TEFt, TEF terminator: Kan r , kanamycin resistance; loxp, site for CRE specific homologous recombination. ( B . The asterisk (*) denotes possible misloading or protein degradation. Note in the RAD51 lane the multiple protein products. Expected protein sizes are listed in Supplemental Table 1. ( C ) Analysis of effects on cell growth by tagging essential genes. A total of 24 strains with essential genes tagged as baits (6×HIS) and preys (3×VSV) were grown to saturation and spotted in 10-fold dilutions on YPD. Pictures were taken after 2 d at 30°C.

    Article Snippet: Transformants that grew on the G418 plates were restreaked and tested for proper integration of the tagging cassette via colony PCR.

    Techniques: Binding Assay, Sequencing, Polymerase Chain Reaction, Generated, Transformation Assay, Homologous Recombination

    L002 reduces hypertension (HTN)-induced murine cardiac fibrosis and hypertrophy: (A) After two weeks of Ang II infusion and L002 treatment, cardiac sections from the four groups were stained with Masson's trichrome to identify collagen deposits. Upper panel shows perivascular deposition of collagen, while the lower panel identifies deposition of cardiac interstitial collagen. (B) Collagen deposition (perivascular + Interstitial) was quantified by ImagePro software program analysis, n = 7–12. (C) M-mode echocardiographic images showing thickness of left ventricular (LV) wall and LV diameters. (D) Quantification of the thickness of LV wall from echocardiography analysis, n = 4–12. (E) Post-mortem heart weight to body weight (HW/BW) were assessed in control and treatment groups. n = 7–12. (F) Shows systolic blood pressures (SBP) after two weeks of Ang II treatment as assessed by tail-cuff methods, n = 4–12. Data represented as mean ± SEM. *p

    Journal: Epigenetics

    Article Title: A novel acetyltransferase p300 inhibitor ameliorates hypertension-associated cardio-renal fibrosis

    doi: 10.1080/15592294.2017.1370173

    Figure Lengend Snippet: L002 reduces hypertension (HTN)-induced murine cardiac fibrosis and hypertrophy: (A) After two weeks of Ang II infusion and L002 treatment, cardiac sections from the four groups were stained with Masson's trichrome to identify collagen deposits. Upper panel shows perivascular deposition of collagen, while the lower panel identifies deposition of cardiac interstitial collagen. (B) Collagen deposition (perivascular + Interstitial) was quantified by ImagePro software program analysis, n = 7–12. (C) M-mode echocardiographic images showing thickness of left ventricular (LV) wall and LV diameters. (D) Quantification of the thickness of LV wall from echocardiography analysis, n = 4–12. (E) Post-mortem heart weight to body weight (HW/BW) were assessed in control and treatment groups. n = 7–12. (F) Shows systolic blood pressures (SBP) after two weeks of Ang II treatment as assessed by tail-cuff methods, n = 4–12. Data represented as mean ± SEM. *p

    Article Snippet: For coupled immunoprecipitation-immunoblot analysis, equal amount of protein from control and L002 or L002, TGF-β treated human cardiac fibroblasts was used for immunoprecipitation with p300 antibody (Millipore, Billerica, MA) and immunoprecipitates were subjected to immunoblot analysis using p300 (Santa Cruz, CA) and phospho-serine/threonine antibody (Abcam, Cambridge, MA).

    Techniques: Staining, Software

    FATp300 inhibitor L002 reduces differentiation, migration and proliferation of cardiac fibroblasts. (A to E) Human cardiac fibroblasts (HCFs) or rat cardiac fibroblasts (RCFs) were cultured and treated in triplicate with L002 or C646 or DMSO in the presence or absence of TGF-β. Total protein were isolated, pooled and processed for Western blot (A, C, D) using indicated antibodies. Experiments were repeated two times. Total RNA were isolated and processed for qPCR in triplicate (B, E) using gene specific primers. (F) For migration study, the scratch wounds were made in monolayer cultures of HCFs. Cells were then pretreated in triplicate with L002 or DMSO for 1 hour followed by TGF-β treatment for 24 h. Photographs were taken at 0 hour and 24-hour post-treatment. (G) For proliferation study, HCFs were pretreated in triplicate with L002 or DMSO for 1 h followed by treatment with TGF-β. Cell numbers were counted at 24 h and 48 h. Data represented as mean ± SEM. **p

    Journal: Epigenetics

    Article Title: A novel acetyltransferase p300 inhibitor ameliorates hypertension-associated cardio-renal fibrosis

    doi: 10.1080/15592294.2017.1370173

    Figure Lengend Snippet: FATp300 inhibitor L002 reduces differentiation, migration and proliferation of cardiac fibroblasts. (A to E) Human cardiac fibroblasts (HCFs) or rat cardiac fibroblasts (RCFs) were cultured and treated in triplicate with L002 or C646 or DMSO in the presence or absence of TGF-β. Total protein were isolated, pooled and processed for Western blot (A, C, D) using indicated antibodies. Experiments were repeated two times. Total RNA were isolated and processed for qPCR in triplicate (B, E) using gene specific primers. (F) For migration study, the scratch wounds were made in monolayer cultures of HCFs. Cells were then pretreated in triplicate with L002 or DMSO for 1 hour followed by TGF-β treatment for 24 h. Photographs were taken at 0 hour and 24-hour post-treatment. (G) For proliferation study, HCFs were pretreated in triplicate with L002 or DMSO for 1 h followed by treatment with TGF-β. Cell numbers were counted at 24 h and 48 h. Data represented as mean ± SEM. **p

    Article Snippet: For coupled immunoprecipitation-immunoblot analysis, equal amount of protein from control and L002 or L002, TGF-β treated human cardiac fibroblasts was used for immunoprecipitation with p300 antibody (Millipore, Billerica, MA) and immunoprecipitates were subjected to immunoblot analysis using p300 (Santa Cruz, CA) and phospho-serine/threonine antibody (Abcam, Cambridge, MA).

