gene exp b2m hs00187842 m1  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher gene exp b2m hs00187842 m1
    Effect of tocilizumab on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. The effect of tocilizumab, a monoclonal antibody inhibiting interleukin 6 receptor (IL-6R), on the expression of the three complement anaphylatoxin receptors [C5aR1 (A) , C5aR2 (B) , and C3aR (C) ] was investigated in patients with non-ST-elevation myocardial infarction (NSTEMI). mRNA levels were quantified by qPCR and related to the reference gene <t>beta-2-microglobulin.</t> The tocilizumab group (gray bars, n = 28) and the placebo group (white bars, n = 32) are presented at four different time-points. Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals ( n = 15) were included as controls. The qPCR results were quantified using the 2 −ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. * P
    Gene Exp B2m Hs00187842 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 39711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp b2m hs00187842 m1/product/Thermo Fisher
    Average 99 stars, based on 39711 article reviews
    Price from $9.99 to $1999.99
    gene exp b2m hs00187842 m1 - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "IL-6 Receptor Inhibition by Tocilizumab Attenuated Expression of C5a Receptor 1 and 2 in Non-ST-Elevation Myocardial Infarction"

    Article Title: IL-6 Receptor Inhibition by Tocilizumab Attenuated Expression of C5a Receptor 1 and 2 in Non-ST-Elevation Myocardial Infarction

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02035

    Effect of tocilizumab on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. The effect of tocilizumab, a monoclonal antibody inhibiting interleukin 6 receptor (IL-6R), on the expression of the three complement anaphylatoxin receptors [C5aR1 (A) , C5aR2 (B) , and C3aR (C) ] was investigated in patients with non-ST-elevation myocardial infarction (NSTEMI). mRNA levels were quantified by qPCR and related to the reference gene beta-2-microglobulin. The tocilizumab group (gray bars, n = 28) and the placebo group (white bars, n = 32) are presented at four different time-points. Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals ( n = 15) were included as controls. The qPCR results were quantified using the 2 −ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. * P
    Figure Legend Snippet: Effect of tocilizumab on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. The effect of tocilizumab, a monoclonal antibody inhibiting interleukin 6 receptor (IL-6R), on the expression of the three complement anaphylatoxin receptors [C5aR1 (A) , C5aR2 (B) , and C3aR (C) ] was investigated in patients with non-ST-elevation myocardial infarction (NSTEMI). mRNA levels were quantified by qPCR and related to the reference gene beta-2-microglobulin. The tocilizumab group (gray bars, n = 28) and the placebo group (white bars, n = 32) are presented at four different time-points. Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals ( n = 15) were included as controls. The qPCR results were quantified using the 2 −ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "Rivastigmine Alleviates Experimentally Induced Colitis in Mice and Rats by Acting at Central and Peripheral Sites to Modulate Immune Responses"

    Article Title: Rivastigmine Alleviates Experimentally Induced Colitis in Mice and Rats by Acting at Central and Peripheral Sites to Modulate Immune Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057668

    Effect of rivastigmine on TNF-α and IL-6 released from peritoneal macrophages of DSS treated mice. Legend as in Figure 3 . Data are mean ± SEM and represent pooled data from 6 animals per group. Significantly different from mice with DSS-induced colitis treated with PBS, ** p
    Figure Legend Snippet: Effect of rivastigmine on TNF-α and IL-6 released from peritoneal macrophages of DSS treated mice. Legend as in Figure 3 . Data are mean ± SEM and represent pooled data from 6 animals per group. Significantly different from mice with DSS-induced colitis treated with PBS, ** p

    Techniques Used: Mouse Assay

    Effect of rivastigmine on TNF-α, MPO, TBARS and ChE activity in rats with DNBS-induced colitis. ChE = ChE activity. Data are mean ± SEM from 13 animals per group. Percent of reduction compared to values in group treated with distilled water and DNBS. Significantly different from the rats treated by rectal administration of rivastigmine (1 mg/kg) * p
    Figure Legend Snippet: Effect of rivastigmine on TNF-α, MPO, TBARS and ChE activity in rats with DNBS-induced colitis. ChE = ChE activity. Data are mean ± SEM from 13 animals per group. Percent of reduction compared to values in group treated with distilled water and DNBS. Significantly different from the rats treated by rectal administration of rivastigmine (1 mg/kg) * p

    Techniques Used: Activity Assay

    3) Product Images from "Inhibition of Plk1 and Cyclin B1 Expression Results in Panobinostat-Induced G2 Delay and Mitotic Defects"

    Article Title: Inhibition of Plk1 and Cyclin B1 Expression Results in Panobinostat-Induced G2 Delay and Mitotic Defects

    Journal: Scientific Reports

    doi: 10.1038/srep02640

    Reduction of G2-specific Cyclin B1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) Cyclin B1 mRNA expression was assessed by qRT-PCR. The data shown represent Cyclin B1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels that were run using identical conditions. Cyclin B1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Cyclin B1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.
    Figure Legend Snippet: Reduction of G2-specific Cyclin B1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) Cyclin B1 mRNA expression was assessed by qRT-PCR. The data shown represent Cyclin B1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels that were run using identical conditions. Cyclin B1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Cyclin B1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.

    Techniques Used: Expressing, Blocking Assay, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot, Software

    Reduction of G2-specific Plk1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) PLK1 mRNA expression was assessed by qRT-PCR. The data shown represent PLK1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels run using identical conditions. The upper panel was probed with Plk1 antibody and the lower panel probed with β-actin antibody. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Plk1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.
    Figure Legend Snippet: Reduction of G2-specific Plk1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) PLK1 mRNA expression was assessed by qRT-PCR. The data shown represent PLK1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels run using identical conditions. The upper panel was probed with Plk1 antibody and the lower panel probed with β-actin antibody. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Plk1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.

    Techniques Used: Expressing, Blocking Assay, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot, Software

    Panobinostat induced p21 Waf1/Cip1 expression. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) p21 Waf1/Cip1 mRNA expression was assessed by qRT-PCR. The data shown represent p21 Waf1/Cip1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels runs using identical conditions and p21 Waf1/Cip1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) p21 Waf1/Cip1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.
    Figure Legend Snippet: Panobinostat induced p21 Waf1/Cip1 expression. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) p21 Waf1/Cip1 mRNA expression was assessed by qRT-PCR. The data shown represent p21 Waf1/Cip1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels runs using identical conditions and p21 Waf1/Cip1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) p21 Waf1/Cip1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.

    Techniques Used: Expressing, Blocking Assay, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot, Software

    Loss of E2F1 recruitment to the Plk1 and Cyclin B1 promoters in response to panobinostat. (A) FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA. E2F1, E2F2, E2F3 and E2F5 mRNA expression was assessed by qRT-PCR. The data shown represent mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B and C) Asynchronous FaDu cells were incubated with 100 nM panobinostat or excipient control for 12 hours and crosslinked. Chromatin was then extracted and sheared and clarified chromatin preparations were immunoprecipitated with anti-FLAG (negative), E2F1, E2F2 or E2F3 antibody. The percentage of bound PLK1 promoter (B) or Cyclin B1 promoter (C) was then assessed by qRT-PCR and shown as the mean and standard deviation of three independent experiments.
    Figure Legend Snippet: Loss of E2F1 recruitment to the Plk1 and Cyclin B1 promoters in response to panobinostat. (A) FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA. E2F1, E2F2, E2F3 and E2F5 mRNA expression was assessed by qRT-PCR. The data shown represent mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B and C) Asynchronous FaDu cells were incubated with 100 nM panobinostat or excipient control for 12 hours and crosslinked. Chromatin was then extracted and sheared and clarified chromatin preparations were immunoprecipitated with anti-FLAG (negative), E2F1, E2F2 or E2F3 antibody. The percentage of bound PLK1 promoter (B) or Cyclin B1 promoter (C) was then assessed by qRT-PCR and shown as the mean and standard deviation of three independent experiments.

    Techniques Used: Blocking Assay, Expressing, Quantitative RT-PCR, Standard Deviation, Incubation, Immunoprecipitation

    4) Product Images from "Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability"

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv933

    Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P
    Figure Legend Snippet: Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P

    Techniques Used: Amplification, Fluorescence In Situ Hybridization, Labeling, Real-time Polymerase Chain Reaction, CNV Assay, Transduction

    Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    5) Product Images from "Adiponectin Induces Oncostatin M Expression in Osteoblasts through the PI3K/Akt Signaling Pathway"

    Article Title: Adiponectin Induces Oncostatin M Expression in Osteoblasts through the PI3K/Akt Signaling Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17010029

    Adiponectin increases OSM expression through nuclear factor-κB (NF-κB) pathway. ( A ) Osteoblasts were pretreated with pyrrolidine dithiocarbamate (PDTC) (10 μM) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (10 μM) or transfected with p65 siRNA (0.5 nM) followed by stimulation with adiponectin (100 ng/mL), the mRNA expression of OSM were analyzed by qPCR ( n = 5); ( B ) Cells were transfected with p65 siRNA for 24 h, the protein level of p65 was measured by Western blot (upper-panel), and supernatant medium was collected to measure OSM expression by ELISA assay (lower-panel) ( n = 5); ( C ) Cells were pretreated with PDTC and TPCK for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 6); ( D ) Cells were incubated with adiponectin in time intervals, and phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 5); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), or Akt inhibitor (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 6). Results are expressed as mean ± standard error of mean S.E.M. *, p
    Figure Legend Snippet: Adiponectin increases OSM expression through nuclear factor-κB (NF-κB) pathway. ( A ) Osteoblasts were pretreated with pyrrolidine dithiocarbamate (PDTC) (10 μM) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (10 μM) or transfected with p65 siRNA (0.5 nM) followed by stimulation with adiponectin (100 ng/mL), the mRNA expression of OSM were analyzed by qPCR ( n = 5); ( B ) Cells were transfected with p65 siRNA for 24 h, the protein level of p65 was measured by Western blot (upper-panel), and supernatant medium was collected to measure OSM expression by ELISA assay (lower-panel) ( n = 5); ( C ) Cells were pretreated with PDTC and TPCK for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 6); ( D ) Cells were incubated with adiponectin in time intervals, and phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 5); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), or Akt inhibitor (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 6). Results are expressed as mean ± standard error of mean S.E.M. *, p

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

    Involvement of Akt in adiponectin-induced OSM expression in osteoblasts. ( A ) Osteoblasts were pretreated with Akt inhibitors (Akti) (20 μM) or transfected with Akt siRNA (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with Akt siRNA (0.5 nM) for 24h, the protein level of Akt was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISA assay ( lower-panel ) ( n = 5); ( C ) Cells were pretreated with Akti (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 4); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-Akt expression was investigated by Western blot ( n = 5). Results are expressed as mean ± S.E.M. *, p
    Figure Legend Snippet: Involvement of Akt in adiponectin-induced OSM expression in osteoblasts. ( A ) Osteoblasts were pretreated with Akt inhibitors (Akti) (20 μM) or transfected with Akt siRNA (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with Akt siRNA (0.5 nM) for 24h, the protein level of Akt was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISA assay ( lower-panel ) ( n = 5); ( C ) Cells were pretreated with Akti (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 4); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-Akt expression was investigated by Western blot ( n = 5). Results are expressed as mean ± S.E.M. *, p

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

    Signaling pathways of phosphatidylinositol 3-kinase (PI3K) involved in potentiating action of adiponectin. ( A ) Osteoblasts were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), or transfected with p85 short interference RNA (siRNA) (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with p85 siRNA (0.5 nM) for 24 h, the protein level of PI3K was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISAassay ( lower-panel ) ( n = 4); ( C ) Cells were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 6). Results are expressed as mean ± S.E.M. *, p
    Figure Legend Snippet: Signaling pathways of phosphatidylinositol 3-kinase (PI3K) involved in potentiating action of adiponectin. ( A ) Osteoblasts were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), or transfected with p85 short interference RNA (siRNA) (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with p85 siRNA (0.5 nM) for 24 h, the protein level of PI3K was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISAassay ( lower-panel ) ( n = 4); ( C ) Cells were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 6). Results are expressed as mean ± S.E.M. *, p

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

    Adiponectin increases oncostatin M (OSM) production in human osteoblasts. ( A ) Osteoblastic cells were incubated with various concentrations of adiponectin (3–200 ng/mL) in OSM mRNA expression and were measured by quantitative polymerase chain reaction (qPCR) ( n = 6); ( B ) Cells were incubated with various concentrations of adiponectin (3–200 ng/mL) and OSM protein levels were measured by Western blot ( n = 4); ( C ) Osteoblasts were stimulated with adiponectin (3–200 ng/mL) for 24 h and the supernatant medium was collected and analyzed by enzyme-linked immunosorbent assay (ELISA) ( n = 5). Results are expressed as mean ± standard error of mean S.E.M. *, p
    Figure Legend Snippet: Adiponectin increases oncostatin M (OSM) production in human osteoblasts. ( A ) Osteoblastic cells were incubated with various concentrations of adiponectin (3–200 ng/mL) in OSM mRNA expression and were measured by quantitative polymerase chain reaction (qPCR) ( n = 6); ( B ) Cells were incubated with various concentrations of adiponectin (3–200 ng/mL) and OSM protein levels were measured by Western blot ( n = 4); ( C ) Osteoblasts were stimulated with adiponectin (3–200 ng/mL) for 24 h and the supernatant medium was collected and analyzed by enzyme-linked immunosorbent assay (ELISA) ( n = 5). Results are expressed as mean ± standard error of mean S.E.M. *, p

    Techniques Used: Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology"

    Article Title: Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology

    Journal: ACS Nano

    doi: 10.1021/acsnano.5b02471

    Evaluation of targeting and therapeutic effects of 3WJ-EGFRapt/anti-miR-21 nanoparticles in vitro . (A) Confocal images showing efficient binding and internalization into MDA-MB-231 cells. Green: cytoplasm; blue: nuclei; and red: RNA nanoparticles. (B) Flow cytometry assay showing the binding to MDA-MB-231 cells. (C) Dual-luciferase assay demonstrating in vitro delivery of anti-miR-21 LNA into MDA-MB-231 cells. (D) qRT-PCR assay depicting the effect of miR-21 knockdown on target gene expression level of PTEN and PDCD4 after treatment. RQ: relative quantification. (E) Caspase-3 assay showing the cellular apoptotic effects of MDA-MB-231 cells after treatment.
    Figure Legend Snippet: Evaluation of targeting and therapeutic effects of 3WJ-EGFRapt/anti-miR-21 nanoparticles in vitro . (A) Confocal images showing efficient binding and internalization into MDA-MB-231 cells. Green: cytoplasm; blue: nuclei; and red: RNA nanoparticles. (B) Flow cytometry assay showing the binding to MDA-MB-231 cells. (C) Dual-luciferase assay demonstrating in vitro delivery of anti-miR-21 LNA into MDA-MB-231 cells. (D) qRT-PCR assay depicting the effect of miR-21 knockdown on target gene expression level of PTEN and PDCD4 after treatment. RQ: relative quantification. (E) Caspase-3 assay showing the cellular apoptotic effects of MDA-MB-231 cells after treatment.

    Techniques Used: In Vitro, Binding Assay, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Luciferase, Quantitative RT-PCR, Expressing, Caspase-3 Assay

    7) Product Images from "MicroRNA-125a influences breast cancer stem cells by targeting leukemia inhibitory factor receptor which regulates the hippo signaling pathway"

    Article Title: MicroRNA-125a influences breast cancer stem cells by targeting leukemia inhibitory factor receptor which regulates the hippo signaling pathway

    Journal: Oncotarget

    doi:

    miR-125a modulation affects malignant and non-malignant breast epithelial stem cells A. qRT-PCR data shows effective inhibition of miR-125a in MCF7 cells using 100nM of antagomirs. B. qRT-PCR data shows effective over expression of miR-125a in MCF12A cells with miR-125a mimic. C. Immunoblotting analysis for LIFR post miR-125a inhibition in MCF7 CSCs. D. Immunoblotting analysis for LIFR in miR-125a over expressing MCF12A stem cells. E. Flow cytometric analysis shows increased percentage of stem cells with miR-125a over expression in MCF12A cells. F. Sphere forming assay demonstrates higher percentage of 3D sphere forming cells in miR-125a over expressing MCF12A. G. Flow cytometric analysis shows decreased percentage of stem cells with miR-125a inhibition in MCF7 cells. H. Sphere forming assay demonstrates lower percentage of 3D sphere forming cells in miR-125a inhibited MCF7 cells. I. Immunostaining of MCF12A spheres for SOX2 expression with over expression of miR-125a compared to mock controls.
    Figure Legend Snippet: miR-125a modulation affects malignant and non-malignant breast epithelial stem cells A. qRT-PCR data shows effective inhibition of miR-125a in MCF7 cells using 100nM of antagomirs. B. qRT-PCR data shows effective over expression of miR-125a in MCF12A cells with miR-125a mimic. C. Immunoblotting analysis for LIFR post miR-125a inhibition in MCF7 CSCs. D. Immunoblotting analysis for LIFR in miR-125a over expressing MCF12A stem cells. E. Flow cytometric analysis shows increased percentage of stem cells with miR-125a over expression in MCF12A cells. F. Sphere forming assay demonstrates higher percentage of 3D sphere forming cells in miR-125a over expressing MCF12A. G. Flow cytometric analysis shows decreased percentage of stem cells with miR-125a inhibition in MCF7 cells. H. Sphere forming assay demonstrates lower percentage of 3D sphere forming cells in miR-125a inhibited MCF7 cells. I. Immunostaining of MCF12A spheres for SOX2 expression with over expression of miR-125a compared to mock controls.

    Techniques Used: Quantitative RT-PCR, Inhibition, Over Expression, Expressing, Flow Cytometry, Immunostaining

    Inverse correlation between miR-125a and LIFR in stem cells A. qRT-PCR data shows expression of miR-125a in MCF7 and human primary breast cancer cells (BC-052) compared to MCF12A cells B. Expression levels of miR-125a in MCF7 and primary breast CSCs as compared to MCF12A stem cells. C. Pictorial representation of the relative levels of miR-125a in malignant and non-malignant stem cells and bulk cells D. Transcript level of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. E. Immunoblotting analysis of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. F. Transcript level of LIFR in MCF7 and human primary breast cancer cells as compared to MCF12A cells G. Immunoblotting analysis of LIFR in MCF7 and primary breast cancer cells as compared to MCF12A cells. H. Predicted interaction of miR-125a with LIFR in silico . Luciferase assay data for HEK293 cells demonstrating a decrease in the luciferase activity with addition of miR-125a mimics to wtLIFR 3′UTR-1 expressing cells but not in wtLIFR 3′UTR-2 and mutLIFR 3′UTR.
    Figure Legend Snippet: Inverse correlation between miR-125a and LIFR in stem cells A. qRT-PCR data shows expression of miR-125a in MCF7 and human primary breast cancer cells (BC-052) compared to MCF12A cells B. Expression levels of miR-125a in MCF7 and primary breast CSCs as compared to MCF12A stem cells. C. Pictorial representation of the relative levels of miR-125a in malignant and non-malignant stem cells and bulk cells D. Transcript level of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. E. Immunoblotting analysis of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. F. Transcript level of LIFR in MCF7 and human primary breast cancer cells as compared to MCF12A cells G. Immunoblotting analysis of LIFR in MCF7 and primary breast cancer cells as compared to MCF12A cells. H. Predicted interaction of miR-125a with LIFR in silico . Luciferase assay data for HEK293 cells demonstrating a decrease in the luciferase activity with addition of miR-125a mimics to wtLIFR 3′UTR-1 expressing cells but not in wtLIFR 3′UTR-2 and mutLIFR 3′UTR.

    Techniques Used: Quantitative RT-PCR, Expressing, In Silico, Luciferase, Activity Assay

    8) Product Images from "MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection"

    Article Title: MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection

    Journal: Nature

    doi: 10.1038/nature14216

    Related to Figure 2 a, qRT-PCR analysis of MAD2L2 expression levels in U2OS cells infected with control or MAD2L2 shRNAs, and used in the repair assays shown in Fig. 2d (Error bars: s.d.). b , c , qRT-PCR analysis of RAD51 ( b ) and MAD2L2 ( c ) expression levels in RAD51-depleted, E6E7-expressing U2OS cells used in the assays shown in Fig. 2e (Error bars: s.d.). d, Clonogenic survival assays of U2OS cells transduced with non-targeting control or 53BP1, RIF1 or MAD2L2 shRNAs and treated with the indicated doses of IR (n=3-4, ± s.e.m.). e, Western blot analysis of 53BP1, MAD2L2 and RIF1 in U2OS cells transduced with the indicated shRNAs. f, CSR in shRNA-transduced primary B cells (n=2, ± s.d.). g, Western blot analysis of MAD2L2 and 53BP1 in CH12F3-2 B cells and mouse primary B cells transduced with the indicated shRNAs. h, MAD2L2 depletion does not affect cellular proliferation in murine B cells. CH12F3-2 cells transduced with control, 53bp1 or Mad2l2 shRNAs were loaded with CFSE and analysed at 0, 24 and 48 h post-stimulation by flow-cytometry. Profiles from all time points are plotted in the same histogram. i, MAD2L2 depletion does not affect the transcription of critical genes implicated in CSR. RT-PCR analysis of AID ( Aicda ) mRNA, IgM ( GLT IgM ) and IgA ( GLT IgA ) germline transcript levels using 2-fold serial dilutions of cDNA made from activated CH12F3-2 B cells transduced with the indicated shRNAs. Gapdh was used as a control for transcript expression.
    Figure Legend Snippet: Related to Figure 2 a, qRT-PCR analysis of MAD2L2 expression levels in U2OS cells infected with control or MAD2L2 shRNAs, and used in the repair assays shown in Fig. 2d (Error bars: s.d.). b , c , qRT-PCR analysis of RAD51 ( b ) and MAD2L2 ( c ) expression levels in RAD51-depleted, E6E7-expressing U2OS cells used in the assays shown in Fig. 2e (Error bars: s.d.). d, Clonogenic survival assays of U2OS cells transduced with non-targeting control or 53BP1, RIF1 or MAD2L2 shRNAs and treated with the indicated doses of IR (n=3-4, ± s.e.m.). e, Western blot analysis of 53BP1, MAD2L2 and RIF1 in U2OS cells transduced with the indicated shRNAs. f, CSR in shRNA-transduced primary B cells (n=2, ± s.d.). g, Western blot analysis of MAD2L2 and 53BP1 in CH12F3-2 B cells and mouse primary B cells transduced with the indicated shRNAs. h, MAD2L2 depletion does not affect cellular proliferation in murine B cells. CH12F3-2 cells transduced with control, 53bp1 or Mad2l2 shRNAs were loaded with CFSE and analysed at 0, 24 and 48 h post-stimulation by flow-cytometry. Profiles from all time points are plotted in the same histogram. i, MAD2L2 depletion does not affect the transcription of critical genes implicated in CSR. RT-PCR analysis of AID ( Aicda ) mRNA, IgM ( GLT IgM ) and IgA ( GLT IgA ) germline transcript levels using 2-fold serial dilutions of cDNA made from activated CH12F3-2 B cells transduced with the indicated shRNAs. Gapdh was used as a control for transcript expression.

    Techniques Used: Quantitative RT-PCR, Expressing, Infection, Transduction, Western Blot, shRNA, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    Related to Figure 1 a , A functional genetic screen for telomere-induced genomic instability regulators (TIGIRs) identifies independent shRNAs against Mad2l2 and the previously identified regulators of NHEJ-mediated telomere fusion Atm , Nbs1 (a.k.a. Nbn ), Rad50 , 53bp1 (a.k.a. Trp53bp1 ) and Rnf8 . Listed are the independent shRNAs enriched > 1.5 fold in at least 2 out of 3 TIGIR-screens, with their average ratio of enrichment over all 3 screens. Ratios reflect shRNA abundance after 12 days of telomere uncapping at 39°C followed by 4 days recovery at 32°C versus shRNA abundance after growth for 4 days at 32°C. b, Additional shRNAs targeting Mad2l2 or known TIGIRs that were enriched > 1.5 fold in 1 of 3 screens, with their ratio. Not shown are additional shRNAs against factors not previously implicated in control of telomere fusion, that were significantly enriched in these TIGIR-screens but await validation. c, qRT-PCR analysis of Mad2l2 expression levels in TRF2ts MEFs transduced with 4 independent shRNAs targeting Mad2l2 and used in Fig. 1b (Error bars: s.d.). d, Survival assay of TRF2ts cells infected with control or Mad2l2 sh4 shRNAs, complemented with empty control or RNAi-resistant Flag-Mad2l2 RR and grown as indicated. e, Western blot showing expression of endogenous MAD2L2 and exogenous Flag-MAD2L2 in TRF2ts cells used in d and in Fig. 1c . f , Photograph of shRNA-transduced TRF2ts cells grown for 12 days at 39°C. Scale bar, 100 μm. g, None of 10 shRNAs targeting Rev1 or Rev3 were significantly enriched in any of 3 independent DDR TIGIR-screens. h, shRNA-mediated Rev1 knockdown does not increase survival upon prolonged telomere uncapping in survival assays of TRF2ts MEFs. Of note, Rev3 knockdown compromised viability and was therefore not informative in these assays. i, qRT-PCR analysis of mouse Rev1 expression levels of cells shown in h (Error bars: s.d.).
    Figure Legend Snippet: Related to Figure 1 a , A functional genetic screen for telomere-induced genomic instability regulators (TIGIRs) identifies independent shRNAs against Mad2l2 and the previously identified regulators of NHEJ-mediated telomere fusion Atm , Nbs1 (a.k.a. Nbn ), Rad50 , 53bp1 (a.k.a. Trp53bp1 ) and Rnf8 . Listed are the independent shRNAs enriched > 1.5 fold in at least 2 out of 3 TIGIR-screens, with their average ratio of enrichment over all 3 screens. Ratios reflect shRNA abundance after 12 days of telomere uncapping at 39°C followed by 4 days recovery at 32°C versus shRNA abundance after growth for 4 days at 32°C. b, Additional shRNAs targeting Mad2l2 or known TIGIRs that were enriched > 1.5 fold in 1 of 3 screens, with their ratio. Not shown are additional shRNAs against factors not previously implicated in control of telomere fusion, that were significantly enriched in these TIGIR-screens but await validation. c, qRT-PCR analysis of Mad2l2 expression levels in TRF2ts MEFs transduced with 4 independent shRNAs targeting Mad2l2 and used in Fig. 1b (Error bars: s.d.). d, Survival assay of TRF2ts cells infected with control or Mad2l2 sh4 shRNAs, complemented with empty control or RNAi-resistant Flag-Mad2l2 RR and grown as indicated. e, Western blot showing expression of endogenous MAD2L2 and exogenous Flag-MAD2L2 in TRF2ts cells used in d and in Fig. 1c . f , Photograph of shRNA-transduced TRF2ts cells grown for 12 days at 39°C. Scale bar, 100 μm. g, None of 10 shRNAs targeting Rev1 or Rev3 were significantly enriched in any of 3 independent DDR TIGIR-screens. h, shRNA-mediated Rev1 knockdown does not increase survival upon prolonged telomere uncapping in survival assays of TRF2ts MEFs. Of note, Rev3 knockdown compromised viability and was therefore not informative in these assays. i, qRT-PCR analysis of mouse Rev1 expression levels of cells shown in h (Error bars: s.d.).

    Techniques Used: Functional Assay, Non-Homologous End Joining, shRNA, Quantitative RT-PCR, Expressing, Transduction, Clonogenic Cell Survival Assay, Infection, Western Blot

    Related to Figure 4 a, Representative images of IF detection of endogenous MAD2L2 and γH2AX or 53BP1 in U2OS cells transduced with control, 53BP1, RNF8, RNF168, RIF1, PTIP or REV3 shRNA lentiviruses, irradiated with 5Gy and processed for IF after 3h (quantifications are shown in Fig. 4e ). b, Western blot or qRT-PCR analysis of PTIP, RNF8, RNF168 and REV3 levels in shRNA-transduced U2OS cells (Error bars: s.d.). c, Quantification and representative images of IF detection of γH2AX and GFPMAD2L2 in Ptip+/+ or Ptip−/− MEFs, 3h after 5Gy (n=2, ± s.d.). d, Representative images of IF for 53BP1 and exogenous Flag-MAD2L2 WT, C70R or L186A in U2OS cells depleted for endogenous MAD2L2 with lentiviral shRNA, processed for IF 3h after 5Gy (quantifications are shown in Extended Data Fig. 7e ). e , Quantification and representative IF images of GFP-MAD2L2 localisation to uncapped telomeres in TRF2ts MEFs transduced with control or 53BP1 shRNAs (n=2, ± s.d.).
    Figure Legend Snippet: Related to Figure 4 a, Representative images of IF detection of endogenous MAD2L2 and γH2AX or 53BP1 in U2OS cells transduced with control, 53BP1, RNF8, RNF168, RIF1, PTIP or REV3 shRNA lentiviruses, irradiated with 5Gy and processed for IF after 3h (quantifications are shown in Fig. 4e ). b, Western blot or qRT-PCR analysis of PTIP, RNF8, RNF168 and REV3 levels in shRNA-transduced U2OS cells (Error bars: s.d.). c, Quantification and representative images of IF detection of γH2AX and GFPMAD2L2 in Ptip+/+ or Ptip−/− MEFs, 3h after 5Gy (n=2, ± s.d.). d, Representative images of IF for 53BP1 and exogenous Flag-MAD2L2 WT, C70R or L186A in U2OS cells depleted for endogenous MAD2L2 with lentiviral shRNA, processed for IF 3h after 5Gy (quantifications are shown in Extended Data Fig. 7e ). e , Quantification and representative IF images of GFP-MAD2L2 localisation to uncapped telomeres in TRF2ts MEFs transduced with control or 53BP1 shRNAs (n=2, ± s.d.).

    Techniques Used: Transduction, shRNA, Irradiation, Western Blot, Quantitative RT-PCR

    Related to Figure 4 a, Frequency distribution (left) and scatter plot (right) of total distances travelled by uncapped telomeres in control or Mad2l2 knockdown cells. b, qRT-PCR analysis of Mad2l2 expression levels and Western blot analysis of 53BP1 proteins levels in TRF2ts cells used in the experiments shown in a and c ( Error bars: ± s.d.). c, Distance travelled by 10 representative uncapped telomeres for each condition. While multiple uncapped telomeres in 53BP1-depleted cells have reduced mobility, this is not seen for uncapped telomeres in MAD2L2-depleted cells. d, Model of the role of MAD2L2 in promoting NHEJ.
    Figure Legend Snippet: Related to Figure 4 a, Frequency distribution (left) and scatter plot (right) of total distances travelled by uncapped telomeres in control or Mad2l2 knockdown cells. b, qRT-PCR analysis of Mad2l2 expression levels and Western blot analysis of 53BP1 proteins levels in TRF2ts cells used in the experiments shown in a and c ( Error bars: ± s.d.). c, Distance travelled by 10 representative uncapped telomeres for each condition. While multiple uncapped telomeres in 53BP1-depleted cells have reduced mobility, this is not seen for uncapped telomeres in MAD2L2-depleted cells. d, Model of the role of MAD2L2 in promoting NHEJ.

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Non-Homologous End Joining

    Related to Figure 3 a, Telomeric single-strand G-overhang assay of TRF2ts MEFs transduced with control or Mad2l2 sh4 shRNAs, showing that the increase in overhang signal upon Mad2l2 knockdown is due to 3′ terminal sequences because the signal is removed by treatment with Escherichia coli 3′ exonuclease EXO1. b, Mad2l2 knockdown causes increased ss telomeric G-overhang signals in TRF2ts; Lig4−/− ;TRF2ts MEFs. c, Quantification of relative telomeric G-overhang signals in TRF2ts; Lig4−/− MEFs transduced with control or Mad2l2 shRNAs and grown at 32°C or for 12 or 24 h at 39°C (n=2, ± s.e.m.). d, qRT-PCR analysis of Mad2l2 expression levels in TRF2ts; Lig4−/− MEFs infected with control or Mad2l2 shRNA lentivirus (Error bars: s.d.). e, Survival assays of TRF2ts MEFs transduced with control or Mad2l2 shRNAs and subsequently with control, Ctip or Exo1 shRNAs. f , Quantification of the survival assays shown in e . g, qRT-PCR analysis of Ctip and EXO1 expression levels of cells shown in e, f, h and in Fig. 3f (Error bars: s.d.). h , Growth curves at 39°C of TRF2ts MEFs transduced with non-targeting control or Mad2l2 shRNAs and subsequently with control, Ctip or Exo1 shRNAs (Error bars: s.e.m.).
    Figure Legend Snippet: Related to Figure 3 a, Telomeric single-strand G-overhang assay of TRF2ts MEFs transduced with control or Mad2l2 sh4 shRNAs, showing that the increase in overhang signal upon Mad2l2 knockdown is due to 3′ terminal sequences because the signal is removed by treatment with Escherichia coli 3′ exonuclease EXO1. b, Mad2l2 knockdown causes increased ss telomeric G-overhang signals in TRF2ts; Lig4−/− ;TRF2ts MEFs. c, Quantification of relative telomeric G-overhang signals in TRF2ts; Lig4−/− MEFs transduced with control or Mad2l2 shRNAs and grown at 32°C or for 12 or 24 h at 39°C (n=2, ± s.e.m.). d, qRT-PCR analysis of Mad2l2 expression levels in TRF2ts; Lig4−/− MEFs infected with control or Mad2l2 shRNA lentivirus (Error bars: s.d.). e, Survival assays of TRF2ts MEFs transduced with control or Mad2l2 shRNAs and subsequently with control, Ctip or Exo1 shRNAs. f , Quantification of the survival assays shown in e . g, qRT-PCR analysis of Ctip and EXO1 expression levels of cells shown in e, f, h and in Fig. 3f (Error bars: s.d.). h , Growth curves at 39°C of TRF2ts MEFs transduced with non-targeting control or Mad2l2 shRNAs and subsequently with control, Ctip or Exo1 shRNAs (Error bars: s.e.m.).

    Techniques Used: Transduction, Quantitative RT-PCR, Expressing, Infection, shRNA

    Related to Figures 3 and 4 a, TSCE analysis in shRNA-transduced TRF2ts MEFs grown at 32°C or for 12 h at 39°C to uncap telomeres (n=2, ± s.d., counting > 1,000 chromosomes per condition, per experiment). TSCE frequency in control cells is set at 1 (corresponding to an average of 6.9% of chromosomes with a TSCE event). Shown on the right are examples of chromosomes without and with TSCE in cells quantified on the left. b, qRT-PCR analysis of Mad2l2 expression levels in 53bp1−/− , Rif1−/− , Ptip−/− and Ptip+/+ MEFs used in the chromosome fusion analysis shown in Fig. 4a and in c (Error bars: s.d.). c, Percentage of chromosomes fused upon TRF2 inhibition in the Ptip+/+ MEFs matching with the Ptip−/− MEFs shown in Fig. 4a (n=2, ± s.e.m.). d, Analysis of different types of telomere fusions in Rif1−/− MEFs. Depletion of MAD2L2 in Rif1−/− MEFs does not reduce inter-chromosomal telomere fusions induced by TRF2 inhibition, indicating epistasis. However, irrespective of TRF2 inhibition, MAD2L2 depletion in Rif1−/− MEFs induces association between sister-telomeres, causing an increase in total fusions scored for MAD2L2-depleted Rif1−/− MEFs, as also visible in Fig. 4a (n=2, ± s.e.m., > 1,300-2,000 chromosomes were analysed per condition, per independent experiment). e, Explanation of scoring different types of telomere fusions shown in d .
    Figure Legend Snippet: Related to Figures 3 and 4 a, TSCE analysis in shRNA-transduced TRF2ts MEFs grown at 32°C or for 12 h at 39°C to uncap telomeres (n=2, ± s.d., counting > 1,000 chromosomes per condition, per experiment). TSCE frequency in control cells is set at 1 (corresponding to an average of 6.9% of chromosomes with a TSCE event). Shown on the right are examples of chromosomes without and with TSCE in cells quantified on the left. b, qRT-PCR analysis of Mad2l2 expression levels in 53bp1−/− , Rif1−/− , Ptip−/− and Ptip+/+ MEFs used in the chromosome fusion analysis shown in Fig. 4a and in c (Error bars: s.d.). c, Percentage of chromosomes fused upon TRF2 inhibition in the Ptip+/+ MEFs matching with the Ptip−/− MEFs shown in Fig. 4a (n=2, ± s.e.m.). d, Analysis of different types of telomere fusions in Rif1−/− MEFs. Depletion of MAD2L2 in Rif1−/− MEFs does not reduce inter-chromosomal telomere fusions induced by TRF2 inhibition, indicating epistasis. However, irrespective of TRF2 inhibition, MAD2L2 depletion in Rif1−/− MEFs induces association between sister-telomeres, causing an increase in total fusions scored for MAD2L2-depleted Rif1−/− MEFs, as also visible in Fig. 4a (n=2, ± s.e.m., > 1,300-2,000 chromosomes were analysed per condition, per independent experiment). e, Explanation of scoring different types of telomere fusions shown in d .

    Techniques Used: shRNA, Quantitative RT-PCR, Expressing, Inhibition

    9) Product Images from "Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver"

    Article Title: Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21525

    Collagen I contributes to the NAFLD-related HCC ( A ) qRT-PCR analysis showed that Collagen I levels were 2.8 fold higher in HCC samples than those in adjacent non-tumor liver tissues. ( B ) Immunofluorescence staining confirmed the higher expression of Collagen I (green) in HCC tissues compared to normal liver tissues. ( C ) Immunofluorescence staining revealed a higher expression of Collagen I (green) in human fatty liver compared to human normal liver. ( D ) In the mouse models of NAFLD/NASH, Collagen I was found to be upregulated. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I to determine the effect of Collagen I on HCC cell proliferation. The vitality of cells at each time point was detected by CCK-8 assay. (E) SMMC-7721 and HepG2 cells cultured on Collagen I proliferated significantly faster than those on either Collagen IV or fibronectin. (F) The effect of Collagen I on cell proliferation appeared to be dose-dependent. Scale bar: 50 μm (B); 100 μm (C, D), * P
    Figure Legend Snippet: Collagen I contributes to the NAFLD-related HCC ( A ) qRT-PCR analysis showed that Collagen I levels were 2.8 fold higher in HCC samples than those in adjacent non-tumor liver tissues. ( B ) Immunofluorescence staining confirmed the higher expression of Collagen I (green) in HCC tissues compared to normal liver tissues. ( C ) Immunofluorescence staining revealed a higher expression of Collagen I (green) in human fatty liver compared to human normal liver. ( D ) In the mouse models of NAFLD/NASH, Collagen I was found to be upregulated. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I to determine the effect of Collagen I on HCC cell proliferation. The vitality of cells at each time point was detected by CCK-8 assay. (E) SMMC-7721 and HepG2 cells cultured on Collagen I proliferated significantly faster than those on either Collagen IV or fibronectin. (F) The effect of Collagen I on cell proliferation appeared to be dose-dependent. Scale bar: 50 μm (B); 100 μm (C, D), * P

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Staining, Expressing, Cell Culture, Concentration Assay, CCK-8 Assay

    The expression of integrin β1 in NAFLD-related HCC ( A – C ) The expression of integrin β1 in HCC. (A) qRT-PCR analysis showed that integrin β1 levels were 1.1 fold higher in HCC samples than those in adjacent non-tumor liver tissues. (B) Correlation analysis indicated that the expression level of integrin β1 were positively correlated with the level of Collagen I, r = 0.43, P = 0.004. (C) Immunohistochemical staining revealed a high expression of integrin β1 in HCC tissues. ( D ) HepG2 cells were seeded into the NLM and FLM and cultured for 15 days. Western blot indicated that cells cultured in FLM had a higher expression of integrin β1 than those in NLM. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I. (E) For SMMC-7721 and HepG2, integrin β1 was higher expressed in Collagen I compared to Collagen IV and fibronectin. (F) Increasing Collagen I concentration could up regulated the expression of integrin β1 in both cell lines. ( G , H ) The expression of integrin β1 in mouse orthotopic tumor model. (G) Western blot indicated that tumors grown in HF- or MCD-fed mice expressed higher level of integrin β1. (H) Immunohistochemical staining of integrin β1 confirmed the same results. ( I – J ) Integrin β1 was knocked down in HCC cells by shRNA technology through lentiviral transduction. (I) Western blot confirmed the stable knock-down of integrin β1 in both cell lines. (J) HepG2 and SMMC-7721 cells cultured on Collagen I grew significantly slowly after knocking down integrin β1. Scale bar: 100 μm (C), 50 μm (H), * P
    Figure Legend Snippet: The expression of integrin β1 in NAFLD-related HCC ( A – C ) The expression of integrin β1 in HCC. (A) qRT-PCR analysis showed that integrin β1 levels were 1.1 fold higher in HCC samples than those in adjacent non-tumor liver tissues. (B) Correlation analysis indicated that the expression level of integrin β1 were positively correlated with the level of Collagen I, r = 0.43, P = 0.004. (C) Immunohistochemical staining revealed a high expression of integrin β1 in HCC tissues. ( D ) HepG2 cells were seeded into the NLM and FLM and cultured for 15 days. Western blot indicated that cells cultured in FLM had a higher expression of integrin β1 than those in NLM. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I. (E) For SMMC-7721 and HepG2, integrin β1 was higher expressed in Collagen I compared to Collagen IV and fibronectin. (F) Increasing Collagen I concentration could up regulated the expression of integrin β1 in both cell lines. ( G , H ) The expression of integrin β1 in mouse orthotopic tumor model. (G) Western blot indicated that tumors grown in HF- or MCD-fed mice expressed higher level of integrin β1. (H) Immunohistochemical staining of integrin β1 confirmed the same results. ( I – J ) Integrin β1 was knocked down in HCC cells by shRNA technology through lentiviral transduction. (I) Western blot confirmed the stable knock-down of integrin β1 in both cell lines. (J) HepG2 and SMMC-7721 cells cultured on Collagen I grew significantly slowly after knocking down integrin β1. Scale bar: 100 μm (C), 50 μm (H), * P

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Cell Culture, Western Blot, Concentration Assay, Mouse Assay, shRNA, Transduction

    10) Product Images from "Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males"

    Article Title: Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males

    Journal: Cell Stem Cell

    doi: 10.1016/j.stem.2018.05.001

    Xist Expression Is Abolished by a Robust Naive Pluripotent Network (A) Schematic illustrating the experiment performed to evaluate the impact of the nPSC culture conditions on the expression of Xist . (B) qRT-PCR analysis of Nanog and Xist in XX1, XX2, XY1, and XY2 ESC lines in SL versus 2iL. P indicates number of passages in 2iL. Error bars represent ± SD. (C) Flow cytometry analysis of male SL Nanog -GFP ESCs and subsequent sorting into three Nanog -GFP populations: low, medium, and high. (D) qRT-PCR analysis of Nanog , Klf4 , Oct4 , and Xist in low, medium, and high Nanog -GFP ESCs. Error bars represent ± SD. (E) Strand-specific RNA-seq showing expression of the positive and negative strands at the Xist locus in male 2iL ESCs. The double-strand Xist probe used in (F) is represented in red. (F) RNA FISH in male and female 2iL ESCs with a double-strand (ds) probe (left) or with a single-strand (ss) probe detecting only Xist (right). The percentage of cells with probe signal is indicated. Female EpiSCs were used as a control for the ss probe. The scale bar represents 5 μm. (G) qRT-PCR analysis of Nanog and Xist in female and male Nanog flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Error bars represent ± SD. (H) qRT-PCR analysis of Xist in XX3 and XY1 ESCs in 2iL, 2i or after 3 and 5 days in 1 μM JAKi + 2i. Error bars represent ± SD. (I) qRT-PCR analysis of Oct4 and Xist in female and male Oct4 flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Female somatic cells were used as control for Xist expression. Error bars represent ± SD.
    Figure Legend Snippet: Xist Expression Is Abolished by a Robust Naive Pluripotent Network (A) Schematic illustrating the experiment performed to evaluate the impact of the nPSC culture conditions on the expression of Xist . (B) qRT-PCR analysis of Nanog and Xist in XX1, XX2, XY1, and XY2 ESC lines in SL versus 2iL. P indicates number of passages in 2iL. Error bars represent ± SD. (C) Flow cytometry analysis of male SL Nanog -GFP ESCs and subsequent sorting into three Nanog -GFP populations: low, medium, and high. (D) qRT-PCR analysis of Nanog , Klf4 , Oct4 , and Xist in low, medium, and high Nanog -GFP ESCs. Error bars represent ± SD. (E) Strand-specific RNA-seq showing expression of the positive and negative strands at the Xist locus in male 2iL ESCs. The double-strand Xist probe used in (F) is represented in red. (F) RNA FISH in male and female 2iL ESCs with a double-strand (ds) probe (left) or with a single-strand (ss) probe detecting only Xist (right). The percentage of cells with probe signal is indicated. Female EpiSCs were used as a control for the ss probe. The scale bar represents 5 μm. (G) qRT-PCR analysis of Nanog and Xist in female and male Nanog flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Error bars represent ± SD. (H) qRT-PCR analysis of Xist in XX3 and XY1 ESCs in 2iL, 2i or after 3 and 5 days in 1 μM JAKi + 2i. Error bars represent ± SD. (I) qRT-PCR analysis of Oct4 and Xist in female and male Oct4 flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Female somatic cells were used as control for Xist expression. Error bars represent ± SD.

    Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, RNA Sequencing Assay, Fluorescence In Situ Hybridization

    Xist Is Transiently and Rapidly Upregulated in Male nPSC Differentiation and Male EpiSC Reprogramming (A) Schematic illustrating three conditions employed to differentiate 2iL and SL nPSCs: suspension culture in serum to generate EBs or adherent monolayer culture in serum-free media ± Fgf2+ActivinA (FA). (B) qRT-PCR analysis of Xist during differentiation of male ESCs in three different conditions. Before differentiation, ESCs were maintained in 2iL or SL conditions, as indicated. Error bars represent ± SD. (C) Strand-specific RNA-seq (negative strand only) showing expression of Xist during differentiation of male 2iL ESCs in three different conditions. Scale represents reads per million (RPM). (D) Heatmap showing expression profile of Xist , differentiation markers, and naive markers during differentiation of male 2iL ESCs, as indicated. Scale represents Z scores of log2-transformed expression values. (E) qRT-PCR analysis of Xist during EB differentiation of male versus female 2iL ESCs. Error bars represent ± SD. (F) Flow cytometry analysis of male GY118F Rex1 +/dGFP EpiSCs following reprogramming induction with GCSF in 2iL. Cells were sorted at different time points, with Rex1 -dGFP reporter activation indicating the subset of cells successfully transitioning to the naive identity. A representative plot from day 3 is shown. (G) qRT-PCR analysis of Xist and naive markers ( Oct4 , Tfcp2l1 , and Esrrb ) in male Rex1 -positive reprogramming intermediates at different time points after induction of reprogramming with 2iL+GCSF/GY118F. Parental EpiSCs (day 0) and ESCs in 2iL were used as controls. Error bars represent ± SD.
    Figure Legend Snippet: Xist Is Transiently and Rapidly Upregulated in Male nPSC Differentiation and Male EpiSC Reprogramming (A) Schematic illustrating three conditions employed to differentiate 2iL and SL nPSCs: suspension culture in serum to generate EBs or adherent monolayer culture in serum-free media ± Fgf2+ActivinA (FA). (B) qRT-PCR analysis of Xist during differentiation of male ESCs in three different conditions. Before differentiation, ESCs were maintained in 2iL or SL conditions, as indicated. Error bars represent ± SD. (C) Strand-specific RNA-seq (negative strand only) showing expression of Xist during differentiation of male 2iL ESCs in three different conditions. Scale represents reads per million (RPM). (D) Heatmap showing expression profile of Xist , differentiation markers, and naive markers during differentiation of male 2iL ESCs, as indicated. Scale represents Z scores of log2-transformed expression values. (E) qRT-PCR analysis of Xist during EB differentiation of male versus female 2iL ESCs. Error bars represent ± SD. (F) Flow cytometry analysis of male GY118F Rex1 +/dGFP EpiSCs following reprogramming induction with GCSF in 2iL. Cells were sorted at different time points, with Rex1 -dGFP reporter activation indicating the subset of cells successfully transitioning to the naive identity. A representative plot from day 3 is shown. (G) qRT-PCR analysis of Xist and naive markers ( Oct4 , Tfcp2l1 , and Esrrb ) in male Rex1 -positive reprogramming intermediates at different time points after induction of reprogramming with 2iL+GCSF/GY118F. Parental EpiSCs (day 0) and ESCs in 2iL were used as controls. Error bars represent ± SD.

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing, Transformation Assay, Flow Cytometry, Cytometry, Activation Assay

    11) Product Images from "Convergent analysis of genome-wide genotyping and transcriptomic data suggests association of zinc finger genes with lithium response in bipolar disorder"

    Article Title: Convergent analysis of genome-wide genotyping and transcriptomic data suggests association of zinc finger genes with lithium response in bipolar disorder

    Journal: American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics

    doi: 10.1002/ajmg.b.32663

    Regional association plot Regional association plot of the region on chromosome 19 in which the two genes ( ZNF493 , ZNF429 ) associated with lithium response and significantly affected by lithium treatment are located. The index SNP is the top-SNP (rs12975981) of ZNF493 and is shown as a purple diamond. Association p-values of SNPs are plotted as points. SNPs are colored based on linkage disequilibrium with the index SNP. cM, centimorgan; Mb, megabase; SNP, single nucleotide polymorphism.
    Figure Legend Snippet: Regional association plot Regional association plot of the region on chromosome 19 in which the two genes ( ZNF493 , ZNF429 ) associated with lithium response and significantly affected by lithium treatment are located. The index SNP is the top-SNP (rs12975981) of ZNF493 and is shown as a purple diamond. Association p-values of SNPs are plotted as points. SNPs are colored based on linkage disequilibrium with the index SNP. cM, centimorgan; Mb, megabase; SNP, single nucleotide polymorphism.

    Techniques Used:

    12) Product Images from "CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster"

    Article Title: CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043144

    Bacterial load is elevated in Chd1 -mutant flies. (A) Bacterial load was analyzed in isolated guts and in whole flies from which intestines had been removed. qPCR was performed with primers targeting 16S rDNA in the absence of infection (−PA) as well as 3 days and 4 days after oral infection with P. aeruginosa. (B) 251659264 P. aeruginosa titers are strongly increased in Chd1 −/− flies after infection. qPCR as in (A) with primers specific for P. aeruginosa . (C) Analysis of the gut-specific bacterium Acetobacter EW911. qPCR as in (A) with primers specific for Acetobacter EW911 . Relative differences of bacterial genes and the fly Rpl32 gene are expressed as 2 −ΔCT values. Values represent mean +/− SD of three independent experiments. Note that SD values are too small to show in the graph.
    Figure Legend Snippet: Bacterial load is elevated in Chd1 -mutant flies. (A) Bacterial load was analyzed in isolated guts and in whole flies from which intestines had been removed. qPCR was performed with primers targeting 16S rDNA in the absence of infection (−PA) as well as 3 days and 4 days after oral infection with P. aeruginosa. (B) 251659264 P. aeruginosa titers are strongly increased in Chd1 −/− flies after infection. qPCR as in (A) with primers specific for P. aeruginosa . (C) Analysis of the gut-specific bacterium Acetobacter EW911. qPCR as in (A) with primers specific for Acetobacter EW911 . Relative differences of bacterial genes and the fly Rpl32 gene are expressed as 2 −ΔCT values. Values represent mean +/− SD of three independent experiments. Note that SD values are too small to show in the graph.

    Techniques Used: Mutagenesis, Isolation, Real-time Polymerase Chain Reaction, Infection

    13) Product Images from "Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells ▿"

    Article Title: Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00221-10

    Analysis of enolase transcripts by quantitative real time RT-PCR. qRT-PCR plot of Pb Eno expression levels of transcripts from yeast cells of P. brasiliensis derived from lungs, livers, and spleens of mice after 7 days of infection. Control systems consisted
    Figure Legend Snippet: Analysis of enolase transcripts by quantitative real time RT-PCR. qRT-PCR plot of Pb Eno expression levels of transcripts from yeast cells of P. brasiliensis derived from lungs, livers, and spleens of mice after 7 days of infection. Control systems consisted

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay, Mouse Assay, Infection

    14) Product Images from "SMG7 is a critical regulator of p53 stability and function in DNA damage stress response"

    Article Title: SMG7 is a critical regulator of p53 stability and function in DNA damage stress response

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2015.42

    SMG7 regulates p21 activation and cell cycle arrest upon DNA damage. ( a ) Total cell extracts from control or irradiated cells (at indicated time points) were analyzed with western blot analysis using antibodies against SMG7, p53, Mdm2, p21 and actin. ( b ) Control or irradiated cells (16 h) were treated with 5-ethynyl-2′-deoxyuridine (EDU) (10 μ M ) for 1 h and then processed for EDU and 7-AAD staining, followed by flow cytometry analysis. ( c ) Total cell extracts from control or Doxorubicin-treated cell (200 ng ml −1 for various time points) were analyzed by western blot as in a . ( d ) Control or Doxorubicin-treated (200 ng ml −1 , 16 h) cells were fed with EDU (10 μ M ) for 1 h and then processed for EDU (green) and DAPI (blue) staining, followed by fluorescence microscopy. ( e ) Quantitation of EDU-positive cells from d . Error bars indicate s.d. calculated from three independent experiments.
    Figure Legend Snippet: SMG7 regulates p21 activation and cell cycle arrest upon DNA damage. ( a ) Total cell extracts from control or irradiated cells (at indicated time points) were analyzed with western blot analysis using antibodies against SMG7, p53, Mdm2, p21 and actin. ( b ) Control or irradiated cells (16 h) were treated with 5-ethynyl-2′-deoxyuridine (EDU) (10 μ M ) for 1 h and then processed for EDU and 7-AAD staining, followed by flow cytometry analysis. ( c ) Total cell extracts from control or Doxorubicin-treated cell (200 ng ml −1 for various time points) were analyzed by western blot as in a . ( d ) Control or Doxorubicin-treated (200 ng ml −1 , 16 h) cells were fed with EDU (10 μ M ) for 1 h and then processed for EDU (green) and DAPI (blue) staining, followed by fluorescence microscopy. ( e ) Quantitation of EDU-positive cells from d . Error bars indicate s.d. calculated from three independent experiments.

    Techniques Used: Activation Assay, Irradiation, Western Blot, Staining, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Quantitation Assay

    In vitro and in vivo interactions between SMG7 and p53. ( a ) Scheme of human SMG7 protein containing the N-terminal 14-3-3-like Tetratricopeptide repeat (TPR) and C-terminal low complex region. ( b ) Immunofluorescence microscopy of H1299 cells transiently expressing FH-SMG7 stained with α-HA antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). ( c ) Cell extracts from H1299 cells transiently expressing p53 with or without FH-SMG7 and the α-Flag immunoprecipitates were analyzed using western blot using α-SMG7 and α-p53 antibodies. ( d , e ) SMG7 binding to p53 in vitro . GST or GST-p53 fusion proteins (full length or various fragments) were used in pulldown assays with purified FH-SMG7 ( d ) or in vitro translated FH-SMG7 ( e ). FH-SMG7 was detected by western blot and GST proteins stained with Ponceau S. ( f , g ) Endogenous interaction of SMG7 with p53 in HCT116 ( f ) and U2OS cells ( g ). Cell extracts and the α-normal mouse IgG or α-p53 immunoprecipitates were analyzed with western blot analysis using α-SMG7 and α-p53 antibodies. ( h ) Cell extracts from control or irradiated cells (at various time points) and the α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated.
    Figure Legend Snippet: In vitro and in vivo interactions between SMG7 and p53. ( a ) Scheme of human SMG7 protein containing the N-terminal 14-3-3-like Tetratricopeptide repeat (TPR) and C-terminal low complex region. ( b ) Immunofluorescence microscopy of H1299 cells transiently expressing FH-SMG7 stained with α-HA antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). ( c ) Cell extracts from H1299 cells transiently expressing p53 with or without FH-SMG7 and the α-Flag immunoprecipitates were analyzed using western blot using α-SMG7 and α-p53 antibodies. ( d , e ) SMG7 binding to p53 in vitro . GST or GST-p53 fusion proteins (full length or various fragments) were used in pulldown assays with purified FH-SMG7 ( d ) or in vitro translated FH-SMG7 ( e ). FH-SMG7 was detected by western blot and GST proteins stained with Ponceau S. ( f , g ) Endogenous interaction of SMG7 with p53 in HCT116 ( f ) and U2OS cells ( g ). Cell extracts and the α-normal mouse IgG or α-p53 immunoprecipitates were analyzed with western blot analysis using α-SMG7 and α-p53 antibodies. ( h ) Cell extracts from control or irradiated cells (at various time points) and the α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated.

    Techniques Used: In Vitro, In Vivo, Immunofluorescence, Microscopy, Expressing, Staining, Western Blot, Binding Assay, Purification, Irradiation

    Knockout of SMG7 abrogates DNA damage-induced p53 stabilization. ( a ) Scheme of AAV-mediated knockout of SMG7 : shown from top to bottom are AAV- SMG7 targeting vector, wild-type SMG7 allele, knockout SMG7 allele containing the IRES-neo cassette and knockout SMG7 allele after removal of the IRES-neo cassette. See details of generation of HCT116 SMG7 −/− cells in Materials and Methods. ( b ) Western blot analysis of SMG7 in HCT116 and two SMG7 −/− clones. Asterisk indicates a nonspecific band. ( c ) Cell extracts from control, irradiated (10 Gy, 4 h) and Doxorubicin-treated cells (200 ng ml −1 , 4 h) were analyzed using western blot analysis with α-SMG7, α-p53 and α-actin antibodies. ( d ) Cell extracts from control and MG132-treated (20 μM, 4 h) cells were analyzed using western blot analysis as in c . ( e ) Control or irradiated cells were harvested at various time points after Cycloheximide (CHX, 50 μg ml −1 ) treatment and cell extracts were analyzed using western blot analysis with α-SMG7, α-p53, α-Mdm2 and α-actin antibodies.
    Figure Legend Snippet: Knockout of SMG7 abrogates DNA damage-induced p53 stabilization. ( a ) Scheme of AAV-mediated knockout of SMG7 : shown from top to bottom are AAV- SMG7 targeting vector, wild-type SMG7 allele, knockout SMG7 allele containing the IRES-neo cassette and knockout SMG7 allele after removal of the IRES-neo cassette. See details of generation of HCT116 SMG7 −/− cells in Materials and Methods. ( b ) Western blot analysis of SMG7 in HCT116 and two SMG7 −/− clones. Asterisk indicates a nonspecific band. ( c ) Cell extracts from control, irradiated (10 Gy, 4 h) and Doxorubicin-treated cells (200 ng ml −1 , 4 h) were analyzed using western blot analysis with α-SMG7, α-p53 and α-actin antibodies. ( d ) Cell extracts from control and MG132-treated (20 μM, 4 h) cells were analyzed using western blot analysis as in c . ( e ) Control or irradiated cells were harvested at various time points after Cycloheximide (CHX, 50 μg ml −1 ) treatment and cell extracts were analyzed using western blot analysis with α-SMG7, α-p53, α-Mdm2 and α-actin antibodies.

    Techniques Used: Knock-Out, Plasmid Preparation, Western Blot, Clone Assay, Irradiation

    Identification of SMG7 as a p53-associated protein. ( a ) Scheme of AAV-mediated epitagging of p53 : (1) AAV- p53 targeting vector; (2) wild-type p53 allele; (3) modified p53 allele containing the Flag-HA cassette; (4) FH-tagged p53 polypeptide. See details of generation of HCT116 p53 +/FH cells in Materials and Methods. ( b ) Cell extracts from control or irradiated cells (10 Gy, 4 h) and the corresponding α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( c ) Silver staining of affinity-purified p53-containing protein complex from irradiated cells (upper panel). Cell extracts and the Flag/HA immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( d ) Several representative SMG7 peptide sequences identified using mass spectrometric analysis. Numbers indicate the overlapping position of the peptides with SMG7 sequence (NP_963862).
    Figure Legend Snippet: Identification of SMG7 as a p53-associated protein. ( a ) Scheme of AAV-mediated epitagging of p53 : (1) AAV- p53 targeting vector; (2) wild-type p53 allele; (3) modified p53 allele containing the Flag-HA cassette; (4) FH-tagged p53 polypeptide. See details of generation of HCT116 p53 +/FH cells in Materials and Methods. ( b ) Cell extracts from control or irradiated cells (10 Gy, 4 h) and the corresponding α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( c ) Silver staining of affinity-purified p53-containing protein complex from irradiated cells (upper panel). Cell extracts and the Flag/HA immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( d ) Several representative SMG7 peptide sequences identified using mass spectrometric analysis. Numbers indicate the overlapping position of the peptides with SMG7 sequence (NP_963862).

    Techniques Used: Plasmid Preparation, Modification, Irradiation, Western Blot, Silver Staining, Affinity Purification, Sequencing

    15) Product Images from "Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans"

    Article Title: Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans

    Journal: eLife

    doi: 10.7554/eLife.17670

    Comparison between putative human and C. elegans propionate shunts. ( A ) Comparison between C. elegans propionate shunt genes (red) and candidate human shunt genes (green, gray). Green text indicates higher confidence annotations based on patient mutations and metabolomics, or in the case of ADHFE1 one-to-one orthology of unique enzymes in both genomes. 3-HP is marked in magenta to indicate that it is a biomarker for impaired flux in the canonical, vitamin B12-dependent propionate breakdown pathway, such as occurs in patients with propionic or methylmalonic acidemia. ( B ) C. elegans propionate shunt genes and orthologs in mouse and humans are strongly co-expressed as a group compared to 10,000 random permutations of five genes from either the whole genome, a subset of only metabolic genes, or a subset of related metabolic genes from connected pathways, including BCAA breakdown and the TCA cycle. The expression data used for this analysis was compiled and weighted using the SEEK and modSEEK databases. Distributions of co-expression scores are shown for each set of randomizations, and vertical dashed lines indicate actual weighted co-expression score for propionate shunt genes and orthologs in human, mouse and C. elegans . ( C ) The expression of several candidate human genes is activated in response to propionate in HepG2 liver carcinoma cells. qRT-PCR experiment showing the average of four replicate experiments, each containing three technical replicates. DOI: http://dx.doi.org/10.7554/eLife.17670.010
    Figure Legend Snippet: Comparison between putative human and C. elegans propionate shunts. ( A ) Comparison between C. elegans propionate shunt genes (red) and candidate human shunt genes (green, gray). Green text indicates higher confidence annotations based on patient mutations and metabolomics, or in the case of ADHFE1 one-to-one orthology of unique enzymes in both genomes. 3-HP is marked in magenta to indicate that it is a biomarker for impaired flux in the canonical, vitamin B12-dependent propionate breakdown pathway, such as occurs in patients with propionic or methylmalonic acidemia. ( B ) C. elegans propionate shunt genes and orthologs in mouse and humans are strongly co-expressed as a group compared to 10,000 random permutations of five genes from either the whole genome, a subset of only metabolic genes, or a subset of related metabolic genes from connected pathways, including BCAA breakdown and the TCA cycle. The expression data used for this analysis was compiled and weighted using the SEEK and modSEEK databases. Distributions of co-expression scores are shown for each set of randomizations, and vertical dashed lines indicate actual weighted co-expression score for propionate shunt genes and orthologs in human, mouse and C. elegans . ( C ) The expression of several candidate human genes is activated in response to propionate in HepG2 liver carcinoma cells. qRT-PCR experiment showing the average of four replicate experiments, each containing three technical replicates. DOI: http://dx.doi.org/10.7554/eLife.17670.010

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

    16) Product Images from "Identification of Endogenous Reference Genes for the Analysis of microRNA Expression in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy"

    Article Title: Identification of Endogenous Reference Genes for the Analysis of microRNA Expression in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100529

    NormFinder analysis of expression stability of candidate reference genes for microRNA qRT-PCR analysis in the systemic PILO-model samples. Ranking of candidate reference genes based on stability values calculated by NormFinder software.
    Figure Legend Snippet: NormFinder analysis of expression stability of candidate reference genes for microRNA qRT-PCR analysis in the systemic PILO-model samples. Ranking of candidate reference genes based on stability values calculated by NormFinder software.