    Techniques: Migration, Cell Culture, Isolation, Western Blot, Real-time Polymerase Chain Reaction

    L002 reduces TGF-β-induced pro-fibrogenic responses in renal cells, and mechanistic insights. (A) For migration assay, podocytes were pretreated in triplicate with L002 or DMSO for 1 hour. Then scratch wounds were made in monolayer cultures, followed by TGF-β treatment for 24 h. Photographs were taken at 0 hour and 24 hours after treatment. (B-C) Cultures of human (proliferating) podocytes or mesangial cells were treated in triplicate with L002/ C646 or DMSO in the presence or absence of TGF-β. Total protein was isolated, pooled and processed for Western blotting. Experiments were repeated twice. (D) Effect of L002 on p300 levels. Renal sections from saline, Ang II and Ang II, L002 groups were immuno-stained with anti-p300 antibody and quantified. Saline (n = 3, 12–15 fields); Ang II (n = 5, 20–25 fields); Ang II, L002 (n = 5, 20–25 fields). Data represented as mean ± SEM. ***p

    Journal: Epigenetics

    Article Title: A novel acetyltransferase p300 inhibitor ameliorates hypertension-associated cardio-renal fibrosis

    doi: 10.1080/15592294.2017.1370173

    Figure Lengend Snippet: L002 reduces TGF-β-induced pro-fibrogenic responses in renal cells, and mechanistic insights. (A) For migration assay, podocytes were pretreated in triplicate with L002 or DMSO for 1 hour. Then scratch wounds were made in monolayer cultures, followed by TGF-β treatment for 24 h. Photographs were taken at 0 hour and 24 hours after treatment. (B-C) Cultures of human (proliferating) podocytes or mesangial cells were treated in triplicate with L002/ C646 or DMSO in the presence or absence of TGF-β. Total protein was isolated, pooled and processed for Western blotting. Experiments were repeated twice. (D) Effect of L002 on p300 levels. Renal sections from saline, Ang II and Ang II, L002 groups were immuno-stained with anti-p300 antibody and quantified. Saline (n = 3, 12–15 fields); Ang II (n = 5, 20–25 fields); Ang II, L002 (n = 5, 20–25 fields). Data represented as mean ± SEM. ***p

    Article Snippet: For coupled immunoprecipitation-immunoblot analysis, equal amount of protein from control and L002 or L002, TGF-β treated human cardiac fibroblasts was used for immunoprecipitation with p300 antibody (Millipore, Billerica, MA) and immunoprecipitates were subjected to immunoblot analysis using p300 (Santa Cruz, CA) and phospho-serine/threonine antibody (Abcam, Cambridge, MA).

    Techniques: Migration, Isolation, Western Blot, Staining

    L002 reduces hypertension induced renal fibrosis and pro-fibrogenic responses in renal cells: (A) After two weeks of Ang II infusion and L002 treatment, renal sections were stained with Masson's trichrome to identify collagen deposits. Upper panel shows perivascular deposition of collagen, while the lower panel identifies interstitial collagen. (B) Collagen deposition (perivascular + Interstitial) was quantified by ImagePro software program analysis, n = 6–12. (C-E) Cultures of human (proliferating) podocytes and differentiated (diff.) podocytes were treated in triplicate with L002 or DMSO in the presence or absence of TGF-β. Total protein were isolated from three wells, pooled and processed for Western blot (C, E) using specific antibodies. Experiments were repeated twice. Total RNA were isolated from three wells and processed for qPCR in triplicate (D) using gene specific primers. Data presented as mean ± SEM *p

    Journal: Epigenetics

    Article Title: A novel acetyltransferase p300 inhibitor ameliorates hypertension-associated cardio-renal fibrosis

    doi: 10.1080/15592294.2017.1370173

    Figure Lengend Snippet: L002 reduces hypertension induced renal fibrosis and pro-fibrogenic responses in renal cells: (A) After two weeks of Ang II infusion and L002 treatment, renal sections were stained with Masson's trichrome to identify collagen deposits. Upper panel shows perivascular deposition of collagen, while the lower panel identifies interstitial collagen. (B) Collagen deposition (perivascular + Interstitial) was quantified by ImagePro software program analysis, n = 6–12. (C-E) Cultures of human (proliferating) podocytes and differentiated (diff.) podocytes were treated in triplicate with L002 or DMSO in the presence or absence of TGF-β. Total protein were isolated from three wells, pooled and processed for Western blot (C, E) using specific antibodies. Experiments were repeated twice. Total RNA were isolated from three wells and processed for qPCR in triplicate (D) using gene specific primers. Data presented as mean ± SEM *p

    Article Snippet: For coupled immunoprecipitation-immunoblot analysis, equal amount of protein from control and L002 or L002, TGF-β treated human cardiac fibroblasts was used for immunoprecipitation with p300 antibody (Millipore, Billerica, MA) and immunoprecipitates were subjected to immunoblot analysis using p300 (Santa Cruz, CA) and phospho-serine/threonine antibody (Abcam, Cambridge, MA).

    Techniques: Staining, Software, Isolation, Western Blot, Real-time Polymerase Chain Reaction