    Techniques Used: Expressing, Quantitative RT-PCR, Software

    Levels of five non-coding RNAs candidate reference genes for microRNA qRT-PCR analysis in the hippocampus of the systemic PILO-injected and control rats. Values are given in the form of RT-qPCR threshold cycle numbers (Ct values), mean SD (n = 6). ANOVA, * , ** , *** .
    Figure Legend Snippet: Levels of five non-coding RNAs candidate reference genes for microRNA qRT-PCR analysis in the hippocampus of the systemic PILO-injected and control rats. Values are given in the form of RT-qPCR threshold cycle numbers (Ct values), mean SD (n = 6). ANOVA, * , ** , *** .

    Techniques Used: Quantitative RT-PCR, Injection

    Selection of the most suitable reference genes for microRNA qRT-PCR analysis in the systemic PILO-model samples using geNorm analysis. A) Expression stability measurements (M) for the five reference genes analyzed. The x-axis from left to right indicates the ranking of the genes according to their expression stability; lower M values indicate higher expression stability. B) Determination of the optimal number of reference genes for normalization was conducted. The software calculates the normalization factor from at least two genes at which the variable V defines the pair-wise variation between two sequential normalization factors.
    Figure Legend Snippet: Selection of the most suitable reference genes for microRNA qRT-PCR analysis in the systemic PILO-model samples using geNorm analysis. A) Expression stability measurements (M) for the five reference genes analyzed. The x-axis from left to right indicates the ranking of the genes according to their expression stability; lower M values indicate higher expression stability. B) Determination of the optimal number of reference genes for normalization was conducted. The software calculates the normalization factor from at least two genes at which the variable V defines the pair-wise variation between two sequential normalization factors.

    Techniques Used: Selection, Quantitative RT-PCR, Expressing, Software

    17) Product Images from "Topical nicotinic receptor activation improves wound bacterial infection outcomes and TLR2-mediated inflammation in diabetic mouse wounds"

    Article Title: Topical nicotinic receptor activation improves wound bacterial infection outcomes and TLR2-mediated inflammation in diabetic mouse wounds

    Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

    doi: 10.1111/wrr.12674

    Topical nAChR activation decreases IL-6 production in MRSA infected diabetic mouse wounds, and slightly increases IL-10 in MRSA infected control mouse wounds. The concentrations of ( A ) IL-6, ( B ) IL-17A, ( C ) TNF-α and ( D ) IL-10 at day 1 post-wounding were measured by ELISA in homogenates of MRSA infected skin wounds with or without nicotine treatment of control mice ( Lepr db/+ ) or diabetic mice ( Lepr db/db ) ( n =8 for each group). Data are presented as the mean ± sem. Statistical analyses were performed using Mann-Whitney one-tailed t-test ( * , p
    Figure Legend Snippet: Topical nAChR activation decreases IL-6 production in MRSA infected diabetic mouse wounds, and slightly increases IL-10 in MRSA infected control mouse wounds. The concentrations of ( A ) IL-6, ( B ) IL-17A, ( C ) TNF-α and ( D ) IL-10 at day 1 post-wounding were measured by ELISA in homogenates of MRSA infected skin wounds with or without nicotine treatment of control mice ( Lepr db/+ ) or diabetic mice ( Lepr db/db ) ( n =8 for each group). Data are presented as the mean ± sem. Statistical analyses were performed using Mann-Whitney one-tailed t-test ( * , p

    Techniques Used: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay, MANN-WHITNEY, One-tailed Test

    18) Product Images from "Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability"

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv933

    Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P
    Figure Legend Snippet: Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P

    Techniques Used: Amplification, Fluorescence In Situ Hybridization, Labeling, Real-time Polymerase Chain Reaction, CNV Assay, Transduction

    Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    19) Product Images from "Keratinocyte nicotinic acetylcholine receptor activation modulates early TLR2-mediated wound healing responses"

    Article Title: Keratinocyte nicotinic acetylcholine receptor activation modulates early TLR2-mediated wound healing responses

    Journal: International immunopharmacology

    doi: 10.1016/j.intimp.2015.05.047

    Nicotinic nAChR activation impairs TLR2 signaling during TLR2-mediated keratinocyte wound healing. (A, B) Western blot analyses for TLR2 signaling pathway in cytosolic and nuclear extracts from wounded NHEKs stimulated with vehicle, MALP2 (100ng ml −1
    Figure Legend Snippet: Nicotinic nAChR activation impairs TLR2 signaling during TLR2-mediated keratinocyte wound healing. (A, B) Western blot analyses for TLR2 signaling pathway in cytosolic and nuclear extracts from wounded NHEKs stimulated with vehicle, MALP2 (100ng ml −1

    Techniques Used: Activation Assay, Western Blot

    Nicotinic AChR activation dampens TLR2 agonist-mediated NHEKs migration during in vitro wound healing to suppress cathelicidin expression and pro-inflammatory cytokine production. (A) Scratch migration assay. MALP2-mediated keratinocytes migration at
    Figure Legend Snippet: Nicotinic AChR activation dampens TLR2 agonist-mediated NHEKs migration during in vitro wound healing to suppress cathelicidin expression and pro-inflammatory cytokine production. (A) Scratch migration assay. MALP2-mediated keratinocytes migration at

    Techniques Used: Activation Assay, Migration, In Vitro, Expressing

    Nicotinic nAChR activation suppresses CD14 and MyD88 expression in TLR2-mediated keratinocyte wound healing. (A) Quantitative RT-PCR analyses of TLR2 and CD14 in wounded NHEKs stimulated with nicotine in presence of MALP2 (100ng ml −1 ) ( n =3). Representative
    Figure Legend Snippet: Nicotinic nAChR activation suppresses CD14 and MyD88 expression in TLR2-mediated keratinocyte wound healing. (A) Quantitative RT-PCR analyses of TLR2 and CD14 in wounded NHEKs stimulated with nicotine in presence of MALP2 (100ng ml −1 ) ( n =3). Representative

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

    20) Product Images from "Compartment-Specific Modulation of GABAergic Synaptic Transmission by TRPV1 Channels in the Dentate Gyrus"

    Article Title: Compartment-Specific Modulation of GABAergic Synaptic Transmission by TRPV1 Channels in the Dentate Gyrus

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3635-14.2014

    TRPV1 activation depresses inhibitory transmission in a CB1 receptor-independent manner. A , Representative traces (left) and summary data (right) showing that bath application of 2 μ m CAP, while blocking CB1R with AM251 (4 μ m ), depresses
    Figure Legend Snippet: TRPV1 activation depresses inhibitory transmission in a CB1 receptor-independent manner. A , Representative traces (left) and summary data (right) showing that bath application of 2 μ m CAP, while blocking CB1R with AM251 (4 μ m ), depresses

    Techniques Used: Activation Assay, Transmission Assay, Blocking Assay

    Mechanism underlying TRPV1-mediated depression of inhibitory transmission. A , Representative traces (left) and summary plot (right) showing that loading DGCs with BAPTA (20 m m ) completely eliminated, whereas pre-incubation with 30 μ m CPA significantly
    Figure Legend Snippet: Mechanism underlying TRPV1-mediated depression of inhibitory transmission. A , Representative traces (left) and summary plot (right) showing that loading DGCs with BAPTA (20 m m ) completely eliminated, whereas pre-incubation with 30 μ m CPA significantly

    Techniques Used: Transmission Assay, Incubation

    TRPV1-mediated depression of inhibition is postsynaptic. A , Top, Representative traces of two consecutive GABAergic IPSCs (100 ms interstimulus interval) before (black) and after (gray) bath application of WIN, AEA, and CAP. Bottom, Summary plots showing
    Figure Legend Snippet: TRPV1-mediated depression of inhibition is postsynaptic. A , Top, Representative traces of two consecutive GABAergic IPSCs (100 ms interstimulus interval) before (black) and after (gray) bath application of WIN, AEA, and CAP. Bottom, Summary plots showing

    Techniques Used: Inhibition, Mass Spectrometry

    In vivo knockdown of TRPV1 abolishes TRPV1-mediated depression of synaptic transmission in the DG. A , Differential interference contrast (top) and fluorescence (bottom) images showing whole-cell recordings from Trpv1 shRNA-expressing (GFP + ) DGCs. R, recording
    Figure Legend Snippet: In vivo knockdown of TRPV1 abolishes TRPV1-mediated depression of synaptic transmission in the DG. A , Differential interference contrast (top) and fluorescence (bottom) images showing whole-cell recordings from Trpv1 shRNA-expressing (GFP + ) DGCs. R, recording

    Techniques Used: In Vivo, Transmission Assay, Fluorescence, shRNA, Expressing

    Functional expression of TRPV1 channels at inhibitory synapses in the dentate gyrus. A , Pharmacological activation of TRPV1 receptors with the agonist CAP (2 μ m ) depressed GABAergic IPSCs in a compartment-specific manner. Left, Representative
    Figure Legend Snippet: Functional expression of TRPV1 channels at inhibitory synapses in the dentate gyrus. A , Pharmacological activation of TRPV1 receptors with the agonist CAP (2 μ m ) depressed GABAergic IPSCs in a compartment-specific manner. Left, Representative

    Techniques Used: Functional Assay, Expressing, Activation Assay

    21) Product Images from "Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors"

    Article Title: Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

    Journal: Molecular Therapy

    doi: 10.1038/mt.2013.201

    Gene expression studies of iE2 cells. ( a ) Expression of (mouse) transgenes in iE2 cells after long-term expansion at days (d) 118 and 207 detected by RT-PCR. P: plasmids with the transgene cDNA. ( b ) Genomic PCR detecting the mouse Sox2 and Klf4 cDNA in genomic DNA. mSox2cDNA is present in iE2 genome (upper panel) but mKlf4 cDNA is absent in iE2 genome (lower panel). iPSC is a human iPSC line derived using pMX-mSox2, mKlf4, mOct4, and mMyc vectors. ( c ) Puromycin resistance of iE2 cells during expansion suggesting continuous expression of p53shRNA from the integrated retroviral vector. ( d ) qRT-PCR analysis of human TP53 gene expression of primary day 19 culture-expanded cord blood (CB) erythroblasts (pCBE19), iE2 and iPSC cells. ( e ) iE2 cells upregulate endogenous human SOX2 gene expression and maintain c-MYC gene expression at both days 118 and 207 of expansion culture. ( f ) qRT-PCR analysis of human MYB gene expression of primary day 19 culture-expanded CB erythroblasts (pCBE19) and iE2 cells. ( g ) Dendrogram and heatmap of unsupervised hierarchical clustering analysis of global gene expression profiles of various cell types. Distance numbers on the top right represent the value of Pearson distance (1-R) determined by Pearson correlation algorithms. iE2 cells are clustering with pCBE19 cells while TF-1 cells are clustering with CD34 + HSPCs. ESC/iPSCs are clustering together and separated from all blood cells.
    Figure Legend Snippet: Gene expression studies of iE2 cells. ( a ) Expression of (mouse) transgenes in iE2 cells after long-term expansion at days (d) 118 and 207 detected by RT-PCR. P: plasmids with the transgene cDNA. ( b ) Genomic PCR detecting the mouse Sox2 and Klf4 cDNA in genomic DNA. mSox2cDNA is present in iE2 genome (upper panel) but mKlf4 cDNA is absent in iE2 genome (lower panel). iPSC is a human iPSC line derived using pMX-mSox2, mKlf4, mOct4, and mMyc vectors. ( c ) Puromycin resistance of iE2 cells during expansion suggesting continuous expression of p53shRNA from the integrated retroviral vector. ( d ) qRT-PCR analysis of human TP53 gene expression of primary day 19 culture-expanded cord blood (CB) erythroblasts (pCBE19), iE2 and iPSC cells. ( e ) iE2 cells upregulate endogenous human SOX2 gene expression and maintain c-MYC gene expression at both days 118 and 207 of expansion culture. ( f ) qRT-PCR analysis of human MYB gene expression of primary day 19 culture-expanded CB erythroblasts (pCBE19) and iE2 cells. ( g ) Dendrogram and heatmap of unsupervised hierarchical clustering analysis of global gene expression profiles of various cell types. Distance numbers on the top right represent the value of Pearson distance (1-R) determined by Pearson correlation algorithms. iE2 cells are clustering with pCBE19 cells while TF-1 cells are clustering with CD34 + HSPCs. ESC/iPSCs are clustering together and separated from all blood cells.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Quantitative RT-PCR

    22) Product Images from "MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex"

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0275-4

    MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Recombinant, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Quantitative RT-PCR

    23) Product Images from "R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease"

    Article Title: R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170418

    R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P
    Figure Legend Snippet: R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P

    Techniques Used: Mouse Assay, Injection, Staining, Generated, Real-time Polymerase Chain Reaction, Purification, Expressing, MANN-WHITNEY

    24) Product Images from "R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease"

    Article Title: R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170418

    R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P
    Figure Legend Snippet: R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P

    Techniques Used: Mouse Assay, Injection, Staining, Generated, Real-time Polymerase Chain Reaction, Purification, Expressing, MANN-WHITNEY

    25) Product Images from "Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR"

    Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0200-6

    Quantitative analysis of Commd1 expression and Zrsr1 -DMR methylation in ΔPA blastocysts and the ΔPA adult brain. a Allelic expression of Commd1 was analyzed in Commd1 + (B6)/ + (BALB) (WT), Commd1 PA(B6)/ + (BALB) (PA), and Commd1 ΔPA(B6)/ + (BALB) (ΔPA) blastocysts in triplicate (shown with n) by pyrosequencing. Alleles were discriminated between using an SNP between B6 (Mat) and BALB/c (Pat) located in exon 2 (rs26846230; C in B6, T in BALB/c). The allelic expression ratios are presented relative to paternal expression in each blastocyst sample. b Methylation at Zrsr1 -DMR was analyzed in the blastocysts used in a . The 343-bp region in the DMR containing 25 CpG sites (B6; maternal allele) or 24 CpG sites (BALB/c; paternal allele) was analyzed via bisulfite sequencing. The alleles were discriminated between using an SNP (rs26846192; C in B6, A in BALB/c) located in a CpG site in the B6 sequence. Closed and open circles depict methylated and unmethylated CpGs, respectively. c Methylation at Zrsr1 -DMR was analyzed in the brains of ΔPA and WT adult F1 mice as described in b . d Allelic expression of Commd1 in the adult F1 brain was quantitatively analyzed in triplicate (shown with n) by pyrosequencing and is presented as in a . e Total Commd1 expression in an adult F1 brain of each genotype was analyzed in triplicate (shown with n) via TaqMan RT-PCR. Total expression is presented relative to the WT expression level. Asterisks (*) indicate statistical significance ( P
    Figure Legend Snippet: Quantitative analysis of Commd1 expression and Zrsr1 -DMR methylation in ΔPA blastocysts and the ΔPA adult brain. a Allelic expression of Commd1 was analyzed in Commd1 + (B6)/ + (BALB) (WT), Commd1 PA(B6)/ + (BALB) (PA), and Commd1 ΔPA(B6)/ + (BALB) (ΔPA) blastocysts in triplicate (shown with n) by pyrosequencing. Alleles were discriminated between using an SNP between B6 (Mat) and BALB/c (Pat) located in exon 2 (rs26846230; C in B6, T in BALB/c). The allelic expression ratios are presented relative to paternal expression in each blastocyst sample. b Methylation at Zrsr1 -DMR was analyzed in the blastocysts used in a . The 343-bp region in the DMR containing 25 CpG sites (B6; maternal allele) or 24 CpG sites (BALB/c; paternal allele) was analyzed via bisulfite sequencing. The alleles were discriminated between using an SNP (rs26846192; C in B6, A in BALB/c) located in a CpG site in the B6 sequence. Closed and open circles depict methylated and unmethylated CpGs, respectively. c Methylation at Zrsr1 -DMR was analyzed in the brains of ΔPA and WT adult F1 mice as described in b . d Allelic expression of Commd1 in the adult F1 brain was quantitatively analyzed in triplicate (shown with n) by pyrosequencing and is presented as in a . e Total Commd1 expression in an adult F1 brain of each genotype was analyzed in triplicate (shown with n) via TaqMan RT-PCR. Total expression is presented relative to the WT expression level. Asterisks (*) indicate statistical significance ( P

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

    Structure of the Zrsr1 / Commd1 locus and analysis of Commd1 expression and Zrsr1 -DMR methylation in the oocyte. a Zrsr1 , an approximately 2.8-kb intronless gene, and the first two exons of Commd1 are represented by gray and white boxes, respectively. Distances from the Zrsr1 gene to Commd1 exon 1 and exon 2 are indicated above the gene with double-headed arrows. The schematic is not drawn to scale. Arrows above (maternal allele) and below (paternal allele) exon 1 and the Zrsr1 gene represent the direction of transcription and the allelic expression status of the genes. The open and closed circles at the Zrsr1 promoter indicate unmethylation and methylation, respectively. A schematic of the targeting vector is shown under the gene. The closed and hatched boxes represent the truncation cassette and the neo-selection marker gene, respectively. These elements are flanked by the 5.2 kb left arm containing exon 1 and the 5.1 kb right arm, which contains part of intron 1. The truncation cassette is flanked by loxP sites, represented by gray arrowheads enclosed in open rectangles. Expected transcription patterns of the WT and PA alleles are shown above the gene schematic with thick lines and dotted lines corresponding to exons and introns, respectively. b RT-PCR analysis of Commd1 expression in growing oocytes prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. c Analysis of methylation at Zrsr1 -DMR in growing and fully grown oocytes used in b . The 223-bp region in the DMR containing 14 CpGs was analyzed via bisulfite sequencing. Each row represents a dataset from one clone, and each circle represents one CpG site. Closed and open circles depict methylated and unmethylated CpGs, respectively
    Figure Legend Snippet: Structure of the Zrsr1 / Commd1 locus and analysis of Commd1 expression and Zrsr1 -DMR methylation in the oocyte. a Zrsr1 , an approximately 2.8-kb intronless gene, and the first two exons of Commd1 are represented by gray and white boxes, respectively. Distances from the Zrsr1 gene to Commd1 exon 1 and exon 2 are indicated above the gene with double-headed arrows. The schematic is not drawn to scale. Arrows above (maternal allele) and below (paternal allele) exon 1 and the Zrsr1 gene represent the direction of transcription and the allelic expression status of the genes. The open and closed circles at the Zrsr1 promoter indicate unmethylation and methylation, respectively. A schematic of the targeting vector is shown under the gene. The closed and hatched boxes represent the truncation cassette and the neo-selection marker gene, respectively. These elements are flanked by the 5.2 kb left arm containing exon 1 and the 5.1 kb right arm, which contains part of intron 1. The truncation cassette is flanked by loxP sites, represented by gray arrowheads enclosed in open rectangles. Expected transcription patterns of the WT and PA alleles are shown above the gene schematic with thick lines and dotted lines corresponding to exons and introns, respectively. b RT-PCR analysis of Commd1 expression in growing oocytes prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. c Analysis of methylation at Zrsr1 -DMR in growing and fully grown oocytes used in b . The 223-bp region in the DMR containing 14 CpGs was analyzed via bisulfite sequencing. Each row represents a dataset from one clone, and each circle represents one CpG site. Closed and open circles depict methylated and unmethylated CpGs, respectively

    Techniques Used: Expressing, Methylation, Plasmid Preparation, Selection, Marker, Reverse Transcription Polymerase Chain Reaction, Positive Control, Molecular Weight, Methylation Sequencing

    26) Product Images from "Soluble ST2 protein inhibits LPS stimulation on monocyte-derived dendritic cells"

    Article Title: Soluble ST2 protein inhibits LPS stimulation on monocyte-derived dendritic cells

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2012.29

    TLR4 expression is suppressed by sST2. ( a ) Relative quantity of TLR4 mRNA was analyzed by real-time PCR after various stimuli (sST2, LPS, sST2/LPS) and normalized against endogenous β-actin. The data are presented as the means of three experiments±s.d.
    Figure Legend Snippet: TLR4 expression is suppressed by sST2. ( a ) Relative quantity of TLR4 mRNA was analyzed by real-time PCR after various stimuli (sST2, LPS, sST2/LPS) and normalized against endogenous β-actin. The data are presented as the means of three experiments±s.d.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    27) Product Images from "Anthropoid primate–specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length"

    Article Title: Anthropoid primate–specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2006337

    THE1B 5′ insertion site creates novel binding site for transcription factor DLX3. (A) Top left, sequence of 5′ insertion site of THE1B near CRH ]. This DLX3 binding site is formed by the insertion of THE1B (right of black bar) into the genome (left of black bar). (C) ChIP for DLX3 (black bars) and RNA polymerase II (white bar) with quantitative real-time PCR. DLX3 is significantly associated with the 5′ end of THE1B and positive control (JRE) in human term placenta. ChIP-qPCR data were normalized to the IgG control for each target and presented relative to negative control (JRE distal). ( n = 3 for all, P
    Figure Legend Snippet: THE1B 5′ insertion site creates novel binding site for transcription factor DLX3. (A) Top left, sequence of 5′ insertion site of THE1B near CRH ]. This DLX3 binding site is formed by the insertion of THE1B (right of black bar) into the genome (left of black bar). (C) ChIP for DLX3 (black bars) and RNA polymerase II (white bar) with quantitative real-time PCR. DLX3 is significantly associated with the 5′ end of THE1B and positive control (JRE) in human term placenta. ChIP-qPCR data were normalized to the IgG control for each target and presented relative to negative control (JRE distal). ( n = 3 for all, P

    Techniques Used: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    BAC transgenic mice exhibit placental CRH expression and delayed parturition, which are eliminated by THE1B deletion. (A) Schematic of human BAC clone RP11-366K18 (gray) with CRH shown to scale (red). (B) Expression of human CRH measured by qPCR in adult tissues and placenta. ( n = 2–4 per group, P
    Figure Legend Snippet: BAC transgenic mice exhibit placental CRH expression and delayed parturition, which are eliminated by THE1B deletion. (A) Schematic of human BAC clone RP11-366K18 (gray) with CRH shown to scale (red). (B) Expression of human CRH measured by qPCR in adult tissues and placenta. ( n = 2–4 per group, P

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

    28) Product Images from "Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins"

    Article Title: Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

    Journal: mBio

    doi: 10.1128/mBio.02170-16

    Modulation of miR-203a-3p targets and analysis of miR-203a-3p expression. Effects of a miR-203a-3p mimic (MIM, green) or an LNA inhibitor (light blue) on TP63 (A) and BMI1 (B) levels in HPV16 E6/E7-expressing HFKs. Expression of 18S rRNA was used as an internal control, and values were normalized to a negative control (C) mimic or LNA. TP63 and BMI1 expression was assessed by RT-PCR. (C) Expression of miR-203a-3p in three independently derived HFK populations expressing HPV16 E6, E7, or E6/E7 or a control vector via TaqMan miR assay. Expression of the noncoding snRNA U6 spliceosomal RNA was used as an internal control. Results represent averages of at least three independent experiments.
    Figure Legend Snippet: Modulation of miR-203a-3p targets and analysis of miR-203a-3p expression. Effects of a miR-203a-3p mimic (MIM, green) or an LNA inhibitor (light blue) on TP63 (A) and BMI1 (B) levels in HPV16 E6/E7-expressing HFKs. Expression of 18S rRNA was used as an internal control, and values were normalized to a negative control (C) mimic or LNA. TP63 and BMI1 expression was assessed by RT-PCR. (C) Expression of miR-203a-3p in three independently derived HFK populations expressing HPV16 E6, E7, or E6/E7 or a control vector via TaqMan miR assay. Expression of the noncoding snRNA U6 spliceosomal RNA was used as an internal control. Results represent averages of at least three independent experiments.

    Techniques Used: Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation

    29) Product Images from "A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization"

    Article Title: A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

    Journal: Journal of Virology

    doi: 10.1128/JVI.01379-12

    Levels of viral DNA synthesis in infected cells. Viral DNA synthesis in each experiment was determined by quantitative real-time PCR at each of the time points indicated. The amount of viral DNA assayed is represented as copies of the viral gene UL83 per copy of the cellular adipsin gene.
    Figure Legend Snippet: Levels of viral DNA synthesis in infected cells. Viral DNA synthesis in each experiment was determined by quantitative real-time PCR at each of the time points indicated. The amount of viral DNA assayed is represented as copies of the viral gene UL83 per copy of the cellular adipsin gene.

    Techniques Used: DNA Synthesis, Infection, Real-time Polymerase Chain Reaction

    Levels of capsid proteins in infected cells. (A) Schematic of locus in the HCMV virus genome from which UL83 to UL85 are produced. Filled arrows above the genome represent proteins encoded within the genome. Black lines below the genome represent transcripts from which those proteins are produced. pA, polyadenylation signal. (B) Western blotting of infected cell lysate. HFF cells were infected at an MOI of 1 with the indicated viruses, and cell lysates were prepared for Western blotting at the indicated time points (indicated above the figure). (C) Determination of relative protein levels in cells. A 2-fold dilution series of protein from lane 4 of panel B (lanes 1 to 3) was analyzed by Western blotting compared to undiluted protein from lane 7 of panel B (lane 4). Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular mass markers (kDa) are indicated to the left of each figure.
    Figure Legend Snippet: Levels of capsid proteins in infected cells. (A) Schematic of locus in the HCMV virus genome from which UL83 to UL85 are produced. Filled arrows above the genome represent proteins encoded within the genome. Black lines below the genome represent transcripts from which those proteins are produced. pA, polyadenylation signal. (B) Western blotting of infected cell lysate. HFF cells were infected at an MOI of 1 with the indicated viruses, and cell lysates were prepared for Western blotting at the indicated time points (indicated above the figure). (C) Determination of relative protein levels in cells. A 2-fold dilution series of protein from lane 4 of panel B (lanes 1 to 3) was analyzed by Western blotting compared to undiluted protein from lane 7 of panel B (lane 4). Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular mass markers (kDa) are indicated to the left of each figure.

    Techniques Used: Infection, Produced, Western Blot

    30) Product Images from "Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts"

    Article Title: Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts

    Journal: Scientific Reports

    doi: 10.1038/srep28538

    Synergetic effect of mixed sheets consisting of PBMNCs and fibroblasts. ( a ) Comparison of VEGF secretion. The VEGF concentration in the culture medium was measured in PBMNCs, fibroblast sheets, and mixed cell sheets for 3 days. Normoxic condition: 37 °C, 20% O 2 for 3 days. Hypoxic condition: 37 °C, 20% O 2 for 2 days followed by 33 °C, 2% O 2 for 1 day. ( b ) Secretion from PBMNCs increased VEGF production by fibroblasts. Fibroblasts were cultured for 48 h with or without the PBMNC-conditioned medium, and the VEGF concentration in the supernatant was analyzed by ELISA. ( c ) TGF-β1 concentration in fibroblasts and PBMNC culture medium at 48 h. ( d ) PDGF-BB concentration in fibroblasts and PBMNC culture medium at 48 h. ( e ) Neutralizing antibody against TGF-β1 and PDGF-BB inhibited VEGF production in fibroblasts. The PBMNC-conditioned medium was co-cultured with a neutralizing antibody against TGF-β1 or PDGF-BB, and the PBMNC-conditioned medium was added to fibroblasts. After 48 h, the VEGF concentration was measured by ELISA. ( f ) TGF-β1 and PDGF-BB recombinant proteins elevated VEGF production by fibroblasts. ( g ) The PBMNC-conditioned medium increased the expression levels of VEGF, collagen I, collagen III, α-SMA, and Axin2 mRNA. Fibroblasts were cultured with the PBMNC-conditioned or control medium for 48 h. The mRNA expression levels were determined using real-time PCR. ACTB was used as an endogenous control. The expression levels were compared with that in control medium, which is presented as 1.
    Figure Legend Snippet: Synergetic effect of mixed sheets consisting of PBMNCs and fibroblasts. ( a ) Comparison of VEGF secretion. The VEGF concentration in the culture medium was measured in PBMNCs, fibroblast sheets, and mixed cell sheets for 3 days. Normoxic condition: 37 °C, 20% O 2 for 3 days. Hypoxic condition: 37 °C, 20% O 2 for 2 days followed by 33 °C, 2% O 2 for 1 day. ( b ) Secretion from PBMNCs increased VEGF production by fibroblasts. Fibroblasts were cultured for 48 h with or without the PBMNC-conditioned medium, and the VEGF concentration in the supernatant was analyzed by ELISA. ( c ) TGF-β1 concentration in fibroblasts and PBMNC culture medium at 48 h. ( d ) PDGF-BB concentration in fibroblasts and PBMNC culture medium at 48 h. ( e ) Neutralizing antibody against TGF-β1 and PDGF-BB inhibited VEGF production in fibroblasts. The PBMNC-conditioned medium was co-cultured with a neutralizing antibody against TGF-β1 or PDGF-BB, and the PBMNC-conditioned medium was added to fibroblasts. After 48 h, the VEGF concentration was measured by ELISA. ( f ) TGF-β1 and PDGF-BB recombinant proteins elevated VEGF production by fibroblasts. ( g ) The PBMNC-conditioned medium increased the expression levels of VEGF, collagen I, collagen III, α-SMA, and Axin2 mRNA. Fibroblasts were cultured with the PBMNC-conditioned or control medium for 48 h. The mRNA expression levels were determined using real-time PCR. ACTB was used as an endogenous control. The expression levels were compared with that in control medium, which is presented as 1.

    Techniques Used: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Real-time Polymerase Chain Reaction

    Immunostaining against markers of the wound healing process 3, 7, and 14 days after mixed cell sheet transplantation and trafermin treatment. ( a ) TGF-β1. ( b ) α-SMA. ( c ) Collagen III. Bar shows 200 μm.
    Figure Legend Snippet: Immunostaining against markers of the wound healing process 3, 7, and 14 days after mixed cell sheet transplantation and trafermin treatment. ( a ) TGF-β1. ( b ) α-SMA. ( c ) Collagen III. Bar shows 200 μm.

    Techniques Used: Immunostaining, Transplantation Assay

    31) Product Images from "Merkel cell polyomavirus Tumor antigens expressed in Merkel cell carcinoma function independently of the ubiquitin ligases Fbw7 and β-TrCP"

    Article Title: Merkel cell polyomavirus Tumor antigens expressed in Merkel cell carcinoma function independently of the ubiquitin ligases Fbw7 and β-TrCP

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007543

    MCPyV LT and 57kT bind Fbw7α independently of its WD40 domain and MCPyV LT S239. (A, B) MCPyV LT was pulled-down with XT10 from whole cell lysates of 293A cells transfected with individual or combinations of full-length MCPyV LT S239A (5μg), MCPyV LT-t (10.5μg), MCPyV 57kT (5μg), Fbw7 (4.5μg), ΔFbox (3μg), or R505L (3μg), and immunoblotted with anti-FLAG to detect co-immunoprecipitated Fbw7. (B) A plasmid encoding only the sequence of MCPyV 57kT was similarly pulled-down and co-immunoprecipitated Fbw7 was detected. Asterisks (*) denote non-specific bands.
    Figure Legend Snippet: MCPyV LT and 57kT bind Fbw7α independently of its WD40 domain and MCPyV LT S239. (A, B) MCPyV LT was pulled-down with XT10 from whole cell lysates of 293A cells transfected with individual or combinations of full-length MCPyV LT S239A (5μg), MCPyV LT-t (10.5μg), MCPyV 57kT (5μg), Fbw7 (4.5μg), ΔFbox (3μg), or R505L (3μg), and immunoblotted with anti-FLAG to detect co-immunoprecipitated Fbw7. (B) A plasmid encoding only the sequence of MCPyV 57kT was similarly pulled-down and co-immunoprecipitated Fbw7 was detected. Asterisks (*) denote non-specific bands.

    Techniques Used: Transfection, Immunoprecipitation, Plasmid Preparation, Sequencing

    MCPyV LT is not bound or destabilized by the WD40 domain of Fbw7. (A) MCPyV LT was pulled-down from whole cell lysates of 293A cells transfected with MCPyV LT (5μg), LT-t (10.5μg) and either the FLAG-Fbw7α (4.5μg), or HA-Fbw7α construct (8μg). In addition, the FLAG-tagged WD40 mutant (FLAG-Fbw7α-R505L) (3μg), and the HA-tagged WD40 mutant provided by Kwun et al. (HA-Fbw7α-R465C) (8μg) were tested for their ability to co-immunoprecipitate with MCPyV LT and LT-t (17). (B) Consistent amounts of MCPyV LT (5μg) were co-expressed with increasing amounts of Fbw7 (2.5μg, 5μg, 10μg) or the degradation incompetent Fbw7 ΔFbox mutant. Immunoblotting with 2T2 was performed to compare MCPyV LT protein levels in each condition.
    Figure Legend Snippet: MCPyV LT is not bound or destabilized by the WD40 domain of Fbw7. (A) MCPyV LT was pulled-down from whole cell lysates of 293A cells transfected with MCPyV LT (5μg), LT-t (10.5μg) and either the FLAG-Fbw7α (4.5μg), or HA-Fbw7α construct (8μg). In addition, the FLAG-tagged WD40 mutant (FLAG-Fbw7α-R505L) (3μg), and the HA-tagged WD40 mutant provided by Kwun et al. (HA-Fbw7α-R465C) (8μg) were tested for their ability to co-immunoprecipitate with MCPyV LT and LT-t (17). (B) Consistent amounts of MCPyV LT (5μg) were co-expressed with increasing amounts of Fbw7 (2.5μg, 5μg, 10μg) or the degradation incompetent Fbw7 ΔFbox mutant. Immunoblotting with 2T2 was performed to compare MCPyV LT protein levels in each condition.

    Techniques Used: Transfection, Construct, Mutagenesis

    SV40 LT, but not the MCPyV T antigens, co-immunoprecipitate with Fbw7α. (A) The SV40 LT antigen was pulled down with an anti-HA antibody from whole cell lysates of 293A cells expressing individual or combinations of HA-SV40 LT or the T701A mutant (5μg), wild-type FLAG-Fbw7 (4.5μg), or FLAG-Fbw7 ΔFbox/R505L mutants (3μg). Detection of co-immunoprecipitated Fbw7 was performed by immunoprecipitating with anti-HA, followed by immunoblotting with anti-FLAG. (B) The reciprocal IP to Fig 1A was performed with HA-SV40 LT, the MCPyV T antigens (HA-LT (5μg), HA-LT-t (10.5μg), and untagged ST (1μg)) and Fbw7, in which Fbw7 was pulled down (FLAG) and immunoblotted for interacting T antigens (anti-HA/2T2 (common-T antibody)). (C) An identical co-immunoprecipitation as Fig 1B was performed, except Fbw7 with an N-terminal Myc tag was pulled down from cellular lysates using a Myc tag specific antibody (9E10). (D) An identical co-immunoprecipitation as Fig 1B was performed with untagged SV40 and MCPyV T antigens. SV40 and MCPyV T antigens were detected by XT10 immunoblotting. (E) A co-immunoprecipitation between MCPyV T antigens (LT and ST) and Fbw7 was also performed through pull-down of the T antigens (XT10—common-T antibody) and detection of co-immunoprecipitated Fbw7 (anti-FLAG). Asterisks (*) denote non-specific bands.
    Figure Legend Snippet: SV40 LT, but not the MCPyV T antigens, co-immunoprecipitate with Fbw7α. (A) The SV40 LT antigen was pulled down with an anti-HA antibody from whole cell lysates of 293A cells expressing individual or combinations of HA-SV40 LT or the T701A mutant (5μg), wild-type FLAG-Fbw7 (4.5μg), or FLAG-Fbw7 ΔFbox/R505L mutants (3μg). Detection of co-immunoprecipitated Fbw7 was performed by immunoprecipitating with anti-HA, followed by immunoblotting with anti-FLAG. (B) The reciprocal IP to Fig 1A was performed with HA-SV40 LT, the MCPyV T antigens (HA-LT (5μg), HA-LT-t (10.5μg), and untagged ST (1μg)) and Fbw7, in which Fbw7 was pulled down (FLAG) and immunoblotted for interacting T antigens (anti-HA/2T2 (common-T antibody)). (C) An identical co-immunoprecipitation as Fig 1B was performed, except Fbw7 with an N-terminal Myc tag was pulled down from cellular lysates using a Myc tag specific antibody (9E10). (D) An identical co-immunoprecipitation as Fig 1B was performed with untagged SV40 and MCPyV T antigens. SV40 and MCPyV T antigens were detected by XT10 immunoblotting. (E) A co-immunoprecipitation between MCPyV T antigens (LT and ST) and Fbw7 was also performed through pull-down of the T antigens (XT10—common-T antibody) and detection of co-immunoprecipitated Fbw7 (anti-FLAG). Asterisks (*) denote non-specific bands.

    Techniques Used: Expressing, Mutagenesis, Immunoprecipitation

    32) Product Images from "Viral proteins as a potential driver of histone depletion in dinoflagellates"

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03993-4

    DVNP expression results in transcriptional impairment in S. cerevisiae . a Spike-in normalized average gene plot showing the enrichment of two biological replicates of Rpb3 ChIPs from DVNP-3HA-NLS-expressing and control cells. b Differences in Rpb3 following DVNP expression. Values represent the mean Rpb3 occupancy calculated over gene bodies after averaging biological replicates ( n . The P -value was calculated using a two sided Welch’s t -test. Box plot notches represent an estimate of the 95% confidence interval of the median
    Figure Legend Snippet: DVNP expression results in transcriptional impairment in S. cerevisiae . a Spike-in normalized average gene plot showing the enrichment of two biological replicates of Rpb3 ChIPs from DVNP-3HA-NLS-expressing and control cells. b Differences in Rpb3 following DVNP expression. Values represent the mean Rpb3 occupancy calculated over gene bodies after averaging biological replicates ( n . The P -value was calculated using a two sided Welch’s t -test. Box plot notches represent an estimate of the 95% confidence interval of the median

    Techniques Used: Expressing

    33) Product Images from "SOX2 as a New Regulator of HPV16 Transcription"

    Article Title: SOX2 as a New Regulator of HPV16 Transcription

    Journal: Viruses

    doi: 10.3390/v9070175

    SOX2 binds to HPV16-LCR putative binding sites in vivo. Chromatin immunoprecipitation assay (ChIP) of SOX2 were performed evaluating the HPV16-LCR region (LCR) from CaSki cells. Anti-p65 and -CTCF antibodies were employed as positive and negative controls, respectively. Non-specific IgG antibody was included as a negative control. Quantitative PCR (qPCR) analysis was performed on the DNA obtained from the ChIP assay evaluating the HPV16-LCR region (black bars), and the Lefty (gray bars) gene promoter region, as a positive control of SOX2 binding ( n = 2). The graphic represents the percentage of input recovered after immunoprecipitation with anti-SOX2, -p65 or -CTCF antibodies. Three independent biological experiments were performed. The standard error of the mean of triplicate qPCR measurements is shown. ** p
    Figure Legend Snippet: SOX2 binds to HPV16-LCR putative binding sites in vivo. Chromatin immunoprecipitation assay (ChIP) of SOX2 were performed evaluating the HPV16-LCR region (LCR) from CaSki cells. Anti-p65 and -CTCF antibodies were employed as positive and negative controls, respectively. Non-specific IgG antibody was included as a negative control. Quantitative PCR (qPCR) analysis was performed on the DNA obtained from the ChIP assay evaluating the HPV16-LCR region (black bars), and the Lefty (gray bars) gene promoter region, as a positive control of SOX2 binding ( n = 2). The graphic represents the percentage of input recovered after immunoprecipitation with anti-SOX2, -p65 or -CTCF antibodies. Three independent biological experiments were performed. The standard error of the mean of triplicate qPCR measurements is shown. ** p

    Techniques Used: Binding Assay, In Vivo, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Positive Control, Immunoprecipitation

    34) Product Images from "MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex"

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0275-4

    MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Recombinant, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Quantitative RT-PCR

    35) Product Images from "PDE4D regulates Spine Plasticity and Memory in the Retrosplenial Cortex"

    Article Title: PDE4D regulates Spine Plasticity and Memory in the Retrosplenial Cortex

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22193-0

    Object exploration (OE) training strongly activates RSC. ( A ) The training protocol used to test the effect of exploration on gene expression involves 2 d of habituation to the training boxes and a 15 min exposure to the objects on training day, a procedure that will produce a memory for both the objects (object recognition memory) and their spatial location (object location memory). ( B ) In RSC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos, Arc ) after OE training, but return to baseline within 3 hrs thereby recapitulating the time course seen after cFC training (n = 8). ( C ) Correlation of RSC induction of the 66 genes shown in Fig. 1d after cFC and OE training. The OE induced gene expression changes in RSC closely recapitulate those seen 30 mins after cFC training.
    Figure Legend Snippet: Object exploration (OE) training strongly activates RSC. ( A ) The training protocol used to test the effect of exploration on gene expression involves 2 d of habituation to the training boxes and a 15 min exposure to the objects on training day, a procedure that will produce a memory for both the objects (object recognition memory) and their spatial location (object location memory). ( B ) In RSC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos, Arc ) after OE training, but return to baseline within 3 hrs thereby recapitulating the time course seen after cFC training (n = 8). ( C ) Correlation of RSC induction of the 66 genes shown in Fig. 1d after cFC and OE training. The OE induced gene expression changes in RSC closely recapitulate those seen 30 mins after cFC training.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Blocking NMDA-R impairs memory and blocks IEG induction in HC and RSC. MK801 (0.1 mg/kg, i.p.) injected 30 mins before cFC training does not affect the response to foot-shock during conditioning ( A ), but it significantly impairs memory when tested 24 hrs but not 1 hr after training ( B ), n = 8 per treatment group and time-point). The same dose blocks training-induced IEG induction ( Fos , Arc and Npas4 ) in RSC (C, n = 8) and HC (D, n = 6). Mean ± s.e.m. are shown. Significant differences from vehicle control are indicated by an asterisk (*).
    Figure Legend Snippet: Blocking NMDA-R impairs memory and blocks IEG induction in HC and RSC. MK801 (0.1 mg/kg, i.p.) injected 30 mins before cFC training does not affect the response to foot-shock during conditioning ( A ), but it significantly impairs memory when tested 24 hrs but not 1 hr after training ( B ), n = 8 per treatment group and time-point). The same dose blocks training-induced IEG induction ( Fos , Arc and Npas4 ) in RSC (C, n = 8) and HC (D, n = 6). Mean ± s.e.m. are shown. Significant differences from vehicle control are indicated by an asterisk (*).

    Techniques Used: Blocking Assay, Injection

    Gene-responses following cFC are highly correlated in hippocampus (HC) and retrosplenial cortex (RSC). ( A ) In HC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos , Arc ) after cFC training, and return to baseline within 3 hrs. ( B ) The time-course of IEG induction observed in HC is recapitulated in RSC (n = 8 mice per group for HC and RSC). ( C ) Venn diagram showing the number of confirmed hits of two replicate RNAseq studies comparing mRNA expression in naïve and fear conditioned mice. All but one gene ( Errf1 ) found in HC are detected in RSC. ( D ) Correlation of mRNA induction by fear conditioning in HP and RSC. The gene-responses of the 66 confirmed hits in RSC and HC are highly correlated. The greater number of calls in RSC is explained by an overall larger gene-response in this structure.
    Figure Legend Snippet: Gene-responses following cFC are highly correlated in hippocampus (HC) and retrosplenial cortex (RSC). ( A ) In HC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos , Arc ) after cFC training, and return to baseline within 3 hrs. ( B ) The time-course of IEG induction observed in HC is recapitulated in RSC (n = 8 mice per group for HC and RSC). ( C ) Venn diagram showing the number of confirmed hits of two replicate RNAseq studies comparing mRNA expression in naïve and fear conditioned mice. All but one gene ( Errf1 ) found in HC are detected in RSC. ( D ) Correlation of mRNA induction by fear conditioning in HP and RSC. The gene-responses of the 66 confirmed hits in RSC and HC are highly correlated. The greater number of calls in RSC is explained by an overall larger gene-response in this structure.

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

    36) Product Images from "RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development"

    Article Title: RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170464

    Regulators of spine maturation are distinct from regulators of spine precursor formation. A) Representative Images of GFP-expressing DIV-16 neurons transfected with the indicated shRNA targeting sequence for 48 hours. B) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine density later in synaptic development (DIV-16). Spine density is expressed as the percentage of the average control spine density. n = 44 control, 16 Ophn-1 shRNA #1, 5 Ophn-1 shRNA #2, 17 β -pix shRNA #1, 7 β -pix shRNA #2, 15 Frabin shRNA #1, 8 Frabin shRNA #2 neurons (Spine density was not significantly different from control as determined by t-test, except for β -pix shRNA #1 which was determined by Mann-Whitney Rank Sum Test). C) Arhgap23 shRNAs significantly increase spine density later during synaptic development (DIV-16). n = 44 control (same as B), 22 Arhgap23 shRNA #1, and 12 Arhgap23 shRNA #2 neurons; p = 0.02 for Control vs Arhgap23 shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.002 for Control vs Arhgap23 shRNA #2 (Mann-Whitney Rank Sum Test). D) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine length later in synaptic development (DIV-16) neurons. Cumulative distribution plot of spine length in DIV-16 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. n = 3273 control, 651 Ophn-1 shRNA #1, 130 Ophn-1 shRNA #2, 729 β -pix shRNA #1, 449 β -pix shRNA #2, 556 Frabin shRNA #1, 688 Frabin shRNA #2 spines (Spine length was not significantly different from control as determined by Mann-Whitney Rank Sum test). E) Arhgap23 and Vav2 shRNAs significantly increase spine length later in neuronal development (DIV-16). n = 3273 control (same as D), 1207 Arhgap23 shRNA #1, 1182 Arhgap23 shRNA #2, 962 Vav2 shRNA #1, and 551 Vav2 shRNA #2 spines; p
    Figure Legend Snippet: Regulators of spine maturation are distinct from regulators of spine precursor formation. A) Representative Images of GFP-expressing DIV-16 neurons transfected with the indicated shRNA targeting sequence for 48 hours. B) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine density later in synaptic development (DIV-16). Spine density is expressed as the percentage of the average control spine density. n = 44 control, 16 Ophn-1 shRNA #1, 5 Ophn-1 shRNA #2, 17 β -pix shRNA #1, 7 β -pix shRNA #2, 15 Frabin shRNA #1, 8 Frabin shRNA #2 neurons (Spine density was not significantly different from control as determined by t-test, except for β -pix shRNA #1 which was determined by Mann-Whitney Rank Sum Test). C) Arhgap23 shRNAs significantly increase spine density later during synaptic development (DIV-16). n = 44 control (same as B), 22 Arhgap23 shRNA #1, and 12 Arhgap23 shRNA #2 neurons; p = 0.02 for Control vs Arhgap23 shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.002 for Control vs Arhgap23 shRNA #2 (Mann-Whitney Rank Sum Test). D) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine length later in synaptic development (DIV-16) neurons. Cumulative distribution plot of spine length in DIV-16 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. n = 3273 control, 651 Ophn-1 shRNA #1, 130 Ophn-1 shRNA #2, 729 β -pix shRNA #1, 449 β -pix shRNA #2, 556 Frabin shRNA #1, 688 Frabin shRNA #2 spines (Spine length was not significantly different from control as determined by Mann-Whitney Rank Sum test). E) Arhgap23 and Vav2 shRNAs significantly increase spine length later in neuronal development (DIV-16). n = 3273 control (same as D), 1207 Arhgap23 shRNA #1, 1182 Arhgap23 shRNA #2, 962 Vav2 shRNA #1, and 551 Vav2 shRNA #2 spines; p

    Techniques Used: Expressing, Transfection, shRNA, Sequencing, MANN-WHITNEY

    37) Product Images from "NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes"

    Article Title: NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19051365

    Expression of NOX isotypes in the epithelial and myeloid compartments of the small intestine. ( a ) RT-PCR. CX3CR1-GFP + and epithelial cells were sorted from the small intestine to examine their expression of NOX enzymes in the steady-state or after activation with PMA. Bar graph indicates the relative expression of Nox2 and Nox4 relative to HPRT. No significant difference was observed upon PMA treatment. Results shown are representative of three independent experiments; columns and error bars indicate mean +/− SEM. ( b ) Immunofluorescence micrograph of the small intestine from a CX3CR1-GFP mouse, immunostained for Nox4. Nox4 is expressed at high levels in GFP + and GFP − cells in the LP, and at moderate levels in epithelial cells. Scale bar is 20 µm. ( c ) Immunofluorescence micrograph of the small intestine before (top) and after (bottom) incubation with pHrodo bioparticles. At physiologic pH the pHrodo beads are non-fluorescent. pHrodo accumulation is seen as bright clusters in GFP + and GFP − cells in the LP, and diffusely throughout the epithelium. In the steady-state, no pHrodo signal is observed. pHrodo bioparticles are rapidly taken up and found in acidic compartments of GFP + and GFP − cells. Immunofluorescence images are representative of three independent experiments; scale bar is 20 µm.
    Figure Legend Snippet: Expression of NOX isotypes in the epithelial and myeloid compartments of the small intestine. ( a ) RT-PCR. CX3CR1-GFP + and epithelial cells were sorted from the small intestine to examine their expression of NOX enzymes in the steady-state or after activation with PMA. Bar graph indicates the relative expression of Nox2 and Nox4 relative to HPRT. No significant difference was observed upon PMA treatment. Results shown are representative of three independent experiments; columns and error bars indicate mean +/− SEM. ( b ) Immunofluorescence micrograph of the small intestine from a CX3CR1-GFP mouse, immunostained for Nox4. Nox4 is expressed at high levels in GFP + and GFP − cells in the LP, and at moderate levels in epithelial cells. Scale bar is 20 µm. ( c ) Immunofluorescence micrograph of the small intestine before (top) and after (bottom) incubation with pHrodo bioparticles. At physiologic pH the pHrodo beads are non-fluorescent. pHrodo accumulation is seen as bright clusters in GFP + and GFP − cells in the LP, and diffusely throughout the epithelium. In the steady-state, no pHrodo signal is observed. pHrodo bioparticles are rapidly taken up and found in acidic compartments of GFP + and GFP − cells. Immunofluorescence images are representative of three independent experiments; scale bar is 20 µm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Immunofluorescence, Incubation

    38) Product Images from "REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors"

    Article Title: REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors

    Journal: Nature neuroscience

    doi: 10.1038/nn.3214

    Maternal deprivation disrupts the increase in REST, epigenetic remodeling and decrease in GluN2B during postnatal development a , Diagram illustrating the maternal deprivation paradigm. b , Representative Western blot of hippocampal nuclear fraction. c , Summary plot ( left panel ) showing a robust decrease in REST protein expression in the hippocampus of maternally-deprived vs . normally-reared pups (n = 3). In the dentate gyrus, whereas REST mRNA decreases (n = 3), GluN2B mRNA increases ( right panel; n = 3). d , Abundance of REST and e , H3K27me3 at the grin2b promoter are decreased relative to age-matched, normally-reared rats ( n = 3). f , Representative Western blots ( left panel ) and summary data ( right panel ), showing that whereas maternal deprivation increases GluN2B protein in total lysate, endoplasmic reticulum (ER) and postsynaptic density (PSD) fractions, GluN2A and GluA2 are unchanged in all fractions. GluN1 was reduced in the ER fraction and increased in the PSD in the hippocampus from maternally-deprived pups vs. normally-reared pups. All samples were assessed at P28–31 (n = 3). Total lysate samples were normalized to β-actin, ER samples were normalized to GRP78, an ER marker, and PSD samples were normalized to PSD-95, which is not altered after maternally-deprived ( see Supplementary Table 9 and full length blots in Supplementary Fig. 12 ).
    Figure Legend Snippet: Maternal deprivation disrupts the increase in REST, epigenetic remodeling and decrease in GluN2B during postnatal development a , Diagram illustrating the maternal deprivation paradigm. b , Representative Western blot of hippocampal nuclear fraction. c , Summary plot ( left panel ) showing a robust decrease in REST protein expression in the hippocampus of maternally-deprived vs . normally-reared pups (n = 3). In the dentate gyrus, whereas REST mRNA decreases (n = 3), GluN2B mRNA increases ( right panel; n = 3). d , Abundance of REST and e , H3K27me3 at the grin2b promoter are decreased relative to age-matched, normally-reared rats ( n = 3). f , Representative Western blots ( left panel ) and summary data ( right panel ), showing that whereas maternal deprivation increases GluN2B protein in total lysate, endoplasmic reticulum (ER) and postsynaptic density (PSD) fractions, GluN2A and GluA2 are unchanged in all fractions. GluN1 was reduced in the ER fraction and increased in the PSD in the hippocampus from maternally-deprived pups vs. normally-reared pups. All samples were assessed at P28–31 (n = 3). Total lysate samples were normalized to β-actin, ER samples were normalized to GRP78, an ER marker, and PSD samples were normalized to PSD-95, which is not altered after maternally-deprived ( see Supplementary Table 9 and full length blots in Supplementary Fig. 12 ).

    Techniques Used: Western Blot, Expressing, Marker

    RNAi-mediated depletion of REST increases GluN2B mRNA and alters NMDAR properties a , Diagram illustrating RNAi injection and time course of assays (qRT-PCR or patch recording). Lentiviral mediated RNAi directed against REST (RESTi) and nontargeting RNAi were delivered by means of sterotaxic injection into the hippocampus of rats at P10 or P24, and evaluated at indicated time points. b , Differential interference contrast (DIC) and GFP fluorescence images showing viral-mediated transduction of DGCs at P14 ( top ), and a patched DGC at P29 ( bottom ). Scale bars = 50 µm ( top panel ) and 20 µm ( bottom panel ). DGL, dentate granule cell layer; ML, molecular layer; R, recording pipette. c , RESTi delivered at P10 (RESTi-P10) effectively knocks down REST by P14 (n = 3), as assessed by qPCR. Delivery of RESTi at P10 and P24 (RESTi-P24) knocked-down REST measured at P28–32 (n = 3) in dentate granule cells (DGCs). d , RESTi injected at P10, but not P24, significantly increased GluN2B mRNA(n = 3) measured at P28–32. e , RESTi delivered at P10 does not exert any effect on GluN2A (n = 3) or GluN1 (n = 3) mRNA expression measured at P28–32. f , Representative DIC ( top left panel ) and fluorescence ( bottom left panel ) images showing simultaneous whole-cell recordings from RESTi-P10 expressing (GFP + ) and non-transduced control (GFP − ) DGCs. R1 and R2 indicate the recording pipettes from GFP − and GFP + neurons, respectively. Scale bars = 20 µm. Summary data ( middle panel ) and representative traces ( right panels ), showing that delivery of RESTi at P10 ( gray circles; 6 animals/13 cells ) increases NMDAR-mediated transmission relative to that of neighboring non-transduced (control) neurons ( white circles; 6 animals/13 cells ). g , Representative traces ( left ) and summary data ( right ) showing that the NMDAR/AMPAR ratio is higher in DGCs expressing REST RNAi delivered at P10 (6 animals/23 cells) but not at P24 (6 animals/18 cells) relative to non-transduced controls (6 animals/29 cells) or controls expressing nontargeting RNAi neurons (6 animals/18 cells). A second lentiviral mediated RNAi targeted to a different sequence in REST (RESTi*) also increased the NMDAR/AMPAR ratio (2 animals/7 cells). Summary data represent the mean ± s.e.m. *p
    Figure Legend Snippet: RNAi-mediated depletion of REST increases GluN2B mRNA and alters NMDAR properties a , Diagram illustrating RNAi injection and time course of assays (qRT-PCR or patch recording). Lentiviral mediated RNAi directed against REST (RESTi) and nontargeting RNAi were delivered by means of sterotaxic injection into the hippocampus of rats at P10 or P24, and evaluated at indicated time points. b , Differential interference contrast (DIC) and GFP fluorescence images showing viral-mediated transduction of DGCs at P14 ( top ), and a patched DGC at P29 ( bottom ). Scale bars = 50 µm ( top panel ) and 20 µm ( bottom panel ). DGL, dentate granule cell layer; ML, molecular layer; R, recording pipette. c , RESTi delivered at P10 (RESTi-P10) effectively knocks down REST by P14 (n = 3), as assessed by qPCR. Delivery of RESTi at P10 and P24 (RESTi-P24) knocked-down REST measured at P28–32 (n = 3) in dentate granule cells (DGCs). d , RESTi injected at P10, but not P24, significantly increased GluN2B mRNA(n = 3) measured at P28–32. e , RESTi delivered at P10 does not exert any effect on GluN2A (n = 3) or GluN1 (n = 3) mRNA expression measured at P28–32. f , Representative DIC ( top left panel ) and fluorescence ( bottom left panel ) images showing simultaneous whole-cell recordings from RESTi-P10 expressing (GFP + ) and non-transduced control (GFP − ) DGCs. R1 and R2 indicate the recording pipettes from GFP − and GFP + neurons, respectively. Scale bars = 20 µm. Summary data ( middle panel ) and representative traces ( right panels ), showing that delivery of RESTi at P10 ( gray circles; 6 animals/13 cells ) increases NMDAR-mediated transmission relative to that of neighboring non-transduced (control) neurons ( white circles; 6 animals/13 cells ). g , Representative traces ( left ) and summary data ( right ) showing that the NMDAR/AMPAR ratio is higher in DGCs expressing REST RNAi delivered at P10 (6 animals/23 cells) but not at P24 (6 animals/18 cells) relative to non-transduced controls (6 animals/29 cells) or controls expressing nontargeting RNAi neurons (6 animals/18 cells). A second lentiviral mediated RNAi targeted to a different sequence in REST (RESTi*) also increased the NMDAR/AMPAR ratio (2 animals/7 cells). Summary data represent the mean ± s.e.m. *p

    Techniques Used: Injection, Quantitative RT-PCR, Fluorescence, Transduction, Transferring, Real-time Polymerase Chain Reaction, Expressing, Transmission Assay, Sequencing

    REST increases transiently, is recruited to and coincides with epigenetic marks of repression at the grin2b promoter during rat hippocampal postnatal development a , Representative Western blots of whole hippocampal lysates showing that REST increases, GluN2B declines, and GluN2A increases during postnatal development (see full-length blot in Supplementary Fig. 1a ). b , Time course showing that REST protein increases transiently at P14–15 (n = 5). c , GluN2B mRNA exhibits a long-term decline during postnatal development, assessed by RT-qPCR. The decline was highly significant from P15 through P60 ( vs . P3; n = 5). d,e , Time course showing that whereas GluN2B protein declines after P21 (n = 6), GluN2A protein increases markedly from P8 to P16 and remains high up to P60 (n = 3). Data were normalized to corresponding values at P3. f , GluN1 mRNA is not altered during postnatal development (n = 6). g , Representative Western blot ( left panel ) and summary data ( right panel ) show REST protein expression in the nuclear fraction of the hippocampal cell body layer, which is enriched for neurons. Note that REST abundance in the neuronal nuclear fraction increases strikingly by P14–15 (n = 4). Data were normalized to corresponding data at P9. See full-length blot in Supplementary Fig. 12 ). h , Map of the rat grin2b gene indicating location of RE1 motifs contained within the proximal (PR1; gray box ) and distal (PR2; white box ) regions of the grin2b promoter probed by chromatin immunoprecipitation (ChIP). i,j , REST occupancy is markedly enriched at the grin2b proximal (PR1, grey bars ) (n = 6) and distal PR2 ( white bars ) (n = 3) promoters at P15 but declines by P60. k,l , REST is not enriched at RE1 sites within the grin2a (n = 9), nor grin1 (n = 6) promoters. Inset, same data depicted with expanded y-axis. m,n , CoREST (n = 3) and G9a (n = 3) are enriched at PR1 by P15. o,p , Increase in H3K9me3 (n = 3) and H3K27me3 (n = 6) (marks of repression) at P15. q , Decrease in trimethylation of H3K4 (n = 3) (mark of active transcription), at PR1. r , MeCP2 occupancy is enriched at grin2b PR1 by P15 with a sharp increase by P60 (n = 3). All samples were normalized to input and to corresponding values at P3. Summary data represent the mean ± s.e.m. *p
    Figure Legend Snippet: REST increases transiently, is recruited to and coincides with epigenetic marks of repression at the grin2b promoter during rat hippocampal postnatal development a , Representative Western blots of whole hippocampal lysates showing that REST increases, GluN2B declines, and GluN2A increases during postnatal development (see full-length blot in Supplementary Fig. 1a ). b , Time course showing that REST protein increases transiently at P14–15 (n = 5). c , GluN2B mRNA exhibits a long-term decline during postnatal development, assessed by RT-qPCR. The decline was highly significant from P15 through P60 ( vs . P3; n = 5). d,e , Time course showing that whereas GluN2B protein declines after P21 (n = 6), GluN2A protein increases markedly from P8 to P16 and remains high up to P60 (n = 3). Data were normalized to corresponding values at P3. f , GluN1 mRNA is not altered during postnatal development (n = 6). g , Representative Western blot ( left panel ) and summary data ( right panel ) show REST protein expression in the nuclear fraction of the hippocampal cell body layer, which is enriched for neurons. Note that REST abundance in the neuronal nuclear fraction increases strikingly by P14–15 (n = 4). Data were normalized to corresponding data at P9. See full-length blot in Supplementary Fig. 12 ). h , Map of the rat grin2b gene indicating location of RE1 motifs contained within the proximal (PR1; gray box ) and distal (PR2; white box ) regions of the grin2b promoter probed by chromatin immunoprecipitation (ChIP). i,j , REST occupancy is markedly enriched at the grin2b proximal (PR1, grey bars ) (n = 6) and distal PR2 ( white bars ) (n = 3) promoters at P15 but declines by P60. k,l , REST is not enriched at RE1 sites within the grin2a (n = 9), nor grin1 (n = 6) promoters. Inset, same data depicted with expanded y-axis. m,n , CoREST (n = 3) and G9a (n = 3) are enriched at PR1 by P15. o,p , Increase in H3K9me3 (n = 3) and H3K27me3 (n = 6) (marks of repression) at P15. q , Decrease in trimethylation of H3K4 (n = 3) (mark of active transcription), at PR1. r , MeCP2 occupancy is enriched at grin2b PR1 by P15 with a sharp increase by P60 (n = 3). All samples were normalized to input and to corresponding values at P3. Summary data represent the mean ± s.e.m. *p

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

    39) Product Images from "Distinct microRNA-155 expression in the vitreous of patients with primary vitreoretinal lymphoma and uveitis"

    Article Title: Distinct microRNA-155 expression in the vitreous of patients with primary vitreoretinal lymphoma and uveitis

    Journal: American journal of ophthalmology

    doi: 10.1016/j.ajo.2013.12.014

    The Pair Plot of 110 Detectable micro Ribonucleic Acids in Vitreous Samples from Primary Vitreoretinal Lymphoma and Uveitis. The data are the average of 3 biological samples from each condition and normalized against microRNA-16. The level of the majority
    Figure Legend Snippet: The Pair Plot of 110 Detectable micro Ribonucleic Acids in Vitreous Samples from Primary Vitreoretinal Lymphoma and Uveitis. The data are the average of 3 biological samples from each condition and normalized against microRNA-16. The level of the majority

    Techniques Used:

    40) Product Images from "MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer"

    Article Title: MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071480

    Differential expression of laryngeal cancer-associated miRNAs between matched paired samples. Relative expressions of laryngeal cancer-associated miRNAs in 5 paired samples were measured by TaqMan® qRT-PCR analysis and shown in left panels . Expression levels in adjacent noncancerous counterparts (NCs) in each pair are set to be 1 in right panels for clear visualization of significant fold difference in each miRNA. Data are expressed as mean values with standard deviations. (A) Up-regulation of 2 miRNAs (miR-196a and miR-455-5p) was confirmed in cancers when 5 cancers were compared with their NCs. *, p
    Figure Legend Snippet: Differential expression of laryngeal cancer-associated miRNAs between matched paired samples. Relative expressions of laryngeal cancer-associated miRNAs in 5 paired samples were measured by TaqMan® qRT-PCR analysis and shown in left panels . Expression levels in adjacent noncancerous counterparts (NCs) in each pair are set to be 1 in right panels for clear visualization of significant fold difference in each miRNA. Data are expressed as mean values with standard deviations. (A) Up-regulation of 2 miRNAs (miR-196a and miR-455-5p) was confirmed in cancers when 5 cancers were compared with their NCs. *, p

    Techniques Used: Expressing, Quantitative RT-PCR

    41) Product Images from "Overexpression of E3 ubiquitin ligase tripartite motif 32 correlates with a poor prognosis in patients with gastric cancer"

    Article Title: Overexpression of E3 ubiquitin ligase tripartite motif 32 correlates with a poor prognosis in patients with gastric cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.5806

    Differential expression of TRIM32 in gastric cancer tissue specimens. Immunohistochemistry staining of TRIM32 protein in tissue specimens with (A) non-reactive (0), (B) weak positive and (C) strong positive staining. Representative images of stain in
    Figure Legend Snippet: Differential expression of TRIM32 in gastric cancer tissue specimens. Immunohistochemistry staining of TRIM32 protein in tissue specimens with (A) non-reactive (0), (B) weak positive and (C) strong positive staining. Representative images of stain in

    Techniques Used: Expressing, Immunohistochemistry, Staining

    TRIM32 mRNA expression levels in gastric cancer tissues, as detected by reverse transcription-quantitative polymerase chain reaction. (A) Relative TRIM32 expression levels of cancer tissue specimens were significantly increased compared with that of non-cancerous
    Figure Legend Snippet: TRIM32 mRNA expression levels in gastric cancer tissues, as detected by reverse transcription-quantitative polymerase chain reaction. (A) Relative TRIM32 expression levels of cancer tissue specimens were significantly increased compared with that of non-cancerous

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Prognostic value of TRIM32 expression levels in gastric cancer
    Figure Legend Snippet: Prognostic value of TRIM32 expression levels in gastric cancer

    Techniques Used: Expressing

    Downregulation of TRIM32 using siRNA inhibits the proliferation and increases the apoptosis of human gastric cancer cells. (A) MKN45 and MKN74 gastric cancer cells were transfected with control or TRIM32 siRNA. The expression levels of TRIM32 mRNA were
    Figure Legend Snippet: Downregulation of TRIM32 using siRNA inhibits the proliferation and increases the apoptosis of human gastric cancer cells. (A) MKN45 and MKN74 gastric cancer cells were transfected with control or TRIM32 siRNA. The expression levels of TRIM32 mRNA were

    Techniques Used: Transfection, Expressing

    Clinicopathological significance of TRIM32 expression levels in human gastric cancer
    Figure Legend Snippet: Clinicopathological significance of TRIM32 expression levels in human gastric cancer

    Techniques Used: Expressing

    Postoperative survival rate was significantly reduced in patients with TRIM32-high tumors compared with those with TRIM32-low tumors. (A) Overall survival rate of patients with gastric cancer (P=0.008). (B) Relapse-free survival rate of patients with
    Figure Legend Snippet: Postoperative survival rate was significantly reduced in patients with TRIM32-high tumors compared with those with TRIM32-low tumors. (A) Overall survival rate of patients with gastric cancer (P=0.008). (B) Relapse-free survival rate of patients with

    Techniques Used:

    Influence of the TRIM32 expression levels on postoperative recurrence
    Figure Legend Snippet: Influence of the TRIM32 expression levels on postoperative recurrence

    Techniques Used: Expressing

    42) Product Images from "HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells"

    Article Title: HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053974

    HGF induced the migration of human chondrosarcoma cells. (A) Immunohistochemistry showing HGF expression in normal cartilage and chondrosarcoma tissues (arrow shows HGF staining). Quantitative data are shown in the lower panel. (B) Quantitative PCR analysis for HGF mRNA expression in normal cartilage, normal bone, and chondrosarcoma tissues. (C and D) Cells were incubated with HGF (3–30 ng/mL), and in vitro migration and invasion was measured by Transwell assay after 24 h. (E) JJ012 cells were treated with HGF for 24 h, after which the wound-scratching assay was performed. Results are expressed as mean ± S.E. * p
    Figure Legend Snippet: HGF induced the migration of human chondrosarcoma cells. (A) Immunohistochemistry showing HGF expression in normal cartilage and chondrosarcoma tissues (arrow shows HGF staining). Quantitative data are shown in the lower panel. (B) Quantitative PCR analysis for HGF mRNA expression in normal cartilage, normal bone, and chondrosarcoma tissues. (C and D) Cells were incubated with HGF (3–30 ng/mL), and in vitro migration and invasion was measured by Transwell assay after 24 h. (E) JJ012 cells were treated with HGF for 24 h, after which the wound-scratching assay was performed. Results are expressed as mean ± S.E. * p

    Techniques Used: Migration, Immunohistochemistry, Expressing, Staining, Real-time Polymerase Chain Reaction, Incubation, In Vitro, Transwell Assay

    43) Product Images from "Selaginellatamariscina Attenuates Metastasis via Akt Pathways in Oral Cancer Cells"

    Article Title: Selaginellatamariscina Attenuates Metastasis via Akt Pathways in Oral Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068035

    Critical role of transcription factor in STE-induced transcriptional inhibition of MMP-2 and MMP-9 in HSC-3 cells.
    Figure Legend Snippet: Critical role of transcription factor in STE-induced transcriptional inhibition of MMP-2 and MMP-9 in HSC-3 cells.

    Techniques Used: Inhibition

    Effects of STE on the activity and protein level of MMP-2, MMP-9 and the protein level of the endogenous inhibitor TIMP-2 and TIMP-1. (A and B) HSC-3 cells were treated with STE (0-100 µg/mL) for 24 h and then subjected to gelatin zymography to analyze the activity of MMP-2 and MMP-9, respectively. (C) HSC-3 cells were treated with STE (0-100 µg/mL) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2. (D) Quantitative results of MMP-2, MMP-9, TIMP-1 and TIMP-2 protein levels which were adjusted with β-actin protein level. The values represented the means ± SD of at least three independent experiments. *p
    Figure Legend Snippet: Effects of STE on the activity and protein level of MMP-2, MMP-9 and the protein level of the endogenous inhibitor TIMP-2 and TIMP-1. (A and B) HSC-3 cells were treated with STE (0-100 µg/mL) for 24 h and then subjected to gelatin zymography to analyze the activity of MMP-2 and MMP-9, respectively. (C) HSC-3 cells were treated with STE (0-100 µg/mL) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2. (D) Quantitative results of MMP-2, MMP-9, TIMP-1 and TIMP-2 protein levels which were adjusted with β-actin protein level. The values represented the means ± SD of at least three independent experiments. *p

    Techniques Used: Activity Assay, Zymography, Western Blot

    STE suppresses MMP-2 and MMP-9 expression at a transcriptional level.
    Figure Legend Snippet: STE suppresses MMP-2 and MMP-9 expression at a transcriptional level.

    Techniques Used: Expressing

    44) Product Images from "Dysregulation of Mir-196b in Head and Neck Cancers Leads to Pleiotropic Effects in the Tumor Cells and Surrounding Stromal Fibroblasts"

    Article Title: Dysregulation of Mir-196b in Head and Neck Cancers Leads to Pleiotropic Effects in the Tumor Cells and Surrounding Stromal Fibroblasts

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18138-8

    Functional analysis of miR-196a and miR-196b in HNSCC-derived cell lines and CAFs. UT-SCC-42B, FaDu cells and CAFs were transfected with either premiR-196a, premiR-196b or non-targeting control, as indicated. ( A ) Cell proliferation was estimated by Tetrazolium-based MTS assay after 4 days. Data were normalized to the absorbance at day 0 and relative to control-transfected cells. ( B ) Bar chart showing the relative migration of UT-SCC-42B, FaDu or CAFs transfected with premiR-196a, premiR-196b or non-targeting control. ( C ) Representative images from the wound healing assays showing the initial scratch (t = 0) area and the residual area at each established final time (5, 15 or 20 h for UT-SCC-42B, FaDu or CAFs, respectively). ( D ) Bar chart showing the relative invasion of UT-SCC-42B, FaDu or CAFs transfected with premiR-196a, premiR-196b or non-targeting control. ( E ) Representative images from the 3D invasion assays of CAFs spheroids embedded into a collagen matrix. The invasive area was determined by calculating the difference between the final area (t = 20 h) and the initial area (t = 0) using Image J analysis program. All data were normalized to control-transfected cells and expressed as the mean ± SD of at least three independent experiments performed in quadruplicate. * p
    Figure Legend Snippet: Functional analysis of miR-196a and miR-196b in HNSCC-derived cell lines and CAFs. UT-SCC-42B, FaDu cells and CAFs were transfected with either premiR-196a, premiR-196b or non-targeting control, as indicated. ( A ) Cell proliferation was estimated by Tetrazolium-based MTS assay after 4 days. Data were normalized to the absorbance at day 0 and relative to control-transfected cells. ( B ) Bar chart showing the relative migration of UT-SCC-42B, FaDu or CAFs transfected with premiR-196a, premiR-196b or non-targeting control. ( C ) Representative images from the wound healing assays showing the initial scratch (t = 0) area and the residual area at each established final time (5, 15 or 20 h for UT-SCC-42B, FaDu or CAFs, respectively). ( D ) Bar chart showing the relative invasion of UT-SCC-42B, FaDu or CAFs transfected with premiR-196a, premiR-196b or non-targeting control. ( E ) Representative images from the 3D invasion assays of CAFs spheroids embedded into a collagen matrix. The invasive area was determined by calculating the difference between the final area (t = 20 h) and the initial area (t = 0) using Image J analysis program. All data were normalized to control-transfected cells and expressed as the mean ± SD of at least three independent experiments performed in quadruplicate. * p

    Techniques Used: Functional Assay, Derivative Assay, Transfection, MTS Assay, Migration

    Analysis of miR-196b expression in early stages of HNSCC tumorigenesis. miR-196b expression levels were quantified by RT-qPCR in 40 patients with laryngeal precancerous lesions, comprising 17 non-progressing dysplasias (NP), 23 progressing dysplasias (P) and the 23 patient-matched invasive tumors (T) subsequently developed. Normal epithelia from 5 non-oncologic patients were included as (N) healthy controls. *** p
    Figure Legend Snippet: Analysis of miR-196b expression in early stages of HNSCC tumorigenesis. miR-196b expression levels were quantified by RT-qPCR in 40 patients with laryngeal precancerous lesions, comprising 17 non-progressing dysplasias (NP), 23 progressing dysplasias (P) and the 23 patient-matched invasive tumors (T) subsequently developed. Normal epithelia from 5 non-oncologic patients were included as (N) healthy controls. *** p

    Techniques Used: Expressing, Quantitative RT-PCR

    Analysis of miR-196a and miR-196b expression in saliva samples. (A ) miR-196a and ( B ) miR-196b expression levels were quantified by RT-qPCR in saliva samples from 15 HNSCC patients and 11 healthy controls and normalized to RNU48 levels with p values calculated by Student t-test.
    Figure Legend Snippet: Analysis of miR-196a and miR-196b expression in saliva samples. (A ) miR-196a and ( B ) miR-196b expression levels were quantified by RT-qPCR in saliva samples from 15 HNSCC patients and 11 healthy controls and normalized to RNU48 levels with p values calculated by Student t-test.

    Techniques Used: Expressing, Quantitative RT-PCR

    Analysis of miR-196a and miR-196b target genes in HNSCC cells and CAFs. mRNA expression levels of potential/validated miR-196a/b target genes were measured by RT-qPCR in ( A ) UT-SCC-42B, (B ) FaDu and ( C ) CAFs after 72 h transfection with either premiR-196a, premiR-196b or non-targeting control. Data were normalized to RPL19 levels and relative to control-transfected cells. The graphs represent the mean ± SD, calculated from at least three independent experiments performed in triplicate. ND, not detected. *p
    Figure Legend Snippet: Analysis of miR-196a and miR-196b target genes in HNSCC cells and CAFs. mRNA expression levels of potential/validated miR-196a/b target genes were measured by RT-qPCR in ( A ) UT-SCC-42B, (B ) FaDu and ( C ) CAFs after 72 h transfection with either premiR-196a, premiR-196b or non-targeting control. Data were normalized to RPL19 levels and relative to control-transfected cells. The graphs represent the mean ± SD, calculated from at least three independent experiments performed in triplicate. ND, not detected. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection

    Analysis of miR-196b expression in HNSCC tissue specimens. miR-196b expression levels were quantified by RT-qPCR in 19 fresh primary tumors (T) and patient-matched normal counterparts (NC). Data were normalized to RNU48 levels, and relative to the normal mucosa from non-oncologic patients (N).
    Figure Legend Snippet: Analysis of miR-196b expression in HNSCC tissue specimens. miR-196b expression levels were quantified by RT-qPCR in 19 fresh primary tumors (T) and patient-matched normal counterparts (NC). Data were normalized to RNU48 levels, and relative to the normal mucosa from non-oncologic patients (N).

    Techniques Used: Expressing, Quantitative RT-PCR

    Analysis of signaling pathways targeted by miR-196a and miR-196b. Total protein lysates from UT-SCC-42B and FaDu cells transfected with either premiR-196a, premiR-196b or non-targeting control were applied to a proteome phospho-kinase array. ( A ) Relative signal intensity values (to control-transfected cells) are displayed as a heat map. ( B ) Western blot validation of the array data. ( C ) and ( D ) Quantification of IRDye fluorescent signals. *p
    Figure Legend Snippet: Analysis of signaling pathways targeted by miR-196a and miR-196b. Total protein lysates from UT-SCC-42B and FaDu cells transfected with either premiR-196a, premiR-196b or non-targeting control were applied to a proteome phospho-kinase array. ( A ) Relative signal intensity values (to control-transfected cells) are displayed as a heat map. ( B ) Western blot validation of the array data. ( C ) and ( D ) Quantification of IRDye fluorescent signals. *p

    Techniques Used: Transfection, Western Blot

    45) Product Images from "Pathogenesis of Plasmodium berghei ANKA infection in the gerbil (Meriones unguiculatus) as an experimental model for severe malaria"

    Article Title: Pathogenesis of Plasmodium berghei ANKA infection in the gerbil (Meriones unguiculatus) as an experimental model for severe malaria

    Journal: Parasite

    doi: 10.1051/parasite/2017040

    Quantitation of cytokine mRNA in the spleen, brain and blood. Gerbils were euthanized under anesthesia at days 1, 3, 5, 7, 9 and 11 after intraperitoneal inoculation with PbA. mRNA levels were measured by reverse transcription (RT)-PCR, and values were expressed as mean (±S.E.M., N = 5) fold changes compared with values from control, uninfected gerbils. a. IL-4 mRNA expression. b. IL-10 mRNA expression. c. IL-6 mRNA expression. d. IFN mRNA expression. e. TNF mRNA expression. All data are representative of two independent experiments and compared by one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-hoc tests for differences between groups.
    Figure Legend Snippet: Quantitation of cytokine mRNA in the spleen, brain and blood. Gerbils were euthanized under anesthesia at days 1, 3, 5, 7, 9 and 11 after intraperitoneal inoculation with PbA. mRNA levels were measured by reverse transcription (RT)-PCR, and values were expressed as mean (±S.E.M., N = 5) fold changes compared with values from control, uninfected gerbils. a. IL-4 mRNA expression. b. IL-10 mRNA expression. c. IL-6 mRNA expression. d. IFN mRNA expression. e. TNF mRNA expression. All data are representative of two independent experiments and compared by one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-hoc tests for differences between groups.

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

    46) Product Images from "Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes"

    Article Title: Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes

    Journal: Prostate Cancer

    doi: 10.1155/2017/4893921

    Quantification of mRNA expression in primary normal prostate (PNF-08) cells and the DU145 and LNCaP PCa cell lines. The expression of ten mRNAs that were assumed to be elevated or reduced according to their presence or absence in the Ago complex was assessed by qRT-PCR. YWHAE, TRFC, CORO1C, and PRDX3 were predicted to be elevated while, NLK, RAB1B, DEDD, CUL5, EI24, and S100A16 were predicted to be reduced in PCa cells as compared to PNF-08 cells.
    Figure Legend Snippet: Quantification of mRNA expression in primary normal prostate (PNF-08) cells and the DU145 and LNCaP PCa cell lines. The expression of ten mRNAs that were assumed to be elevated or reduced according to their presence or absence in the Ago complex was assessed by qRT-PCR. YWHAE, TRFC, CORO1C, and PRDX3 were predicted to be elevated while, NLK, RAB1B, DEDD, CUL5, EI24, and S100A16 were predicted to be reduced in PCa cells as compared to PNF-08 cells.

    Techniques Used: Expressing, Quantitative RT-PCR

    47) Product Images from "Notch3 Signaling-Mediated Melanoma-Endothelial Crosstalk Regulates Melanoma Stem-Like Cell Homeostasis and Niche Morphogenesis"

    Article Title: Notch3 Signaling-Mediated Melanoma-Endothelial Crosstalk Regulates Melanoma Stem-Like Cell Homeostasis and Niche Morphogenesis

    Journal: Laboratory investigation; a journal of technical methods and pathology

    doi: 10.1038/labinvest.2017.1

    Notch3 up-regulation in co-cultured melanoma cells and ECs in the 2D niche model. Flow cytometry segregated melanoma cells and ECs from the 2D niche model were subjected to Stem cell and Notch signaling pathway-specific PCR arrays (SA Biosciences) using their mono-culture counterparts as control. Notch3 was consistently up-regulated both in co-cultured melanoma cells and ECs. The concomitant up-regulation of Notch downstream effectors, Hes1 and Hey1, further confirmed activated Notch signaling in co-cultured melanoma cells. B. qRT-PCR validated Notch3 induction in co-cultured melanoma cells following sorting by flow cytometry. Data was expressed as fold change of the ratio between Notch3 and GAPDH mRNA in co-cultured melanoma cells, normalized to that of their mono-culture counterpart. C. The magnitude of Notch3 induction in co-cultured melanoma cells was most evident when direct cell-cell contact is allowed, while EC ECM or soluble factors in transwell co-cultures produced only limited/partial result. *, P
    Figure Legend Snippet: Notch3 up-regulation in co-cultured melanoma cells and ECs in the 2D niche model. Flow cytometry segregated melanoma cells and ECs from the 2D niche model were subjected to Stem cell and Notch signaling pathway-specific PCR arrays (SA Biosciences) using their mono-culture counterparts as control. Notch3 was consistently up-regulated both in co-cultured melanoma cells and ECs. The concomitant up-regulation of Notch downstream effectors, Hes1 and Hey1, further confirmed activated Notch signaling in co-cultured melanoma cells. B. qRT-PCR validated Notch3 induction in co-cultured melanoma cells following sorting by flow cytometry. Data was expressed as fold change of the ratio between Notch3 and GAPDH mRNA in co-cultured melanoma cells, normalized to that of their mono-culture counterpart. C. The magnitude of Notch3 induction in co-cultured melanoma cells was most evident when direct cell-cell contact is allowed, while EC ECM or soluble factors in transwell co-cultures produced only limited/partial result. *, P

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Quantitative RT-PCR, Produced

    Effective and functional Notch3 KD depletes MSLCs and VM-engaging melanoma cells in 1205 Lu xenografts. A. Western blot analyses demonstrated that stem cell markers (e.g., CD133 and CD271) as well as VM maker, CD144, were down-regulated in Notch3 KD 1205 Lu tumors, compared to non-target control (A). Consistent with the observed VM inhibition by Notch3 KD above, the expression of Tie-1, a VM-associated gene, was also down-regulated in Nocth3 KD 1205Lu xenografts by qRT-PCR (B). Of note, the efficacy of Notch3 KD was maintained in the tumor xenografts as shown by Western blotting (A). On the other hand, in A375 melanoma cells where no change in tumorigenicity was appreciated following Notch3 KD, the expression of stem-like cell marker, CD271, was not affected (C). Attempts to further validate CD133 and CD144 in Notch3 KD A375 xenografts using Western blot analysis (C8161 lysate included as a positive control) were unsuccessful due to sensitivity issue ( Fig. 6C ; note that CD133 and CD144 signals were undetectable both in the control and KD xenografts despite maximal loading and prolonged overnight exposure). *, P
    Figure Legend Snippet: Effective and functional Notch3 KD depletes MSLCs and VM-engaging melanoma cells in 1205 Lu xenografts. A. Western blot analyses demonstrated that stem cell markers (e.g., CD133 and CD271) as well as VM maker, CD144, were down-regulated in Notch3 KD 1205 Lu tumors, compared to non-target control (A). Consistent with the observed VM inhibition by Notch3 KD above, the expression of Tie-1, a VM-associated gene, was also down-regulated in Nocth3 KD 1205Lu xenografts by qRT-PCR (B). Of note, the efficacy of Notch3 KD was maintained in the tumor xenografts as shown by Western blotting (A). On the other hand, in A375 melanoma cells where no change in tumorigenicity was appreciated following Notch3 KD, the expression of stem-like cell marker, CD271, was not affected (C). Attempts to further validate CD133 and CD144 in Notch3 KD A375 xenografts using Western blot analysis (C8161 lysate included as a positive control) were unsuccessful due to sensitivity issue ( Fig. 6C ; note that CD133 and CD144 signals were undetectable both in the control and KD xenografts despite maximal loading and prolonged overnight exposure). *, P

    Techniques Used: Functional Assay, Western Blot, Inhibition, Expressing, Quantitative RT-PCR, Marker, Positive Control

    Two dimensional (2D) melanoma-EC co-culture model recapitulates MSLC niche in vitro . A. Schematic representation of the 2D MSLC niche model in vitro . GFP-labeled CD133 − non-MSLCs were co-cultured with RFP-labeled human umbilical vein endothelial cells (HUVECs) at 1:1 or 1:4 ratios for 5 days. B. In this model, ECs aligned to form branching tubular networks, reminiscent of the vascular niche in vivo (Magnification, ×100; scale bar, 200 μm). Co-cultured melanoma cells were then segregated from ECs by flow cytometry. C. MSLC (e.g., CD133 and CD271) and VM (e.g., CD144) markers were up-regulated in co-cultured melanoma cells compared to their mono-culture counter parts using qRT-PCR, simulating “dynamic stemness” and VM morphogenesis in vitro . D. Such a niche-inducing phenomenon was most pronounced when melanoma-EC contact is permitted, while EC extracellular matrix (ECM) or soluble factors in transwell cultures alone exhibited limited/partial effects. *, P
    Figure Legend Snippet: Two dimensional (2D) melanoma-EC co-culture model recapitulates MSLC niche in vitro . A. Schematic representation of the 2D MSLC niche model in vitro . GFP-labeled CD133 − non-MSLCs were co-cultured with RFP-labeled human umbilical vein endothelial cells (HUVECs) at 1:1 or 1:4 ratios for 5 days. B. In this model, ECs aligned to form branching tubular networks, reminiscent of the vascular niche in vivo (Magnification, ×100; scale bar, 200 μm). Co-cultured melanoma cells were then segregated from ECs by flow cytometry. C. MSLC (e.g., CD133 and CD271) and VM (e.g., CD144) markers were up-regulated in co-cultured melanoma cells compared to their mono-culture counter parts using qRT-PCR, simulating “dynamic stemness” and VM morphogenesis in vitro . D. Such a niche-inducing phenomenon was most pronounced when melanoma-EC contact is permitted, while EC extracellular matrix (ECM) or soluble factors in transwell cultures alone exhibited limited/partial effects. *, P

    Techniques Used: Co-Culture Assay, In Vitro, Labeling, Cell Culture, In Vivo, Flow Cytometry, Cytometry, Quantitative RT-PCR

    48) Product Images from "Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b"

    Article Title: Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07169-w

    miR-196a and miR-196b specifically targeted ANXA1 expression in HNSCC-derived cell lines. miR-196a and miR-196b expression levels were quantified by RT-qPCR in SCC42B ( A ) and FaDu ( B ) cells transfected with either premiR-196a, premiR-196b or non-targeting control. Data were normalized to RNU48 levels and relative to control-transfected cells. mRNA expression levels of ANXA1 and ANXA2 were measured by qRT-PCR in SCC42B ( C ), and FaDu ( D ) after 72 h transfection with either premiR-196a, premiR-196b or non-targeting control. Data were normalized to RPL19 levels and relative to control-transfected cells. The graphs represent the mean ± SD, calculated from at least three independent experiments performed in triplicate. ANXA1 protein expression was analyzed by Western blot in SCC42B ( E ) and FaDu ( F ) cells after 72 h transfection with either premiR-196a, premiR-196b or non-targeting control. The graphs represent the mean ± SD, calculated from at least three independent experiments. *p
    Figure Legend Snippet: miR-196a and miR-196b specifically targeted ANXA1 expression in HNSCC-derived cell lines. miR-196a and miR-196b expression levels were quantified by RT-qPCR in SCC42B ( A ) and FaDu ( B ) cells transfected with either premiR-196a, premiR-196b or non-targeting control. Data were normalized to RNU48 levels and relative to control-transfected cells. mRNA expression levels of ANXA1 and ANXA2 were measured by qRT-PCR in SCC42B ( C ), and FaDu ( D ) after 72 h transfection with either premiR-196a, premiR-196b or non-targeting control. Data were normalized to RPL19 levels and relative to control-transfected cells. The graphs represent the mean ± SD, calculated from at least three independent experiments performed in triplicate. ANXA1 protein expression was analyzed by Western blot in SCC42B ( E ) and FaDu ( F ) cells after 72 h transfection with either premiR-196a, premiR-196b or non-targeting control. The graphs represent the mean ± SD, calculated from at least three independent experiments. *p

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

    Analysis of miR-196a and miR-196b expression and ANXA1 mRNA levels in HNSCC tissue specimens. ( A ) miR-196a and ( B ) miR-196b expression levels were quantified by RT-qPCR in 11 fresh primary tumors (T) and patient-matched normal epithelia (N). Data were normalized to RNU48 levels, and relative to the normal mucosa from non-oncologic patients. ( C ) ANXA1 mRNA levels were quantified by RT-qPCR and data normalized to RPL19 levels. p -values obtained from the Wilcoxon test provided statistical confirmation of the significant changes.
    Figure Legend Snippet: Analysis of miR-196a and miR-196b expression and ANXA1 mRNA levels in HNSCC tissue specimens. ( A ) miR-196a and ( B ) miR-196b expression levels were quantified by RT-qPCR in 11 fresh primary tumors (T) and patient-matched normal epithelia (N). Data were normalized to RNU48 levels, and relative to the normal mucosa from non-oncologic patients. ( C ) ANXA1 mRNA levels were quantified by RT-qPCR and data normalized to RPL19 levels. p -values obtained from the Wilcoxon test provided statistical confirmation of the significant changes.

    Techniques Used: Expressing, Quantitative RT-PCR

    ANXA1 is directly targeted by both miR-196a and miR-196b in HNSCC cells. The constructs psi-CHECK-2-ANXA1–3′UTR-wt ( A ) and psi-CHECK-2-ANXA1–3′UTR-mut ( B ) were co-transfected with the indicated specific premiR precursors and miRNA inhibitors into SCC38, SCC42B, and FaDu cells. Renilla luciferase activity under the control of ANXA1 3′-UTR was measured and normalized to firefly luciferase, relative to control-transfected cells. The graphs represent the mean percentage ± SD, calculated from at least three independent experiments performed in quadruplicate. *p
    Figure Legend Snippet: ANXA1 is directly targeted by both miR-196a and miR-196b in HNSCC cells. The constructs psi-CHECK-2-ANXA1–3′UTR-wt ( A ) and psi-CHECK-2-ANXA1–3′UTR-mut ( B ) were co-transfected with the indicated specific premiR precursors and miRNA inhibitors into SCC38, SCC42B, and FaDu cells. Renilla luciferase activity under the control of ANXA1 3′-UTR was measured and normalized to firefly luciferase, relative to control-transfected cells. The graphs represent the mean percentage ± SD, calculated from at least three independent experiments performed in quadruplicate. *p

    Techniques Used: Construct, Transfection, Luciferase, Activity Assay

    49) Product Images from "Identification of microRNA signature in the progression of gestational trophoblastic disease"

    Article Title: Identification of microRNA signature in the progression of gestational trophoblastic disease

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0108-2

    Validation of miR-371a-5p potential target genes BCCIP , BNIP3L , and SOX2 a qRT-PCR quantification of the mRNA levels of BCCIP , BNIP3L , and SOX2 genes. BeWo, JAR, and JEG-3 cells were transfected with miR-371a-5p mimic/inhibitor or control mimic/inhibitor. Cells were harvested at 36 h post transfection, and α-tubulin was used as the endogenous reference gene. b Western blotting analysis of BCCIP, BNIP3L, and SOX2. Cells were harvested at 48 h post transfection, and α-tubulin was used as the endogenous control. c Direct interaction of miR-371a-5p and BCCIP . Upper: bioinformatic predicted miR-371a-5p targeting site in 3′-UTR of BCCIP . Lower: luciferase activity assay. At 48 h post co-transfection with BCCIP–WT or BCCIP–MUT and miR-371a-5p mimic/inhibitor or control mimic/inhibitor, luciferase activities were measured in HTR-8/SVneo cells. Renilla luciferase activities in corresponding controls were normalized to 1. Mck stands for control mimic and ick stands for control inhibitor. N = 9, * P
    Figure Legend Snippet: Validation of miR-371a-5p potential target genes BCCIP , BNIP3L , and SOX2 a qRT-PCR quantification of the mRNA levels of BCCIP , BNIP3L , and SOX2 genes. BeWo, JAR, and JEG-3 cells were transfected with miR-371a-5p mimic/inhibitor or control mimic/inhibitor. Cells were harvested at 36 h post transfection, and α-tubulin was used as the endogenous reference gene. b Western blotting analysis of BCCIP, BNIP3L, and SOX2. Cells were harvested at 48 h post transfection, and α-tubulin was used as the endogenous control. c Direct interaction of miR-371a-5p and BCCIP . Upper: bioinformatic predicted miR-371a-5p targeting site in 3′-UTR of BCCIP . Lower: luciferase activity assay. At 48 h post co-transfection with BCCIP–WT or BCCIP–MUT and miR-371a-5p mimic/inhibitor or control mimic/inhibitor, luciferase activities were measured in HTR-8/SVneo cells. Renilla luciferase activities in corresponding controls were normalized to 1. Mck stands for control mimic and ick stands for control inhibitor. N = 9, * P

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

    Validation of miR-518a-3p potential target genes MST1 and EFNA4 a qRT-PCR quantification of the mRNA level of MST1 and EFNA4 genes. BeWo, JAR, and JEG-3 cells were transfected with miR-518a-3p mimic/inhibitor or controls. Cells were harvested at 36 h post transfection, and α-tubulin served as the endogenous reference gene. b Western blotting analysis of MST1 and EFNA4. Cells were harvested at 48 h post transfection, and α-tubulin was taken as the endogenous control. c Direct interaction of miR-518a-3p and MST1 . Upper: bioinformatic predicted miR-518a-3p targeting site in 3′-UTR of MST1 . Lower: luciferase activity assay. At 48 h post co-transfection with MST1-WT or MST1-MUT and miR-518a-3p mimic/inhibitor or control mimic/inhibitor, luciferase activities were measured in HTR-8/SVneo cells. Renilla luciferase activities in corresponding controls were normalized to 1. Mck stands for control mimic and ick stands for control inhibitor. N = 9, ** P
    Figure Legend Snippet: Validation of miR-518a-3p potential target genes MST1 and EFNA4 a qRT-PCR quantification of the mRNA level of MST1 and EFNA4 genes. BeWo, JAR, and JEG-3 cells were transfected with miR-518a-3p mimic/inhibitor or controls. Cells were harvested at 36 h post transfection, and α-tubulin served as the endogenous reference gene. b Western blotting analysis of MST1 and EFNA4. Cells were harvested at 48 h post transfection, and α-tubulin was taken as the endogenous control. c Direct interaction of miR-518a-3p and MST1 . Upper: bioinformatic predicted miR-518a-3p targeting site in 3′-UTR of MST1 . Lower: luciferase activity assay. At 48 h post co-transfection with MST1-WT or MST1-MUT and miR-518a-3p mimic/inhibitor or control mimic/inhibitor, luciferase activities were measured in HTR-8/SVneo cells. Renilla luciferase activities in corresponding controls were normalized to 1. Mck stands for control mimic and ick stands for control inhibitor. N = 9, ** P

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

    MiR-371a-5p and miR-518a-3p were upregulated in GTN tissues and choriocarcinoma cells a Validation of the microarray profiling data. Expression levels of the most differentially expressed 16 miRNAs were examined by qRT-PCR in 34 cases of GTN and 60 cases of CHM trophoblastic tissues and then normalized to the housekeeping gene RNU6 with the 2 −ΔΔCt method. Differences were evaluated by using Mann–Whitney U -test. Fold changes were 0.26 for miR-370-3p, 217.56 for miR-371a-5p, 19.5 for miR-518a-3p, 9.63 for miR-519d-3p, 6.41 for miR-520a-3p, and 2.52 for miR-934. b miR-371a-5p and miR-518a-3p expression levels quantified by qRT-PCR in villous primary human trophoblasts (PHT), immortalized normal trophoblastic cell line HTR-8/SVneo, and choriocarcinoma cell lines BeWo, JAR, and JEG-3. Relative expressions in choriocarcinoma cells were normalized against corresponding miRNA levels in PHT cells. * P
    Figure Legend Snippet: MiR-371a-5p and miR-518a-3p were upregulated in GTN tissues and choriocarcinoma cells a Validation of the microarray profiling data. Expression levels of the most differentially expressed 16 miRNAs were examined by qRT-PCR in 34 cases of GTN and 60 cases of CHM trophoblastic tissues and then normalized to the housekeeping gene RNU6 with the 2 −ΔΔCt method. Differences were evaluated by using Mann–Whitney U -test. Fold changes were 0.26 for miR-370-3p, 217.56 for miR-371a-5p, 19.5 for miR-518a-3p, 9.63 for miR-519d-3p, 6.41 for miR-520a-3p, and 2.52 for miR-934. b miR-371a-5p and miR-518a-3p expression levels quantified by qRT-PCR in villous primary human trophoblasts (PHT), immortalized normal trophoblastic cell line HTR-8/SVneo, and choriocarcinoma cell lines BeWo, JAR, and JEG-3. Relative expressions in choriocarcinoma cells were normalized against corresponding miRNA levels in PHT cells. * P

    Techniques Used: Microarray, Expressing, Quantitative RT-PCR, MANN-WHITNEY

    50) Product Images from "Activation of Peroxisome Proliferator-Activated Receptor ? Inhibits Streptozotocin-Induced Diabetic Nephropathy Through Anti-Inflammatory Mechanisms in Mice"

    Article Title: Activation of Peroxisome Proliferator-Activated Receptor ? Inhibits Streptozotocin-Induced Diabetic Nephropathy Through Anti-Inflammatory Mechanisms in Mice

    Journal: Diabetes

    doi: 10.2337/db10-1361

    Time course of changes in UAE and PPARδ mRNA and protein expression in the kidneys. A : The UAE increased progressively in the untreated diabetic (DM) group during the 8-week observation period after the induction of diabetes. GW0742 treatment (DM+GW0742) significantly reduced UAE at 8 weeks compared with the DM group. Control, nondiabetic control mice. Data are means ± SE. * P
    Figure Legend Snippet: Time course of changes in UAE and PPARδ mRNA and protein expression in the kidneys. A : The UAE increased progressively in the untreated diabetic (DM) group during the 8-week observation period after the induction of diabetes. GW0742 treatment (DM+GW0742) significantly reduced UAE at 8 weeks compared with the DM group. Control, nondiabetic control mice. Data are means ± SE. * P

    Techniques Used: Expressing, Mouse Assay

    PPARδ activation suppresses diabetes–induced renal inflammation and macrophage infiltration. Quantitative RT-PCR analysis of the expression of two macrophage markers (CD14 and CD11c) shows that GW0742 inhibited diabetes–induced macrophage infiltration into the kidney. Similarly, GW0742 suppressed MCP-1, CCR-2, TGF-β, and OPN mRNA levels in the kidney. mRNA levels are normalized to GAPDH. Data are means ± SE. * P
    Figure Legend Snippet: PPARδ activation suppresses diabetes–induced renal inflammation and macrophage infiltration. Quantitative RT-PCR analysis of the expression of two macrophage markers (CD14 and CD11c) shows that GW0742 inhibited diabetes–induced macrophage infiltration into the kidney. Similarly, GW0742 suppressed MCP-1, CCR-2, TGF-β, and OPN mRNA levels in the kidney. mRNA levels are normalized to GAPDH. Data are means ± SE. * P

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

    Schematic diagram showing the mechanisms involved in the renoprotective effects of PPARδ in diabetic nephropathy. The anti-inflammatory transcriptional repressor Bcl-6 represses the expression of MCP-1. PPARδ activation by GW0742 releases Bcl-6, which is associated with suppression of MCP-1, to attenuate macrophage infiltration, inflammatory gene expression, and type IV collagen accumulation in the kidney.
    Figure Legend Snippet: Schematic diagram showing the mechanisms involved in the renoprotective effects of PPARδ in diabetic nephropathy. The anti-inflammatory transcriptional repressor Bcl-6 represses the expression of MCP-1. PPARδ activation by GW0742 releases Bcl-6, which is associated with suppression of MCP-1, to attenuate macrophage infiltration, inflammatory gene expression, and type IV collagen accumulation in the kidney.

    Techniques Used: Expressing, Activation Assay

    51) Product Images from "Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells"

    Article Title: Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells

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

    doi: 10.1152/ajplung.00054.2016

    Direct association of Sirt-2 with the IKBα promoter is responsible for the activation of NF-κB-dependent transcription in HLMVEC. To investigate how Sirt-2 affects IKBα promoter activity and the recruitment of other factors, we
    Figure Legend Snippet: Direct association of Sirt-2 with the IKBα promoter is responsible for the activation of NF-κB-dependent transcription in HLMVEC. To investigate how Sirt-2 affects IKBα promoter activity and the recruitment of other factors, we

    Techniques Used: Activation Assay, Activity Assay

    52) Product Images from "High Level of Plasma Matrix Metalloproteinase-11 Is Associated with Clinicopathological Characteristics in Patients with Oral Squamous Cell Carcinoma"

    Article Title: High Level of Plasma Matrix Metalloproteinase-11 Is Associated with Clinicopathological Characteristics in Patients with Oral Squamous Cell Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113129

    ELISA-determined plasma MMP-11 level of OSCC patients at various clinical features. (A) MMP-11 levels were compared according to stage and results showed that MMP-11 levels were significantly higher in tumor stage II, III and IV when compared with stage I patients. (B) MMP-11 levels were compared according to tumor T status and results showed that MMP-11 levels were significantly higher in advanced tumor extent when compared with T1 patients. (C) MMP-11 levels were compared according to N status and results showed that MMP-11 levels were significantly higher in N2, but not in N1 when compared with patients without nodal metastasis.
    Figure Legend Snippet: ELISA-determined plasma MMP-11 level of OSCC patients at various clinical features. (A) MMP-11 levels were compared according to stage and results showed that MMP-11 levels were significantly higher in tumor stage II, III and IV when compared with stage I patients. (B) MMP-11 levels were compared according to tumor T status and results showed that MMP-11 levels were significantly higher in advanced tumor extent when compared with T1 patients. (C) MMP-11 levels were compared according to N status and results showed that MMP-11 levels were significantly higher in N2, but not in N1 when compared with patients without nodal metastasis.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    MMP-11 expression in oral cancer cell lines and the MMP-11 knockdown in SCC9 cells reduce cell migration. (A) Western blot analysis showing the expression of MMP-11 protein in five oral cancer cell lines. (B) MMP-11 mRNA levels in five oral cancer cell lines were determined by quantitative real-time PCR using β-actin as an endogenous control. Results are shown as mean ± SD of three independent experiments. (C) Endogenous MMP-11 expression is silenced by infection with 4 different shRNA constructs (#1 and #2, #3 and #4) for MMP-11 in SCC9 oral cancer cells. Knockdown efficiency was analyzed by Western blotting. Results showed an approximately 80% reduction of #1 shRNA of MMP-11. *p
    Figure Legend Snippet: MMP-11 expression in oral cancer cell lines and the MMP-11 knockdown in SCC9 cells reduce cell migration. (A) Western blot analysis showing the expression of MMP-11 protein in five oral cancer cell lines. (B) MMP-11 mRNA levels in five oral cancer cell lines were determined by quantitative real-time PCR using β-actin as an endogenous control. Results are shown as mean ± SD of three independent experiments. (C) Endogenous MMP-11 expression is silenced by infection with 4 different shRNA constructs (#1 and #2, #3 and #4) for MMP-11 in SCC9 oral cancer cells. Knockdown efficiency was analyzed by Western blotting. Results showed an approximately 80% reduction of #1 shRNA of MMP-11. *p

    Techniques Used: Expressing, Migration, Western Blot, Real-time Polymerase Chain Reaction, Infection, shRNA, Construct

    53) Product Images from "High Glucose Increases Metallothionein Expression in Renal Proximal Tubular Epithelial Cells"

    Article Title: High Glucose Increases Metallothionein Expression in Renal Proximal Tubular Epithelial Cells

    Journal: Experimental Diabetes Research

    doi: 10.1155/2011/534872

    High glucose increases MT-1 mRNA and MT-1/-2 protein expression. mProx24 cells were serum-starved for 24 h before stimulation with high glucose or mannitol. (a) Cells were harvested after 24 h, and MT-1 mRNA expression was analyzed by qRT-PCR in three independent experiments and normalized for GAPDH. (b–e) MT-1/-2 protein expression was determined by immunofluorescent staining with anti-MT-1/-2 antibody 24 h after stimulation followed by densitometric analysis. Results are means ± SEM of three independent experiments. * P
    Figure Legend Snippet: High glucose increases MT-1 mRNA and MT-1/-2 protein expression. mProx24 cells were serum-starved for 24 h before stimulation with high glucose or mannitol. (a) Cells were harvested after 24 h, and MT-1 mRNA expression was analyzed by qRT-PCR in three independent experiments and normalized for GAPDH. (b–e) MT-1/-2 protein expression was determined by immunofluorescent staining with anti-MT-1/-2 antibody 24 h after stimulation followed by densitometric analysis. Results are means ± SEM of three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Staining

    54) Product Images from "Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution"

    Article Title: Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112737

    In vitro experiments examining the effects of NGF on LECs. A. Adult human dermal lymphatic endothelial cells (LECs) were seeded in the upper inserts of Boyden chamber migration plates. NGF was added to the lower wells at the indicated concentrations. Migratory cells were quantified. B. Matrigel was placed in tissue culture plate wells. LECs were incubated with the indicated cytokines and then added to wells. Tubes were quantified at four hours. C. RNA was extracted from LECs or positive control adult human skeletal muscle and cDNA was synthesized. Gene expression levels of NGF receptor TrkA were quantified by qRT-PCR. D. Gene expression levels of p75 NTR in human skeletal muscle and LECs. E. RIPA lysates were made from LECs treated with the indicated cytokines for 1, 5, 15, or 30 minutes. Western blot analysis was performed for the indicated proteins using β-actin as a loading control.
    Figure Legend Snippet: In vitro experiments examining the effects of NGF on LECs. A. Adult human dermal lymphatic endothelial cells (LECs) were seeded in the upper inserts of Boyden chamber migration plates. NGF was added to the lower wells at the indicated concentrations. Migratory cells were quantified. B. Matrigel was placed in tissue culture plate wells. LECs were incubated with the indicated cytokines and then added to wells. Tubes were quantified at four hours. C. RNA was extracted from LECs or positive control adult human skeletal muscle and cDNA was synthesized. Gene expression levels of NGF receptor TrkA were quantified by qRT-PCR. D. Gene expression levels of p75 NTR in human skeletal muscle and LECs. E. RIPA lysates were made from LECs treated with the indicated cytokines for 1, 5, 15, or 30 minutes. Western blot analysis was performed for the indicated proteins using β-actin as a loading control.

    Techniques Used: In Vitro, Migration, Incubation, Positive Control, Synthesized, Expressing, Quantitative RT-PCR, Western Blot

    55) Product Images from "MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex"

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0275-4

    MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Methylation, Stable Transfection, Recombinant, Transfection, Immunoprecipitation, In Vivo, Cell Culture, Activation Assay

    PRMT5 and MEP50 expression is upregulated in HH pathway-activated cancers, and PRMT5 inhibition is a potential therapeutic strategy for such cancers. a , b Immunoblot analysis of endogenous GLI1 in H146 and AGS cells stably expressing PRMT5 ( a ) or MEP50 ( b ) siRNAs. c Immunoblot analysis of endogenous GLI1 in H146 cells stably expressing GLI1 siRNA. In a – c , siRNAs were stably expressed via recombinant retroviruses. d Growth curves of PRMT5, MEP50, and GLI1-knockdown H146 SCLC cells. Results are shown in the mean ± s.d. of triplicate experiments. e A quantitative colony formation assay was performed by plating cells at a density of 1 × 10 4 cells in a six-well plate and incubating them for 14 days. Surviving colonies were counted and represented as the mean ± s.d. of three independent wells. In d and e , siMEP50, siPRMT5, or siGLI1 was stably expressed via recombinant retrovirus in H146 cells. f , g IC 50 values of cyclopamine in PRMT5-knockdown or MEP50-knockdown AGS cells. siRNAs were stably expressed via recombinant retroviruses. Cell viability ( e ) is shown as the mean ± s.d. n = 4. IC 50 values of cyclopamine are shown in g . h – j Upregulated expression of PRMT5 , MEP50 , and GLI1 target genes in small cell lung carcinoma ( h ), gastric adenocarcinoma ( i ), and skin basal cell carcinoma ( j ) from the ONCOMINE database ( https://www.oncomine.org/ ). The threshold of data was p ≤ 0.05. Each boxplot shows the log 2 maximum, minimum, and median signal intensity of each mRNA from the corresponding expression array. Bold lines on each boxplot define the median value. P -values and sample numbers are indicated in each panel. Unprocessed original scans of blots are shown in Supplementary Fig. 6 . Source data of d – f is shown in Supplementary Data 2
    Figure Legend Snippet: PRMT5 and MEP50 expression is upregulated in HH pathway-activated cancers, and PRMT5 inhibition is a potential therapeutic strategy for such cancers. a , b Immunoblot analysis of endogenous GLI1 in H146 and AGS cells stably expressing PRMT5 ( a ) or MEP50 ( b ) siRNAs. c Immunoblot analysis of endogenous GLI1 in H146 cells stably expressing GLI1 siRNA. In a – c , siRNAs were stably expressed via recombinant retroviruses. d Growth curves of PRMT5, MEP50, and GLI1-knockdown H146 SCLC cells. Results are shown in the mean ± s.d. of triplicate experiments. e A quantitative colony formation assay was performed by plating cells at a density of 1 × 10 4 cells in a six-well plate and incubating them for 14 days. Surviving colonies were counted and represented as the mean ± s.d. of three independent wells. In d and e , siMEP50, siPRMT5, or siGLI1 was stably expressed via recombinant retrovirus in H146 cells. f , g IC 50 values of cyclopamine in PRMT5-knockdown or MEP50-knockdown AGS cells. siRNAs were stably expressed via recombinant retroviruses. Cell viability ( e ) is shown as the mean ± s.d. n = 4. IC 50 values of cyclopamine are shown in g . h – j Upregulated expression of PRMT5 , MEP50 , and GLI1 target genes in small cell lung carcinoma ( h ), gastric adenocarcinoma ( i ), and skin basal cell carcinoma ( j ) from the ONCOMINE database ( https://www.oncomine.org/ ). The threshold of data was p ≤ 0.05. Each boxplot shows the log 2 maximum, minimum, and median signal intensity of each mRNA from the corresponding expression array. Bold lines on each boxplot define the median value. P -values and sample numbers are indicated in each panel. Unprocessed original scans of blots are shown in Supplementary Fig. 6 . Source data of d – f is shown in Supplementary Data 2

    Techniques Used: Expressing, Inhibition, Stable Transfection, Recombinant, Colony Assay

    GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Incubation

    MEP50/PRMT5 complex supports GLI1 activation through GLI1 stabilisation downstream of the HH signalling pathway. a–c Endogenous GLI1/MEP50/PRMT5 complex in C3H10T1/2 cells. Cells were treated with SAG for 36 h, and complex was detected by immunoprecipitation (IP) with anti-PRMT5 (D5P2T) ( a ), anti-MEP50 (ERP10708 [B]) ( b ), or anti-GLI1 (V812) ( c ) antibodies, followed by immunoblot (IB) with antibodies against indicated proteins. d Dissociation of PRMT5 and GLI1 in stable MEP50 knockdown C3H10T1/2 cells by siMEP50-m2. Cells were treated with SAG for 48 h and treated with 50 μM MG132 for 4 h. GLI1/PRMT5 complex was detected by immunoprecipitation with anti-PRMT5 (D5P2T) or anti-GLI1 (V812) antibodies, followed by immunoblot with indicated antibodies. e Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing MEP50 siRNAs (siMEP50-m1 or siMEP50-m2). f Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing two independent PRMT5 siRNAs. g Immunoblot of nuclear and cytoplasmic GLI1 and MEP50 in stable MEP50-knockdown (siMEP50-m2) or control siGFP-expressing cells treated with 300 nM SAG. Cells were treated with SAG for 24 h and separated into cytosol and nucleus fractions. h Immunoblot analysis of endogenous nuclear and cytoplasmic GLI1 and PRMT5 in stable PRMT5-knockdown (siPRMT5-m2) C3H10T1/2 cells. Cells were treated with 300 nM SAG for 24 h and then separated as in h . i In vivo ubiquitination of GLI1 in C3H10T1/2 cells with or without expression of siMEP50. FLAG-ubiquitin was transfected into C3H10T1/2 cells. After 48 h of transfection, then cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was immunoprecipitated with an anti-GLI1 (C-1) antibody, followed by immunoblotting with indicated antibodies. j In vivo ubiquitination of GLI1 in C3H10T1/2 cells with exogenous expression of PRMT5 or MEP50. FLAG-ubiquitin and HA-PRMT5, HA-PRMT5 G367A/R368A (inactive form of PRMT5), or Myc-MEP50 were transfected into C3H10T1/2 cells. After 48 h, the cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was detected as described in i . In a , i and j , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: MEP50/PRMT5 complex supports GLI1 activation through GLI1 stabilisation downstream of the HH signalling pathway. a–c Endogenous GLI1/MEP50/PRMT5 complex in C3H10T1/2 cells. Cells were treated with SAG for 36 h, and complex was detected by immunoprecipitation (IP) with anti-PRMT5 (D5P2T) ( a ), anti-MEP50 (ERP10708 [B]) ( b ), or anti-GLI1 (V812) ( c ) antibodies, followed by immunoblot (IB) with antibodies against indicated proteins. d Dissociation of PRMT5 and GLI1 in stable MEP50 knockdown C3H10T1/2 cells by siMEP50-m2. Cells were treated with SAG for 48 h and treated with 50 μM MG132 for 4 h. GLI1/PRMT5 complex was detected by immunoprecipitation with anti-PRMT5 (D5P2T) or anti-GLI1 (V812) antibodies, followed by immunoblot with indicated antibodies. e Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing MEP50 siRNAs (siMEP50-m1 or siMEP50-m2). f Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing two independent PRMT5 siRNAs. g Immunoblot of nuclear and cytoplasmic GLI1 and MEP50 in stable MEP50-knockdown (siMEP50-m2) or control siGFP-expressing cells treated with 300 nM SAG. Cells were treated with SAG for 24 h and separated into cytosol and nucleus fractions. h Immunoblot analysis of endogenous nuclear and cytoplasmic GLI1 and PRMT5 in stable PRMT5-knockdown (siPRMT5-m2) C3H10T1/2 cells. Cells were treated with 300 nM SAG for 24 h and then separated as in h . i In vivo ubiquitination of GLI1 in C3H10T1/2 cells with or without expression of siMEP50. FLAG-ubiquitin was transfected into C3H10T1/2 cells. After 48 h of transfection, then cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was immunoprecipitated with an anti-GLI1 (C-1) antibody, followed by immunoblotting with indicated antibodies. j In vivo ubiquitination of GLI1 in C3H10T1/2 cells with exogenous expression of PRMT5 or MEP50. FLAG-ubiquitin and HA-PRMT5, HA-PRMT5 G367A/R368A (inactive form of PRMT5), or Myc-MEP50 were transfected into C3H10T1/2 cells. After 48 h, the cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was detected as described in i . In a , i and j , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Activation Assay, Immunoprecipitation, Expressing, In Vivo, Transfection

    MEP50/PRMT5 complex induces GLI1 methylation. a , b Methylation of GLI1 in MEP50- ( a ) or PRMT5- ( b ) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in ( c ). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. 4 . In c , data represent one of three independent experiments with similar results. In a and d , data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: MEP50/PRMT5 complex induces GLI1 methylation. a , b Methylation of GLI1 in MEP50- ( a ) or PRMT5- ( b ) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in ( c ). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. 4 . In c , data represent one of three independent experiments with similar results. In a and d , data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Methylation, Stable Transfection, Recombinant, Transfection, Cell Culture, Immunoprecipitation, In Vitro, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Staining

    MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Recombinant, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Quantitative RT-PCR

    56) Product Images from "Interspecies Systems Biology Uncovers Metabolites Affecting C. elegans Gene Expression and Life History Traits"

    Article Title: Interspecies Systems Biology Uncovers Metabolites Affecting C. elegans Gene Expression and Life History Traits

    Journal: Cell

    doi: 10.1016/j.cell.2014.01.047

    Chemical Epistasis with Vitamin B12 and Propionic Acid on C. elegans Gene Expression (A) Network of the two vitamin B12-dependent pathways. Metabolites are indicated in rectangles. Green metabolites activate the dietary sensor when supplemented to the bacterial diet. Genes encoding the metabolic enzymes involved are indicated with arrows between metabolites. Genes that, when mutated, activate the dietary sensor are indicated in red. Red hexagons – vitamin B12; green hexagons – vitamin B6; blue indicates folate metabolism. 5-Me-THF = 5-methyltetrahydrofolate; THF = tetrahydrofolate, the biologically active form of folate. (B) Chemical epistasis of combined supplementation of Ado-Cbl and propionic acid on Pacdh-1::GFP dietary sensor animals fed the E. coli OP50 diet. (C) qRT-PCR of 14 Comamonas -upregulated and 14 Comamonas -downregulated genes in wild type animals fed indicated diets and supplemented metabolites. PA = propionic acid. N2 = wild type animals. Changes in expression for each gene are plotted as log 2 fold change compared to respective mRNA levels in wild type animals fed E. coli OP50 (first and third columns) or Comamonas aq. DA1877 (middle column). .
    Figure Legend Snippet: Chemical Epistasis with Vitamin B12 and Propionic Acid on C. elegans Gene Expression (A) Network of the two vitamin B12-dependent pathways. Metabolites are indicated in rectangles. Green metabolites activate the dietary sensor when supplemented to the bacterial diet. Genes encoding the metabolic enzymes involved are indicated with arrows between metabolites. Genes that, when mutated, activate the dietary sensor are indicated in red. Red hexagons – vitamin B12; green hexagons – vitamin B6; blue indicates folate metabolism. 5-Me-THF = 5-methyltetrahydrofolate; THF = tetrahydrofolate, the biologically active form of folate. (B) Chemical epistasis of combined supplementation of Ado-Cbl and propionic acid on Pacdh-1::GFP dietary sensor animals fed the E. coli OP50 diet. (C) qRT-PCR of 14 Comamonas -upregulated and 14 Comamonas -downregulated genes in wild type animals fed indicated diets and supplemented metabolites. PA = propionic acid. N2 = wild type animals. Changes in expression for each gene are plotted as log 2 fold change compared to respective mRNA levels in wild type animals fed E. coli OP50 (first and third columns) or Comamonas aq. DA1877 (middle column). .

    Techniques Used: Expressing, Quantitative RT-PCR

    57) Product Images from "High Glucose Increases Metallothionein Expression in Renal Proximal Tubular Epithelial Cells"

    Article Title: High Glucose Increases Metallothionein Expression in Renal Proximal Tubular Epithelial Cells

    Journal: Experimental Diabetes Research

    doi: 10.1155/2011/534872

    High glucose increases MT-1 mRNA and MT-1/-2 protein expression. mProx24 cells were serum-starved for 24 h before stimulation with high glucose or mannitol. (a) Cells were harvested after 24 h, and MT-1 mRNA expression was analyzed by qRT-PCR in three independent experiments and normalized for GAPDH. (b–e) MT-1/-2 protein expression was determined by immunofluorescent staining with anti-MT-1/-2 antibody 24 h after stimulation followed by densitometric analysis. Results are means ± SEM of three independent experiments. * P
    Figure Legend Snippet: High glucose increases MT-1 mRNA and MT-1/-2 protein expression. mProx24 cells were serum-starved for 24 h before stimulation with high glucose or mannitol. (a) Cells were harvested after 24 h, and MT-1 mRNA expression was analyzed by qRT-PCR in three independent experiments and normalized for GAPDH. (b–e) MT-1/-2 protein expression was determined by immunofluorescent staining with anti-MT-1/-2 antibody 24 h after stimulation followed by densitometric analysis. Results are means ± SEM of three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Staining

    58) Product Images from "Infusion with Human Bone Marrow-derived Mesenchymal Stem Cells Improves β-cell Function in Patients and Non-obese Mice with Severe Diabetes"

    Article Title: Infusion with Human Bone Marrow-derived Mesenchymal Stem Cells Improves β-cell Function in Patients and Non-obese Mice with Severe Diabetes

    Journal: Scientific Reports

    doi: 10.1038/srep37894

    Infused MSCs migrate and accumulate in the pancreatic tissues in diabetic NOD mice. Following infusion with MSCs, some mice from each group were sacrificed and their lung, liver, intestine, kidney and pancreatic tissues were dissected. The levels of human Alu element DNA were determined by quantitative RT-PCR and analyzed, according to the standard curve. Data are expressed as the mean ± SEM of individual groups of mice (n = 6 per group per time points). Statistical significance was analyzed by ANOVA and post hoc Fisher’s least significant difference test. * P
    Figure Legend Snippet: Infused MSCs migrate and accumulate in the pancreatic tissues in diabetic NOD mice. Following infusion with MSCs, some mice from each group were sacrificed and their lung, liver, intestine, kidney and pancreatic tissues were dissected. The levels of human Alu element DNA were determined by quantitative RT-PCR and analyzed, according to the standard curve. Data are expressed as the mean ± SEM of individual groups of mice (n = 6 per group per time points). Statistical significance was analyzed by ANOVA and post hoc Fisher’s least significant difference test. * P

    Techniques Used: Mouse Assay, Quantitative RT-PCR

    59) Product Images from "Transmission dynamics of an emerging infectious disease in wildlife through host reproductive cycles"

    Article Title: Transmission dynamics of an emerging infectious disease in wildlife through host reproductive cycles

    Journal: The ISME journal

    doi: 10.1038/ismej.2010.123

    ( a ) Seasonal abundance of CyHV-3 in gill and brain tissues of common carp: +, CyHV-3 concentrations in gill tissues of fish captured in 2008; × , those in brain tissues in 2008; ○, those in gill tissues in 2009; □, those in brain tissues in 2009. Water temperature changes in 2008 and 2009 are indicated by dashed and solid lines, respectively. ( b ) CyHV-3 concentrations in intestinal contents of common carp captured in 2009. Solid line indicates water temperature.
    Figure Legend Snippet: ( a ) Seasonal abundance of CyHV-3 in gill and brain tissues of common carp: +, CyHV-3 concentrations in gill tissues of fish captured in 2008; × , those in brain tissues in 2008; ○, those in gill tissues in 2009; □, those in brain tissues in 2009. Water temperature changes in 2008 and 2009 are indicated by dashed and solid lines, respectively. ( b ) CyHV-3 concentrations in intestinal contents of common carp captured in 2009. Solid line indicates water temperature.

    Techniques Used: Fluorescence In Situ Hybridization

    Variation in water CyHV-3 concentrations at breeding and non-breeding sites before, during and after mating of common carp in 2009. Mating occurred on the days indicated by arrows. CyHV-3 concentrations of water samples from N1 on 2 April and from N2 on 9 June, 2009 were not determined because of low virus recovery (
    Figure Legend Snippet: Variation in water CyHV-3 concentrations at breeding and non-breeding sites before, during and after mating of common carp in 2009. Mating occurred on the days indicated by arrows. CyHV-3 concentrations of water samples from N1 on 2 April and from N2 on 9 June, 2009 were not determined because of low virus recovery (

    Techniques Used:

    Seroprevalence of CyHV-3 infection in the common carp population of Lake Biwa in 2008 and 2009. Bars indicate numbers of anti-CyHV-3 antibody-positive/negative individuals, and the solid line depicts the proportion (%) of antibody-positive fish for each size class.
    Figure Legend Snippet: Seroprevalence of CyHV-3 infection in the common carp population of Lake Biwa in 2008 and 2009. Bars indicate numbers of anti-CyHV-3 antibody-positive/negative individuals, and the solid line depicts the proportion (%) of antibody-positive fish for each size class.

    Techniques Used: Infection, Fluorescence In Situ Hybridization

    60) Product Images from "Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males"

    Article Title: Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males

    Journal: Cell Stem Cell

    doi: 10.1016/j.stem.2018.05.001

    Xist Expression Is Abolished by a Robust Naive Pluripotent Network (A) Schematic illustrating the experiment performed to evaluate the impact of the nPSC culture conditions on the expression of Xist . (B) qRT-PCR analysis of Nanog and Xist in XX1, XX2, XY1, and XY2 ESC lines in SL versus 2iL. P indicates number of passages in 2iL. Error bars represent ± SD. (C) Flow cytometry analysis of male SL Nanog -GFP ESCs and subsequent sorting into three Nanog -GFP populations: low, medium, and high. (D) qRT-PCR analysis of Nanog , Klf4 , Oct4 , and Xist in low, medium, and high Nanog -GFP ESCs. Error bars represent ± SD. (E) Strand-specific RNA-seq showing expression of the positive and negative strands at the Xist locus in male 2iL ESCs. The double-strand Xist probe used in (F) is represented in red. (F) RNA FISH in male and female 2iL ESCs with a double-strand (ds) probe (left) or with a single-strand (ss) probe detecting only Xist (right). The percentage of cells with probe signal is indicated. Female EpiSCs were used as a control for the ss probe. The scale bar represents 5 μm. (G) qRT-PCR analysis of Nanog and Xist in female and male Nanog flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Error bars represent ± SD. (H) qRT-PCR analysis of Xist in XX3 and XY1 ESCs in 2iL, 2i or after 3 and 5 days in 1 μM JAKi + 2i. Error bars represent ± SD. (I) qRT-PCR analysis of Oct4 and Xist in female and male Oct4 flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Female somatic cells were used as control for Xist expression. Error bars represent ± SD.
    Figure Legend Snippet: Xist Expression Is Abolished by a Robust Naive Pluripotent Network (A) Schematic illustrating the experiment performed to evaluate the impact of the nPSC culture conditions on the expression of Xist . (B) qRT-PCR analysis of Nanog and Xist in XX1, XX2, XY1, and XY2 ESC lines in SL versus 2iL. P indicates number of passages in 2iL. Error bars represent ± SD. (C) Flow cytometry analysis of male SL Nanog -GFP ESCs and subsequent sorting into three Nanog -GFP populations: low, medium, and high. (D) qRT-PCR analysis of Nanog , Klf4 , Oct4 , and Xist in low, medium, and high Nanog -GFP ESCs. Error bars represent ± SD. (E) Strand-specific RNA-seq showing expression of the positive and negative strands at the Xist locus in male 2iL ESCs. The double-strand Xist probe used in (F) is represented in red. (F) RNA FISH in male and female 2iL ESCs with a double-strand (ds) probe (left) or with a single-strand (ss) probe detecting only Xist (right). The percentage of cells with probe signal is indicated. Female EpiSCs were used as a control for the ss probe. The scale bar represents 5 μm. (G) qRT-PCR analysis of Nanog and Xist in female and male Nanog flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Error bars represent ± SD. (H) qRT-PCR analysis of Xist in XX3 and XY1 ESCs in 2iL, 2i or after 3 and 5 days in 1 μM JAKi + 2i. Error bars represent ± SD. (I) qRT-PCR analysis of Oct4 and Xist in female and male Oct4 flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Female somatic cells were used as control for Xist expression. Error bars represent ± SD.

    Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, RNA Sequencing Assay, Fluorescence In Situ Hybridization

    Xist Is Transiently and Rapidly Upregulated in Male nPSC Differentiation and Male EpiSC Reprogramming (A) Schematic illustrating three conditions employed to differentiate 2iL and SL nPSCs: suspension culture in serum to generate EBs or adherent monolayer culture in serum-free media ± Fgf2+ActivinA (FA). (B) qRT-PCR analysis of Xist during differentiation of male ESCs in three different conditions. Before differentiation, ESCs were maintained in 2iL or SL conditions, as indicated. Error bars represent ± SD. (C) Strand-specific RNA-seq (negative strand only) showing expression of Xist during differentiation of male 2iL ESCs in three different conditions. Scale represents reads per million (RPM). (D) Heatmap showing expression profile of Xist , differentiation markers, and naive markers during differentiation of male 2iL ESCs, as indicated. Scale represents Z scores of log2-transformed expression values. (E) qRT-PCR analysis of Xist during EB differentiation of male versus female 2iL ESCs. Error bars represent ± SD. (F) Flow cytometry analysis of male GY118F Rex1 +/dGFP EpiSCs following reprogramming induction with GCSF in 2iL. Cells were sorted at different time points, with Rex1 -dGFP reporter activation indicating the subset of cells successfully transitioning to the naive identity. A representative plot from day 3 is shown. (G) qRT-PCR analysis of Xist and naive markers ( Oct4 , Tfcp2l1 , and Esrrb ) in male Rex1 -positive reprogramming intermediates at different time points after induction of reprogramming with 2iL+GCSF/GY118F. Parental EpiSCs (day 0) and ESCs in 2iL were used as controls. Error bars represent ± SD.
    Figure Legend Snippet: Xist Is Transiently and Rapidly Upregulated in Male nPSC Differentiation and Male EpiSC Reprogramming (A) Schematic illustrating three conditions employed to differentiate 2iL and SL nPSCs: suspension culture in serum to generate EBs or adherent monolayer culture in serum-free media ± Fgf2+ActivinA (FA). (B) qRT-PCR analysis of Xist during differentiation of male ESCs in three different conditions. Before differentiation, ESCs were maintained in 2iL or SL conditions, as indicated. Error bars represent ± SD. (C) Strand-specific RNA-seq (negative strand only) showing expression of Xist during differentiation of male 2iL ESCs in three different conditions. Scale represents reads per million (RPM). (D) Heatmap showing expression profile of Xist , differentiation markers, and naive markers during differentiation of male 2iL ESCs, as indicated. Scale represents Z scores of log2-transformed expression values. (E) qRT-PCR analysis of Xist during EB differentiation of male versus female 2iL ESCs. Error bars represent ± SD. (F) Flow cytometry analysis of male GY118F Rex1 +/dGFP EpiSCs following reprogramming induction with GCSF in 2iL. Cells were sorted at different time points, with Rex1 -dGFP reporter activation indicating the subset of cells successfully transitioning to the naive identity. A representative plot from day 3 is shown. (G) qRT-PCR analysis of Xist and naive markers ( Oct4 , Tfcp2l1 , and Esrrb ) in male Rex1 -positive reprogramming intermediates at different time points after induction of reprogramming with 2iL+GCSF/GY118F. Parental EpiSCs (day 0) and ESCs in 2iL were used as controls. Error bars represent ± SD.

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing, Transformation Assay, Flow Cytometry, Cytometry, Activation Assay

    61) Product Images from "Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model"

    Article Title: Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127319

    P23H mutated mouse Rho transgene copy number. (A) Target and sequence of primers and probes used for qPCR, FAM: 6-carboxyfluorescein and TAM: tetramethylrhodamine. (B) WT endogenous rat Rho (light grey) and P23H mutant mouse Rho (dark grey) copy numbers were evaluated by qPCR and 2 -ΔΔCt method for WT (WT), hemizygous P23H-1 (P23H hemi) and homozygous P23H-1 (P23H homo) rats. All experiments were conducted on the gDNA of 5 rats per group. Three asteriks (***) indicate a p value of
    Figure Legend Snippet: P23H mutated mouse Rho transgene copy number. (A) Target and sequence of primers and probes used for qPCR, FAM: 6-carboxyfluorescein and TAM: tetramethylrhodamine. (B) WT endogenous rat Rho (light grey) and P23H mutant mouse Rho (dark grey) copy numbers were evaluated by qPCR and 2 -ΔΔCt method for WT (WT), hemizygous P23H-1 (P23H hemi) and homozygous P23H-1 (P23H homo) rats. All experiments were conducted on the gDNA of 5 rats per group. Three asteriks (***) indicate a p value of

    Techniques Used: Sequencing, Real-time Polymerase Chain Reaction, Mutagenesis

    62) Product Images from "Preservation of Essential Odor-Guided Behaviors and Odor-Based Reversal Learning after Targeting Adult Brain Serotonin Synthesis"

    Article Title: Preservation of Essential Odor-Guided Behaviors and Odor-Based Reversal Learning after Targeting Adult Brain Serotonin Synthesis

    Journal: eNeuro

    doi: 10.1523/ENEURO.0257-16.2016

    Confirmation of Tph2 targeting and 5-HT depletion in cohorts used for behavior experiments. A , qPCR results displaying a significant reduction in Tph2 expression, relative to Actb , in the DRN region of Tph2 fl/fl mice injected with AAV-Cre ( n = 8) compared with untreated controls ( n = 9). *** p
    Figure Legend Snippet: Confirmation of Tph2 targeting and 5-HT depletion in cohorts used for behavior experiments. A , qPCR results displaying a significant reduction in Tph2 expression, relative to Actb , in the DRN region of Tph2 fl/fl mice injected with AAV-Cre ( n = 8) compared with untreated controls ( n = 9). *** p

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

    63) Product Images from "Depletion of Bone Marrow-derived Macrophages Perturbs the Innate Immune Response to Surgery and Reduces Postoperative Memory Dysfunction"

    Article Title: Depletion of Bone Marrow-derived Macrophages Perturbs the Innate Immune Response to Surgery and Reduces Postoperative Memory Dysfunction

    Journal: Anesthesiology

    doi: 10.1097/ALN.0b013e3182834d94

    Clodrolip effect and early kinetic of the mRNA hippocampal expression of IL-1β, TNF-α, IL-6 and MCP-1 after tibia fracture
    Figure Legend Snippet: Clodrolip effect and early kinetic of the mRNA hippocampal expression of IL-1β, TNF-α, IL-6 and MCP-1 after tibia fracture

    Techniques Used: Expressing

    Effects of systemic macrophage depletion with clodrolip on the IL-6 serum concentration after the tibia fracture
    Figure Legend Snippet: Effects of systemic macrophage depletion with clodrolip on the IL-6 serum concentration after the tibia fracture

    Techniques Used: Concentration Assay

    64) Product Images from "Peri-adolescent asthma symptoms cause adult anxiety-related behavior and neurobiological processes in mice"

    Article Title: Peri-adolescent asthma symptoms cause adult anxiety-related behavior and neurobiological processes in mice

    Journal: Behavioural brain research

    doi: 10.1016/j.bbr.2017.02.046

    Adult lung state and cytokine expression Lung function measures for the four treatment groups: Control (CON), Airway Inflammation (AI), Labored Breathing (LB), and Airway Inflammation + Labored Breathing (AI+LB). Adolescent HDM treatments (AI and AI+LB groups) led to increased mucus ( A) , average inflammation patch length (B) , number of inflammation patches (C) , and gene expression (mRNA) for IL-4 (E), IL-5 (F) , and IL-13 (G) . Mucus and IL-5 expression were both greater in females compared to males. Collagen measures were not significantly different across groups (D) . Main effects of AI and Sex indicated by * ( p
    Figure Legend Snippet: Adult lung state and cytokine expression Lung function measures for the four treatment groups: Control (CON), Airway Inflammation (AI), Labored Breathing (LB), and Airway Inflammation + Labored Breathing (AI+LB). Adolescent HDM treatments (AI and AI+LB groups) led to increased mucus ( A) , average inflammation patch length (B) , number of inflammation patches (C) , and gene expression (mRNA) for IL-4 (E), IL-5 (F) , and IL-13 (G) . Mucus and IL-5 expression were both greater in females compared to males. Collagen measures were not significantly different across groups (D) . Main effects of AI and Sex indicated by * ( p

    Techniques Used: Expressing

    65) Product Images from "Recurrent hotspot mutations in HRAS Q61 and PI3K-AKT pathway genes as drivers of breast adenomyoepitheliomas"

    Article Title: Recurrent hotspot mutations in HRAS Q61 and PI3K-AKT pathway genes as drivers of breast adenomyoepitheliomas

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04128-5

    Expression of mutant HRAS Q61R results in the acquisition of a partial myoepithelial phenotype in non-malignant breast epithelial cells. a Representative western blot (left) analysis of total protein expression of alpha-smooth muscle actin (αSMA), TIMP1, cytokeratin 5 (CK5), E-cadherin, vimentin, and nuclear protein expression of ∆N-p63 and TA-p63 in MCF-10A P , MCF-10A H1047R , and MCF-12A cells stably expressing empty vector (EV), HRAS WT , or mutant HRAS Q61R . α-Tubulin and Histone H3 were used as protein loading controls for total and nuclear protein expression, respectively. Quantification (right) using LI-COR is shown based on experiments done in triplicate. Comparisons of protein levels were performed between HRAS WT and mutant HRAS Q61R , both relative to EV. Error bars, s.d. of mean ( n = 3). n.s. = not significant, * P
    Figure Legend Snippet: Expression of mutant HRAS Q61R results in the acquisition of a partial myoepithelial phenotype in non-malignant breast epithelial cells. a Representative western blot (left) analysis of total protein expression of alpha-smooth muscle actin (αSMA), TIMP1, cytokeratin 5 (CK5), E-cadherin, vimentin, and nuclear protein expression of ∆N-p63 and TA-p63 in MCF-10A P , MCF-10A H1047R , and MCF-12A cells stably expressing empty vector (EV), HRAS WT , or mutant HRAS Q61R . α-Tubulin and Histone H3 were used as protein loading controls for total and nuclear protein expression, respectively. Quantification (right) using LI-COR is shown based on experiments done in triplicate. Comparisons of protein levels were performed between HRAS WT and mutant HRAS Q61R , both relative to EV. Error bars, s.d. of mean ( n = 3). n.s. = not significant, * P

    Techniques Used: Expressing, Mutagenesis, Western Blot, Stable Transfection, Plasmid Preparation

    66) Product Images from "NF-?B-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma"

    Article Title: NF-?B-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078728

    TNF-α Induces IL-6 and LIF Expression in Glioma Cells. A B, U251-MG and X1016 cells were treated with TNF-α (10 ng/ml) for the indicated times. RNA was isolated followed by generation of cDNA, and qRT-PCR was performed for the indicated genes. Data are shown as replicates of three and the experiment repeated with similar results observed. *, p
    Figure Legend Snippet: TNF-α Induces IL-6 and LIF Expression in Glioma Cells. A B, U251-MG and X1016 cells were treated with TNF-α (10 ng/ml) for the indicated times. RNA was isolated followed by generation of cDNA, and qRT-PCR was performed for the indicated genes. Data are shown as replicates of three and the experiment repeated with similar results observed. *, p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    TNF-α Recruits p65 to the IL-6 Promoter and STAT3 to the IL-6 and SOCS3 Promoters. U251-MG cells were grown in the absence or presence of TNF-α (10 ng/ml) for various times, sonicated and soluble chromatin was immunoprecipitated with antibodies specific for p65 or STAT3. Immunoprecipitated DNA was then analyzed by qRT-PCR using primers specific for the IL-6 and SOCS3 promoters. Each sample was normalized to genomic DNA isolated from cells that were cross-linked and processed, yet did not incur the immunoprecipitation step. The results are shown as percentages of input, replicates of three, and error bars represent standard deviation.
    Figure Legend Snippet: TNF-α Recruits p65 to the IL-6 Promoter and STAT3 to the IL-6 and SOCS3 Promoters. U251-MG cells were grown in the absence or presence of TNF-α (10 ng/ml) for various times, sonicated and soluble chromatin was immunoprecipitated with antibodies specific for p65 or STAT3. Immunoprecipitated DNA was then analyzed by qRT-PCR using primers specific for the IL-6 and SOCS3 promoters. Each sample was normalized to genomic DNA isolated from cells that were cross-linked and processed, yet did not incur the immunoprecipitation step. The results are shown as percentages of input, replicates of three, and error bars represent standard deviation.

    Techniques Used: Sonication, Immunoprecipitation, Quantitative RT-PCR, Isolation, Standard Deviation

    Signaling Schematic Illustrating the Cycle of Cooperation Between NF-κB and STAT3. NF-κB and STAT3 are competent to ensure activation of themselves and each other, either in an autocrine and/or paracrine manner. Upon stimulation with TNF-α, the NF-κB pathway becomes activated, as shown by the phosphorylation and nuclear translocation of NF-κB p65 and transcription of NF-κB genes, including IL-6 and LIF. Newly synthesized IL-6 is secreted by the cells, and binds in an autocrine or paracrine manner to the IL-6 receptor. This leads to activation of the IL-6R/gp130 complex and the intracellular kinases JAK1/2. STAT3 proteins then become phosphorylated by JAK1/2, dimerize, enter the nucleus and begin the transcription of STAT3 driven genes such as SOCS3 and cIAP2.
    Figure Legend Snippet: Signaling Schematic Illustrating the Cycle of Cooperation Between NF-κB and STAT3. NF-κB and STAT3 are competent to ensure activation of themselves and each other, either in an autocrine and/or paracrine manner. Upon stimulation with TNF-α, the NF-κB pathway becomes activated, as shown by the phosphorylation and nuclear translocation of NF-κB p65 and transcription of NF-κB genes, including IL-6 and LIF. Newly synthesized IL-6 is secreted by the cells, and binds in an autocrine or paracrine manner to the IL-6 receptor. This leads to activation of the IL-6R/gp130 complex and the intracellular kinases JAK1/2. STAT3 proteins then become phosphorylated by JAK1/2, dimerize, enter the nucleus and begin the transcription of STAT3 driven genes such as SOCS3 and cIAP2.

    Techniques Used: Activation Assay, Translocation Assay, Synthesized

    Combined Pharmacological Inhibition Disrupts NF-κB and STAT3 Signaling In Vitro and Prolongs Survival In Vivo . A B, Xenograft X1046 cells were disaggregated into single cells and briefly propagated as neurospheres in vitro . Cells were pre-treated with AZD1480 (1 µM) and/or WA (5 µM) for 2 h prior to TNF-α (10 ng/ml) for 0.25 or 2 h (A) or 2 h (B). Cells were lysed and immunoblotted with the indicated Ab (A), or RNA was isolated followed by generation of cDNA and qRT-PCR was performed for IL-6 (B). Densitometric values of p-p65, p-IKKα/β and p-STAT3 were normalized to total p65, total IKKα/β and total STAT3, respectively. Data are shown as replicates of three. *, p
    Figure Legend Snippet: Combined Pharmacological Inhibition Disrupts NF-κB and STAT3 Signaling In Vitro and Prolongs Survival In Vivo . A B, Xenograft X1046 cells were disaggregated into single cells and briefly propagated as neurospheres in vitro . Cells were pre-treated with AZD1480 (1 µM) and/or WA (5 µM) for 2 h prior to TNF-α (10 ng/ml) for 0.25 or 2 h (A) or 2 h (B). Cells were lysed and immunoblotted with the indicated Ab (A), or RNA was isolated followed by generation of cDNA and qRT-PCR was performed for IL-6 (B). Densitometric values of p-p65, p-IKKα/β and p-STAT3 were normalized to total p65, total IKKα/β and total STAT3, respectively. Data are shown as replicates of three. *, p

    Techniques Used: Inhibition, In Vitro, In Vivo, Isolation, Quantitative RT-PCR

    67) Product Images from "PDE4D regulates Spine Plasticity and Memory in the Retrosplenial Cortex"

    Article Title: PDE4D regulates Spine Plasticity and Memory in the Retrosplenial Cortex

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22193-0

    Pde4d knockdown (KD) in HC and RSC enhances contextual memory. mRNA levels of Pde4d in hippocampal cultures treated with AAV5 expressing shPde4d or shGFP control. shRNA efficiently knocks down Pde4d mRNA levels in cultured neurons. ( B ) Stereotactic injection of AAV5 coexpressing the shRNAs and eGFP marker results in GFP fluorescence restricted to HC. ( C ) Freezing in the memory test but not during training (shown here as baseline - before first shock, inter trial interval (ITI) - between the two shocks, and post-US – after second shock) is significantly increased after Pde4d KD (n = 19) in HC as compared to control virus (n = 20) and uninjected mice (n = 32). ( D ) Posttraining intrahippocampal (i.h.) injection of the PDE4 inhibitor rolipram enhances contextual memory in a dose dependent manner (n = 25 vehicle, n = 8–16 drug, respectively). ( E ) Stereotactic injection of AAV5 carrying the shRNAs and expressing eGFP results in restricted fluorescence in rostral RSC. ( F ) Freezing in the memory test but not during training is significantly increased after Pde4d KD (n = 23) in RSC as compared to control virus (n = 24). Significant differences from shGFP (panel C/E) or vehicle (panel D) control are indicated by an asterisk (*).
    Figure Legend Snippet: Pde4d knockdown (KD) in HC and RSC enhances contextual memory. mRNA levels of Pde4d in hippocampal cultures treated with AAV5 expressing shPde4d or shGFP control. shRNA efficiently knocks down Pde4d mRNA levels in cultured neurons. ( B ) Stereotactic injection of AAV5 coexpressing the shRNAs and eGFP marker results in GFP fluorescence restricted to HC. ( C ) Freezing in the memory test but not during training (shown here as baseline - before first shock, inter trial interval (ITI) - between the two shocks, and post-US – after second shock) is significantly increased after Pde4d KD (n = 19) in HC as compared to control virus (n = 20) and uninjected mice (n = 32). ( D ) Posttraining intrahippocampal (i.h.) injection of the PDE4 inhibitor rolipram enhances contextual memory in a dose dependent manner (n = 25 vehicle, n = 8–16 drug, respectively). ( E ) Stereotactic injection of AAV5 carrying the shRNAs and expressing eGFP results in restricted fluorescence in rostral RSC. ( F ) Freezing in the memory test but not during training is significantly increased after Pde4d KD (n = 23) in RSC as compared to control virus (n = 24). Significant differences from shGFP (panel C/E) or vehicle (panel D) control are indicated by an asterisk (*).

    Techniques Used: Expressing, shRNA, Cell Culture, Injection, Marker, Fluorescence, Mouse Assay

    Effect of Pde4d KD and fear conditioning on spines in retrosplenial layer 2/3 neurons. ( A ) Representative laser scanning confocal micrographs of dendritic segments and dendritic spines in RSC L2/3 pyramidal neurons. Examples of thin-necked spines (yellow arrow), mushroom (arrowhead), and stubby (double arrowhead) spines in the 3× magnification insert represent the different spine classes. ( B ) Total spine number is increased by Pde4d KD independent of training. ( C ) Fear conditioning did not increase spine density in shGFP control virus injected mice. ( D ) Increased density of mushroom spines is observed after Pde4d KD and training. Stubby spines, in contrast, occur at a higher density in shPde4d virus injected mice irrespective of training when compared shGFP controls shown in panel C. n = 44–56 samples from 8 mice per group. Significant differences from untrained mice (naive) mice are indicated by an asterisk (*), and changes from shGFP control by the greek letter Chi (χ).
    Figure Legend Snippet: Effect of Pde4d KD and fear conditioning on spines in retrosplenial layer 2/3 neurons. ( A ) Representative laser scanning confocal micrographs of dendritic segments and dendritic spines in RSC L2/3 pyramidal neurons. Examples of thin-necked spines (yellow arrow), mushroom (arrowhead), and stubby (double arrowhead) spines in the 3× magnification insert represent the different spine classes. ( B ) Total spine number is increased by Pde4d KD independent of training. ( C ) Fear conditioning did not increase spine density in shGFP control virus injected mice. ( D ) Increased density of mushroom spines is observed after Pde4d KD and training. Stubby spines, in contrast, occur at a higher density in shPde4d virus injected mice irrespective of training when compared shGFP controls shown in panel C. n = 44–56 samples from 8 mice per group. Significant differences from untrained mice (naive) mice are indicated by an asterisk (*), and changes from shGFP control by the greek letter Chi (χ).

    Techniques Used: Injection, Mouse Assay

    Effect of Pde4d KD and fear conditioning on spines in hippocampal CA1. ( A ) Representative laser-scanning confocal micrographs of dorsal HC stratum radiatum used for spine analysis. Dendritic segments of CA1 pyramidal neuron secondary apical branches were sampled 0–50 μm from branchpoint. Examples of thin-necked spines (yellow arrow), mushroom (arrowhead), and stubby (double arrowhead) spines in the 3× magnification inset represent the different spine classes. ( B ) Total spine number is increased by training independent of Pde4d KD. ( C ) The training induced increase mainly involves mushroom spines in the shGFP control virus injected HC. ( D ) The training induced increase involves mushroom and stubby spines after Pde4d KD. n = 47–52 cells from 7–8 mice per group. Significant differences from untrained mice (naive) are indicated by an asterisk (*).
    Figure Legend Snippet: Effect of Pde4d KD and fear conditioning on spines in hippocampal CA1. ( A ) Representative laser-scanning confocal micrographs of dorsal HC stratum radiatum used for spine analysis. Dendritic segments of CA1 pyramidal neuron secondary apical branches were sampled 0–50 μm from branchpoint. Examples of thin-necked spines (yellow arrow), mushroom (arrowhead), and stubby (double arrowhead) spines in the 3× magnification inset represent the different spine classes. ( B ) Total spine number is increased by training independent of Pde4d KD. ( C ) The training induced increase mainly involves mushroom spines in the shGFP control virus injected HC. ( D ) The training induced increase involves mushroom and stubby spines after Pde4d KD. n = 47–52 cells from 7–8 mice per group. Significant differences from untrained mice (naive) are indicated by an asterisk (*).

    Techniques Used: Injection, Mouse Assay

    68) Product Images from "Developmentally Programmed 3? CpG Island Methylation Confers Tissue- and Cell-Type-Specific Transcriptional Activation"

    Article Title: Developmentally Programmed 3? CpG Island Methylation Confers Tissue- and Cell-Type-Specific Transcriptional Activation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01124-12

    Functional characterization of tissue-/cell-type-specifically methylated 3′ CGIs reveals CTCF-dependent enhancer-blocking mechanism for transcriptional regulation. (A) Luciferase reporter assays. (1) Promoter assays for the PRR15 and Hic1 3′
    Figure Legend Snippet: Functional characterization of tissue-/cell-type-specifically methylated 3′ CGIs reveals CTCF-dependent enhancer-blocking mechanism for transcriptional regulation. (A) Luciferase reporter assays. (1) Promoter assays for the PRR15 and Hic1 3′

    Techniques Used: Functional Assay, Methylation, Blocking Assay, Luciferase

    Detailed analysis of tissue-specific PRR15 3′ CGI methylation and gene expression. (A) DNA methylation mapping of the PRR15 locus in human brain and pancreas. A diagram of the CpG map is shown at the top. Each vertical line represents a CpG site
    Figure Legend Snippet: Detailed analysis of tissue-specific PRR15 3′ CGI methylation and gene expression. (A) DNA methylation mapping of the PRR15 locus in human brain and pancreas. A diagram of the CpG map is shown at the top. Each vertical line represents a CpG site

    Techniques Used: Methylation, Expressing, DNA Methylation Assay

    69) Product Images from "Acetylcholine Receptor Activation as a Modulator of Glioblastoma Invasion"

    Article Title: Acetylcholine Receptor Activation as a Modulator of Glioblastoma Invasion

    Journal: Cells

    doi: 10.3390/cells8101203

    AChRs are upregulated in active zones of infiltration in GBM patients. mRNA expression of CHRNA7 , CHRNB2 , CHRM1 , and CHRM3 within cellular tumor (CT), infiltrating tumor (IT), and leading edge (LE) populations from GBM samples in the Ivy GAP dataset. Tukey’s honest significant difference test was used to compare the expression changes in the IT and LE populations in comparison to the expression within the CT population. CHRNA7 : CT vs. LE p
    Figure Legend Snippet: AChRs are upregulated in active zones of infiltration in GBM patients. mRNA expression of CHRNA7 , CHRNB2 , CHRM1 , and CHRM3 within cellular tumor (CT), infiltrating tumor (IT), and leading edge (LE) populations from GBM samples in the Ivy GAP dataset. Tukey’s honest significant difference test was used to compare the expression changes in the IT and LE populations in comparison to the expression within the CT population. CHRNA7 : CT vs. LE p

    Techniques Used: Expressing

    70) Product Images from "Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition"

    Article Title: Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2014.08.026

    Proinflammatory cytokines induce markers of epithelial to mesenchymal transition (EMT) in human esophageal epithelial cells in a partially reversible fashion. (A) Morphologic changes in EPC2-hTERT cells stimulated with TNFα, TGFβ, and IL1β compared to unstimulated cells. Arrows highlight representative spindle-shaped cells. Pictures were taken at 100X. (B) mRNA expression of αSMA, vimentin and E-cadherin in EPC2-hTERT cells stimulated in triplicate following 4-week continuous stimulation with TNFα, TGFβ, or IL1β compared to 3 weeks of cytokine stimulation and 1 week of cytokine-free rescue. * p
    Figure Legend Snippet: Proinflammatory cytokines induce markers of epithelial to mesenchymal transition (EMT) in human esophageal epithelial cells in a partially reversible fashion. (A) Morphologic changes in EPC2-hTERT cells stimulated with TNFα, TGFβ, and IL1β compared to unstimulated cells. Arrows highlight representative spindle-shaped cells. Pictures were taken at 100X. (B) mRNA expression of αSMA, vimentin and E-cadherin in EPC2-hTERT cells stimulated in triplicate following 4-week continuous stimulation with TNFα, TGFβ, or IL1β compared to 3 weeks of cytokine stimulation and 1 week of cytokine-free rescue. * p

    Techniques Used: Expressing

    71) Product Images from "Aberrant WNT/?-catenin signaling in parathyroid carcinoma"

    Article Title: Aberrant WNT/?-catenin signaling in parathyroid carcinoma

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-294

    Effects of 5 μM 5-aza-2'-deoxycytidine (Aza) on in vitro cultured primary parathyroid carcinoma cells (PC1) . A, APC/GAPDH mRNA expression ratio as determined by real-time quantitative RT-PCR analysis. B, Western blotting analysis of APC. Coomassie blue-stained filter is shown as loading control. C, cell viability. D, apoptosis. Camptothecin at 0.1 μg/ml was used as positive control.
    Figure Legend Snippet: Effects of 5 μM 5-aza-2'-deoxycytidine (Aza) on in vitro cultured primary parathyroid carcinoma cells (PC1) . A, APC/GAPDH mRNA expression ratio as determined by real-time quantitative RT-PCR analysis. B, Western blotting analysis of APC. Coomassie blue-stained filter is shown as loading control. C, cell viability. D, apoptosis. Camptothecin at 0.1 μg/ml was used as positive control.

    Techniques Used: In Vitro, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Staining, Positive Control

    72) Product Images from "MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex"

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0275-4

    MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 methylation inhibits the interaction of GLI1 with its E3 ligase complex, ITCH/NUMB, resulting in GLI1 stabilisation. a Interaction of GLI1 and endogenous ITCH or NUMB from stably PRMT5-knockdown or MEP50-knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. MG132 (50 μM) was applied for 4 h before harvesting. b Interaction of GLI1 mutants with endogenous ITCH or NUMB in C3H10T1/2 cells. The cells were transfected as indicated. At 48 h post-transfection, 50 μM MG132 was applied for 4 h, and then the cells were lysed and subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with antibodies against the indicated proteins. c In vivo ubiquitination of HA-GLI1-RK mutants. Cells were transfected and cultured for 24 h, followed by treatment with 50 µM MG132 for 4 h before harvesting. Ubiquitinated GLI1 was detected by immuoprecipitation with an anti-HA (3F10) antibody and immunoblotting with anti-FLAG (upper panel) or anti-HA (lower panel) antibodies. The asterisk denotes non-specific bands. d Schematic diagram of the mechanism of PRMT5/MEP50-mediated GLI1 stabilisation. When the HH signalling pathway inactivates, the ITCH/NUMB E3 ligase complex binds to and ubiquitinates GLI1 for proteasomal degradation. In turn, under HH signalling pathway activation, the MEP50/PRMT5 complex methylates GLI1 to dissociate the ITCH/NUMB complex from GLI1, resulting in GLI1 stabilisation. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Methylation, Stable Transfection, Recombinant, Transfection, Immunoprecipitation, In Vivo, Cell Culture, Activation Assay

    PRMT5 and MEP50 expression is upregulated in HH pathway-activated cancers, and PRMT5 inhibition is a potential therapeutic strategy for such cancers. a , b Immunoblot analysis of endogenous GLI1 in H146 and AGS cells stably expressing PRMT5 ( a ) or MEP50 ( b ) siRNAs. c Immunoblot analysis of endogenous GLI1 in H146 cells stably expressing GLI1 siRNA. In a – c , siRNAs were stably expressed via recombinant retroviruses. d Growth curves of PRMT5, MEP50, and GLI1-knockdown H146 SCLC cells. Results are shown in the mean ± s.d. of triplicate experiments. e A quantitative colony formation assay was performed by plating cells at a density of 1 × 10 4 cells in a six-well plate and incubating them for 14 days. Surviving colonies were counted and represented as the mean ± s.d. of three independent wells. In d and e , siMEP50, siPRMT5, or siGLI1 was stably expressed via recombinant retrovirus in H146 cells. f , g IC 50 values of cyclopamine in PRMT5-knockdown or MEP50-knockdown AGS cells. siRNAs were stably expressed via recombinant retroviruses. Cell viability ( e ) is shown as the mean ± s.d. n = 4. IC 50 values of cyclopamine are shown in g . h – j Upregulated expression of PRMT5 , MEP50 , and GLI1 target genes in small cell lung carcinoma ( h ), gastric adenocarcinoma ( i ), and skin basal cell carcinoma ( j ) from the ONCOMINE database ( https://www.oncomine.org/ ). The threshold of data was p ≤ 0.05. Each boxplot shows the log 2 maximum, minimum, and median signal intensity of each mRNA from the corresponding expression array. Bold lines on each boxplot define the median value. P -values and sample numbers are indicated in each panel. Unprocessed original scans of blots are shown in Supplementary Fig. 6 . Source data of d – f is shown in Supplementary Data 2
    Figure Legend Snippet: PRMT5 and MEP50 expression is upregulated in HH pathway-activated cancers, and PRMT5 inhibition is a potential therapeutic strategy for such cancers. a , b Immunoblot analysis of endogenous GLI1 in H146 and AGS cells stably expressing PRMT5 ( a ) or MEP50 ( b ) siRNAs. c Immunoblot analysis of endogenous GLI1 in H146 cells stably expressing GLI1 siRNA. In a – c , siRNAs were stably expressed via recombinant retroviruses. d Growth curves of PRMT5, MEP50, and GLI1-knockdown H146 SCLC cells. Results are shown in the mean ± s.d. of triplicate experiments. e A quantitative colony formation assay was performed by plating cells at a density of 1 × 10 4 cells in a six-well plate and incubating them for 14 days. Surviving colonies were counted and represented as the mean ± s.d. of three independent wells. In d and e , siMEP50, siPRMT5, or siGLI1 was stably expressed via recombinant retrovirus in H146 cells. f , g IC 50 values of cyclopamine in PRMT5-knockdown or MEP50-knockdown AGS cells. siRNAs were stably expressed via recombinant retroviruses. Cell viability ( e ) is shown as the mean ± s.d. n = 4. IC 50 values of cyclopamine are shown in g . h – j Upregulated expression of PRMT5 , MEP50 , and GLI1 target genes in small cell lung carcinoma ( h ), gastric adenocarcinoma ( i ), and skin basal cell carcinoma ( j ) from the ONCOMINE database ( https://www.oncomine.org/ ). The threshold of data was p ≤ 0.05. Each boxplot shows the log 2 maximum, minimum, and median signal intensity of each mRNA from the corresponding expression array. Bold lines on each boxplot define the median value. P -values and sample numbers are indicated in each panel. Unprocessed original scans of blots are shown in Supplementary Fig. 6 . Source data of d – f is shown in Supplementary Data 2

    Techniques Used: Expressing, Inhibition, Stable Transfection, Recombinant, Colony Assay

    GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Incubation

    MEP50/PRMT5 complex supports GLI1 activation through GLI1 stabilisation downstream of the HH signalling pathway. a–c Endogenous GLI1/MEP50/PRMT5 complex in C3H10T1/2 cells. Cells were treated with SAG for 36 h, and complex was detected by immunoprecipitation (IP) with anti-PRMT5 (D5P2T) ( a ), anti-MEP50 (ERP10708 [B]) ( b ), or anti-GLI1 (V812) ( c ) antibodies, followed by immunoblot (IB) with antibodies against indicated proteins. d Dissociation of PRMT5 and GLI1 in stable MEP50 knockdown C3H10T1/2 cells by siMEP50-m2. Cells were treated with SAG for 48 h and treated with 50 μM MG132 for 4 h. GLI1/PRMT5 complex was detected by immunoprecipitation with anti-PRMT5 (D5P2T) or anti-GLI1 (V812) antibodies, followed by immunoblot with indicated antibodies. e Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing MEP50 siRNAs (siMEP50-m1 or siMEP50-m2). f Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing two independent PRMT5 siRNAs. g Immunoblot of nuclear and cytoplasmic GLI1 and MEP50 in stable MEP50-knockdown (siMEP50-m2) or control siGFP-expressing cells treated with 300 nM SAG. Cells were treated with SAG for 24 h and separated into cytosol and nucleus fractions. h Immunoblot analysis of endogenous nuclear and cytoplasmic GLI1 and PRMT5 in stable PRMT5-knockdown (siPRMT5-m2) C3H10T1/2 cells. Cells were treated with 300 nM SAG for 24 h and then separated as in h . i In vivo ubiquitination of GLI1 in C3H10T1/2 cells with or without expression of siMEP50. FLAG-ubiquitin was transfected into C3H10T1/2 cells. After 48 h of transfection, then cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was immunoprecipitated with an anti-GLI1 (C-1) antibody, followed by immunoblotting with indicated antibodies. j In vivo ubiquitination of GLI1 in C3H10T1/2 cells with exogenous expression of PRMT5 or MEP50. FLAG-ubiquitin and HA-PRMT5, HA-PRMT5 G367A/R368A (inactive form of PRMT5), or Myc-MEP50 were transfected into C3H10T1/2 cells. After 48 h, the cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was detected as described in i . In a , i and j , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: MEP50/PRMT5 complex supports GLI1 activation through GLI1 stabilisation downstream of the HH signalling pathway. a–c Endogenous GLI1/MEP50/PRMT5 complex in C3H10T1/2 cells. Cells were treated with SAG for 36 h, and complex was detected by immunoprecipitation (IP) with anti-PRMT5 (D5P2T) ( a ), anti-MEP50 (ERP10708 [B]) ( b ), or anti-GLI1 (V812) ( c ) antibodies, followed by immunoblot (IB) with antibodies against indicated proteins. d Dissociation of PRMT5 and GLI1 in stable MEP50 knockdown C3H10T1/2 cells by siMEP50-m2. Cells were treated with SAG for 48 h and treated with 50 μM MG132 for 4 h. GLI1/PRMT5 complex was detected by immunoprecipitation with anti-PRMT5 (D5P2T) or anti-GLI1 (V812) antibodies, followed by immunoblot with indicated antibodies. e Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing MEP50 siRNAs (siMEP50-m1 or siMEP50-m2). f Immunoblot of endogenous GLI1 in C3H10T1/2 cells expressing two independent PRMT5 siRNAs. g Immunoblot of nuclear and cytoplasmic GLI1 and MEP50 in stable MEP50-knockdown (siMEP50-m2) or control siGFP-expressing cells treated with 300 nM SAG. Cells were treated with SAG for 24 h and separated into cytosol and nucleus fractions. h Immunoblot analysis of endogenous nuclear and cytoplasmic GLI1 and PRMT5 in stable PRMT5-knockdown (siPRMT5-m2) C3H10T1/2 cells. Cells were treated with 300 nM SAG for 24 h and then separated as in h . i In vivo ubiquitination of GLI1 in C3H10T1/2 cells with or without expression of siMEP50. FLAG-ubiquitin was transfected into C3H10T1/2 cells. After 48 h of transfection, then cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was immunoprecipitated with an anti-GLI1 (C-1) antibody, followed by immunoblotting with indicated antibodies. j In vivo ubiquitination of GLI1 in C3H10T1/2 cells with exogenous expression of PRMT5 or MEP50. FLAG-ubiquitin and HA-PRMT5, HA-PRMT5 G367A/R368A (inactive form of PRMT5), or Myc-MEP50 were transfected into C3H10T1/2 cells. After 48 h, the cells were treated with 50 µM MG132 for 4 h. Endogenous ubiquitinated GLI1 was detected as described in i . In a , i and j , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Activation Assay, Immunoprecipitation, Expressing, In Vivo, Transfection

    MEP50/PRMT5 complex induces GLI1 methylation. a , b Methylation of GLI1 in MEP50- ( a ) or PRMT5- ( b ) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in ( c ). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. 4 . In c , data represent one of three independent experiments with similar results. In a and d , data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6
    Figure Legend Snippet: MEP50/PRMT5 complex induces GLI1 methylation. a , b Methylation of GLI1 in MEP50- ( a ) or PRMT5- ( b ) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in ( c ). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. 4 . In c , data represent one of three independent experiments with similar results. In a and d , data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Techniques Used: Methylation, Stable Transfection, Recombinant, Transfection, Cell Culture, Immunoprecipitation, In Vitro, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Staining

    MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Recombinant, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Quantitative RT-PCR

    73) Product Images from "REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors"

    Article Title: REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors

    Journal: Nature neuroscience

    doi: 10.1038/nn.3214

    Maternal deprivation disrupts the increase in REST, epigenetic remodeling and decrease in GluN2B during postnatal development a , Diagram illustrating the maternal deprivation paradigm. b , Representative Western blot of hippocampal nuclear fraction. c , Summary plot ( left panel ) showing a robust decrease in REST protein expression in the hippocampus of maternally-deprived vs . normally-reared pups (n = 3). In the dentate gyrus, whereas REST mRNA decreases (n = 3), GluN2B mRNA increases ( right panel; n = 3). d , Abundance of REST and e , H3K27me3 at the grin2b promoter are decreased relative to age-matched, normally-reared rats ( n = 3). f , Representative Western blots ( left panel ) and summary data ( right panel ), showing that whereas maternal deprivation increases GluN2B protein in total lysate, endoplasmic reticulum (ER) and postsynaptic density (PSD) fractions, GluN2A and GluA2 are unchanged in all fractions. GluN1 was reduced in the ER fraction and increased in the PSD in the hippocampus from maternally-deprived pups vs. normally-reared pups. All samples were assessed at P28–31 (n = 3). Total lysate samples were normalized to β-actin, ER samples were normalized to GRP78, an ER marker, and PSD samples were normalized to PSD-95, which is not altered after maternally-deprived ( see Supplementary Table 9 and full length blots in Supplementary Fig. 12 ).
    Figure Legend Snippet: Maternal deprivation disrupts the increase in REST, epigenetic remodeling and decrease in GluN2B during postnatal development a , Diagram illustrating the maternal deprivation paradigm. b , Representative Western blot of hippocampal nuclear fraction. c , Summary plot ( left panel ) showing a robust decrease in REST protein expression in the hippocampus of maternally-deprived vs . normally-reared pups (n = 3). In the dentate gyrus, whereas REST mRNA decreases (n = 3), GluN2B mRNA increases ( right panel; n = 3). d , Abundance of REST and e , H3K27me3 at the grin2b promoter are decreased relative to age-matched, normally-reared rats ( n = 3). f , Representative Western blots ( left panel ) and summary data ( right panel ), showing that whereas maternal deprivation increases GluN2B protein in total lysate, endoplasmic reticulum (ER) and postsynaptic density (PSD) fractions, GluN2A and GluA2 are unchanged in all fractions. GluN1 was reduced in the ER fraction and increased in the PSD in the hippocampus from maternally-deprived pups vs. normally-reared pups. All samples were assessed at P28–31 (n = 3). Total lysate samples were normalized to β-actin, ER samples were normalized to GRP78, an ER marker, and PSD samples were normalized to PSD-95, which is not altered after maternally-deprived ( see Supplementary Table 9 and full length blots in Supplementary Fig. 12 ).

    Techniques Used: Western Blot, Expressing, Marker

    74) Product Images from "Recurrent hotspot mutations in HRAS Q61 and PI3K-AKT pathway genes as drivers of breast adenomyoepitheliomas"

    Article Title: Recurrent hotspot mutations in HRAS Q61 and PI3K-AKT pathway genes as drivers of breast adenomyoepitheliomas

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04128-5

    Expression of mutant HRAS Q61R results in the acquisition of a partial myoepithelial phenotype in non-malignant breast epithelial cells. a Representative western blot (left) analysis of total protein expression of alpha-smooth muscle actin (αSMA), TIMP1, cytokeratin 5 (CK5), E-cadherin, vimentin, and nuclear protein expression of ∆N-p63 and TA-p63 in MCF-10A P , MCF-10A H1047R , and MCF-12A cells stably expressing empty vector (EV), HRAS WT , or mutant HRAS Q61R . α-Tubulin and Histone H3 were used as protein loading controls for total and nuclear protein expression, respectively. Quantification (right) using LI-COR is shown based on experiments done in triplicate. Comparisons of protein levels were performed between HRAS WT and mutant HRAS Q61R , both relative to EV. Error bars, s.d. of mean ( n = 3). n.s. = not significant, * P
    Figure Legend Snippet: Expression of mutant HRAS Q61R results in the acquisition of a partial myoepithelial phenotype in non-malignant breast epithelial cells. a Representative western blot (left) analysis of total protein expression of alpha-smooth muscle actin (αSMA), TIMP1, cytokeratin 5 (CK5), E-cadherin, vimentin, and nuclear protein expression of ∆N-p63 and TA-p63 in MCF-10A P , MCF-10A H1047R , and MCF-12A cells stably expressing empty vector (EV), HRAS WT , or mutant HRAS Q61R . α-Tubulin and Histone H3 were used as protein loading controls for total and nuclear protein expression, respectively. Quantification (right) using LI-COR is shown based on experiments done in triplicate. Comparisons of protein levels were performed between HRAS WT and mutant HRAS Q61R , both relative to EV. Error bars, s.d. of mean ( n = 3). n.s. = not significant, * P

    Techniques Used: Expressing, Mutagenesis, Western Blot, Stable Transfection, Plasmid Preparation

    75) Product Images from "Corilagin suppresses cholangiocarcinoma progression through Notch signaling pathway in vitro and in vivo"

    Article Title: Corilagin suppresses cholangiocarcinoma progression through Notch signaling pathway in vitro and in vivo

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2016.3413

    Corilagin induces CCA cell apoptosis. (A and B) Two CCA cell lines (QBC939 and MZ-Cha-1) were treated with corilagin at increasing concentrations for 24 h and analyzed for apoptosis by flow cytometry. (C and D) QBC939 and MZ-Cha-1 cells were harvested and RNA was isolated from each group. SYBR green real-time PCR reactions were performed to detect the alterations of bcl-2 (C) and caspase 3 (D) gene expression treated with corilagin for 24 h. Each column represents the mean of three independent experiments. * P
    Figure Legend Snippet: Corilagin induces CCA cell apoptosis. (A and B) Two CCA cell lines (QBC939 and MZ-Cha-1) were treated with corilagin at increasing concentrations for 24 h and analyzed for apoptosis by flow cytometry. (C and D) QBC939 and MZ-Cha-1 cells were harvested and RNA was isolated from each group. SYBR green real-time PCR reactions were performed to detect the alterations of bcl-2 (C) and caspase 3 (D) gene expression treated with corilagin for 24 h. Each column represents the mean of three independent experiments. * P

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

    Related Articles

    Amplification:

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape
    Article Snippet: The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block. .. PCR reaction settings were as follows: hot start for 15 min at 95 °C, and amplification 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C repeated for 40 cycles with data recorded twice during the extension step.

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: .. Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems). .. Real-time data were collected by CopyCaller v2.0 software.

    Positive Control:

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women
    Article Snippet: The reaction contained 100 ng of DNA, 900 nM of each primer, 250 nM probe, and the TaqMan Universal Master Mix II (Applied Biosystems). .. As a negative control, nuclease-free sterile water was reacted with the primers in the absence of a template, and the MMTV env gene from the C3H strain (GenBank AF228552) was used as a positive control.

    Synthesized:

    Article Title: Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin
    Article Snippet: TaqMan real‐time polymerase chain reaction (qPCR) Gene expression analysis was performed with TaqMan Universal Master Mix II with UNG (Applied Biosystems, MA, USA) in a QuantStudio 12K Flex Real‐Time PCR System (Applied Biosystems). .. The cDNAs thus synthesized were subjected to qPCR using gene‐specific TaqMan probes as listed in Table .

    Blocking Assay:

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape
    Article Snippet: .. The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block. .. All protocols were performed following the manufacturer’s instructions. qPCR reactions for TurboGFP were performed using QuantiTect SYBR Green PCR kit (204143) and run using the Stratagene Mx300P qPCR system (Agilent Genomics).

    Real-time Polymerase Chain Reaction:

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape
    Article Snippet: .. The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block. .. All protocols were performed following the manufacturer’s instructions. qPCR reactions for TurboGFP were performed using QuantiTect SYBR Green PCR kit (204143) and run using the Stratagene Mx300P qPCR system (Agilent Genomics).

    Article Title: miR-182 Regulates Slit2-Mediated Axon Guidance by Modulating the Local Translation of a Specific mRNA
    Article Snippet: Paragraph title: TaqMan qPCR for miR-182 ... The cDNA obtained was used for the TaqMan Micro RNA assay using xtr-miR-182-5p and U6 snRNA-specific primers and probes and the TaqMan Universal Master Mix II (MMIX II) no AmpErase Uracil N-Glycosylase (UNG) (all Thermo Fisher).

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: Paragraph title: Quantitative real-time polymerase chain reaction (qRT-PCR) ... 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions.

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women
    Article Snippet: Paragraph title: Real-time PCR ... The reaction contained 100 ng of DNA, 900 nM of each primer, 250 nM probe, and the TaqMan Universal Master Mix II (Applied Biosystems).

    Article Title: MicroRNA-1 inhibits tumorigenicity of esophageal squamous cell carcinoma and enhances sensitivity to gefitinib
    Article Snippet: Total RNA (500 ng) was quantitated at 260 nm and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China) at 37°C for 15 min and 85°C for 30 sec. qPCR was performed using the SYBR Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.) in the ABI PRISM 7900HT (Applied Biosystems; Thermo Fisher Scientific, Inc.) system. .. The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-1 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin
    Article Snippet: .. TaqMan real‐time polymerase chain reaction (qPCR) Gene expression analysis was performed with TaqMan Universal Master Mix II with UNG (Applied Biosystems, MA, USA) in a QuantStudio 12K Flex Real‐Time PCR System (Applied Biosystems). .. In brief, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) followed by RNA clean‐up using the RNeasy kit (Qiagen, Valencia, CA, USA).

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: Each assay was run as a TaqMan real-time PCR reaction in triplicate, using an FAM dye-based assay targeted to 11p13 and a VIC dye-based assay for the reference gene, RNase P (PN 4316844 from Applied Biosystems). .. Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems).

    Article Title: CIC protein instability contributes to tumorigenesis in glioblastoma
    Article Snippet: .. One μg RNA was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) and quantitative real-time PCR performed was performed using TaqMan universal master mix II (Life Technologies) according the manufacturers protocol with a StepOne Real-Time PCR machine (Life Technologies). .. Human CIC (Hs00209424_m1), PJA1 (Hs00254654_s1), ETV5 (Hs00927557_m1), ETV1 (Hs00951951_m1), and β-actin (Hs01060665_g1) and mouse ETV1 (Mm00514804_m1) were purchased from Life Technologies.

    Article Title: The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR
    Article Snippet: Cards were loaded by mixing 50 µL DNA extract with 50 µL TaqMan Universal Master Mix II (Life Technologies Corporation). .. Cards were run on the ViiA™ 7 real-time PCR system (Life Technologies Corporation) using PCR cycling conditions comprising 30 s at 60°C, 10 min at 95°C followed by 40 two-step cycles of 15 s at 95°C and 1 min at 60°C.

    Article Title: Analyzing the Circulating MicroRNAs in Exosomes/Extracellular Vesicles from Serum or Plasma by qRT-PCR
    Article Snippet: Paragraph title: 2.5 cDNA and Quantitative Real-Time PCR (qRT-PCR) Profiling Reagents and Solutions ... TaqMan® Universal Master Mix, No AmpErase® UNG (Applied Biosystems).

    Article Title: Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR
    Article Snippet: .. Real-time PCR was performed in 25-μl reaction mixtures containing 12.5 μl of TaqMan universal master mix (Applied Biosystems), 300 nM concentrations of each primer, 250 nM WT probe, 200 nM vaccine probe, and 5 μl of sample DNA. .. Thermocycling was performed on the Prism 7900HT (Applied Biosystems) and consisted of 2 min at 50°C, 10 min at 95°C for AmpliTaq Gold activation, and 40 cycles of 95°C for 15 s and 60°C for 1 min.

    Multiplex Assay:

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape
    Article Snippet: Quantitative PCR miTRAP cDNA for use with TaqMan miRNA multiplex arrays was first preamplified using TaqMan PreAmp Master Mix (ThermoFisher, 4,391,128) and custom miRNA PreAmp primer pool. .. The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block.

    Incubation:

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: Then, the cells were incubated with the nanoparticles containing anti-miRNA (100 nM) in 1 mL of medium for 48 h. The expression levels of miR-210 were evaluated by TaqMan qRT-PCR. .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions.

    Expressing:

    Article Title: Participation of the miR-22-HDAC4-DLCO Axis in Patients with COPD by Tobacco and Biomass
    Article Snippet: Validation of Samples by TaqMan RT-qPCR The cDNA was obtained from serum using the RT (Reverse Transciption) kit and TaqMan Universal Master Mix II with UNG (Uracil-N-glycosylate; Applied Biosystems-Thermo Fisher Scientific). .. The expression level of miRNA was evaluated using the comparative threshold cycle method (ΔCt) and was normalized with a corresponding sequence of C. elegans miRNA as an exogenous normalizer in gene expression (spike-in cel-miR-39).

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: Then, the cells were incubated with the nanoparticles containing anti-miRNA (100 nM) in 1 mL of medium for 48 h. The expression levels of miR-210 were evaluated by TaqMan qRT-PCR. .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions.

    Article Title: MicroRNA-1 inhibits tumorigenicity of esophageal squamous cell carcinoma and enhances sensitivity to gefitinib
    Article Snippet: PIK3CA expression levels were normalized to β-actin expression using the 2−ΔΔCt method ( ). .. The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-1 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin
    Article Snippet: .. TaqMan real‐time polymerase chain reaction (qPCR) Gene expression analysis was performed with TaqMan Universal Master Mix II with UNG (Applied Biosystems, MA, USA) in a QuantStudio 12K Flex Real‐Time PCR System (Applied Biosystems). .. In brief, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) followed by RNA clean‐up using the RNeasy kit (Qiagen, Valencia, CA, USA).

    Concentration Assay:

    Article Title: Participation of the miR-22-HDAC4-DLCO Axis in Patients with COPD by Tobacco and Biomass
    Article Snippet: Validation of Samples by TaqMan RT-qPCR The cDNA was obtained from serum using the RT (Reverse Transciption) kit and TaqMan Universal Master Mix II with UNG (Uracil-N-glycosylate; Applied Biosystems-Thermo Fisher Scientific). .. The relative concentration of miR-22 was described as a fold change (2-ΔCt), by using the equation ΔCt = (Ct miRNA-Ct spike).

    Cell Culture:

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: The mirVana miRNA Isolation Kit (Ambion, USA) was used for total RNA extraction from cultured cells. .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions.

    Generated:

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women
    Article Snippet: The reaction contained 100 ng of DNA, 900 nM of each primer, 250 nM probe, and the TaqMan Universal Master Mix II (Applied Biosystems). .. To calculate the sensitivity of the test and the viral copy number, we generated a complete standard curve with the plasmid DNA from the MMTV C3H strain by preparing serial dilutions (10-1 – 10-7 ) for the positive control reaction with 1 ng of template.

    Polymerase Chain Reaction:

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape
    Article Snippet: The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block. .. All protocols were performed following the manufacturer’s instructions. qPCR reactions for TurboGFP were performed using QuantiTect SYBR Green PCR kit (204143) and run using the Stratagene Mx300P qPCR system (Agilent Genomics).

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: .. Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems). .. Real-time data were collected by CopyCaller v2.0 software.

    Article Title: The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR
    Article Snippet: The 13 additional PCR reactions in each channel, plus the manufacturers card control PCR, were filled with assays required for an alternate study. .. Cards were loaded by mixing 50 µL DNA extract with 50 µL TaqMan Universal Master Mix II (Life Technologies Corporation).

    Article Title: Analyzing the Circulating MicroRNAs in Exosomes/Extracellular Vesicles from Serum or Plasma by qRT-PCR
    Article Snippet: TaqMan® Universal Master Mix, No AmpErase® UNG (Applied Biosystems). .. 384-well PCR plates with optical adhesive film.

    Article Title: Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR
    Article Snippet: Real-time PCR was performed in 25-μl reaction mixtures containing 12.5 μl of TaqMan universal master mix (Applied Biosystems), 300 nM concentrations of each primer, 250 nM WT probe, 200 nM vaccine probe, and 5 μl of sample DNA. .. VZV DNA quantification was done by comparing the cycle threshold ( CT ) value (PCR cycle at which the reporter fluorescence reaches 20 standard deviations above background emissions) of the samples to the CT versus plasmid quantity standard curve.

    Fluorescence:

    Article Title: Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR
    Article Snippet: Real-time PCR was performed in 25-μl reaction mixtures containing 12.5 μl of TaqMan universal master mix (Applied Biosystems), 300 nM concentrations of each primer, 250 nM WT probe, 200 nM vaccine probe, and 5 μl of sample DNA. .. VZV DNA quantification was done by comparing the cycle threshold ( CT ) value (PCR cycle at which the reporter fluorescence reaches 20 standard deviations above background emissions) of the samples to the CT versus plasmid quantity standard curve.

    Isolation:

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: The mirVana miRNA Isolation Kit (Ambion, USA) was used for total RNA extraction from cultured cells. .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions.

    Article Title: CIC protein instability contributes to tumorigenesis in glioblastoma
    Article Snippet: Total RNA was isolated using the Qiagen RNeasy mini kit according to the manufacturer’s recommendations. .. One μg RNA was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) and quantitative real-time PCR performed was performed using TaqMan universal master mix II (Life Technologies) according the manufacturers protocol with a StepOne Real-Time PCR machine (Life Technologies).

    Size-exclusion Chromatography:

    Article Title: MicroRNA-1 inhibits tumorigenicity of esophageal squamous cell carcinoma and enhances sensitivity to gefitinib
    Article Snippet: Total RNA (500 ng) was quantitated at 260 nm and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China) at 37°C for 15 min and 85°C for 30 sec. qPCR was performed using the SYBR Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.) in the ABI PRISM 7900HT (Applied Biosystems; Thermo Fisher Scientific, Inc.) system. .. The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-1 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Sequencing:

    Article Title: Participation of the miR-22-HDAC4-DLCO Axis in Patients with COPD by Tobacco and Biomass
    Article Snippet: Validation of Samples by TaqMan RT-qPCR The cDNA was obtained from serum using the RT (Reverse Transciption) kit and TaqMan Universal Master Mix II with UNG (Uracil-N-glycosylate; Applied Biosystems-Thermo Fisher Scientific). .. The expression level of miRNA was evaluated using the comparative threshold cycle method (ΔCt) and was normalized with a corresponding sequence of C. elegans miRNA as an exogenous normalizer in gene expression (spike-in cel-miR-39).

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: Each probe was designed based on a genomic sequence (Homo_sapiens.GRCh37/hg19) using Applied Biosystems proprietary software. .. Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems).

    Article Title: Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR
    Article Snippet: Primer and probe design was performed by using Primer Express software, version 3.0 (Applied Biosystems), and sequence homogeneity was confirmed by comparison to all available sequences on the GenBank database by using BLAST ( ). .. Real-time PCR was performed in 25-μl reaction mixtures containing 12.5 μl of TaqMan universal master mix (Applied Biosystems), 300 nM concentrations of each primer, 250 nM WT probe, 200 nM vaccine probe, and 5 μl of sample DNA.

    Quantitative RT-PCR:

    Article Title: Participation of the miR-22-HDAC4-DLCO Axis in Patients with COPD by Tobacco and Biomass
    Article Snippet: .. Validation of Samples by TaqMan RT-qPCR The cDNA was obtained from serum using the RT (Reverse Transciption) kit and TaqMan Universal Master Mix II with UNG (Uracil-N-glycosylate; Applied Biosystems-Thermo Fisher Scientific). .. Pre-designed commercial assay of TaqMan probes from Thermo Fisher Scientific were specific to miR-22 (hsa-miR-22-3p ID 000398).

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions. .. MiRNA expression levels were expressed relative to the internal control according to the comparative threshold cycle (Ct) method.

    Article Title: MicroRNA-1 inhibits tumorigenicity of esophageal squamous cell carcinoma and enhances sensitivity to gefitinib
    Article Snippet: Paragraph title: Quantitative RT-PCR ... The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-1 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: CIC protein instability contributes to tumorigenesis in glioblastoma
    Article Snippet: Paragraph title: Quantitative real-time RT-PCR ... One μg RNA was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) and quantitative real-time PCR performed was performed using TaqMan universal master mix II (Life Technologies) according the manufacturers protocol with a StepOne Real-Time PCR machine (Life Technologies).

    Article Title: Analyzing the Circulating MicroRNAs in Exosomes/Extracellular Vesicles from Serum or Plasma by qRT-PCR
    Article Snippet: Paragraph title: 2.5 cDNA and Quantitative Real-Time PCR (qRT-PCR) Profiling Reagents and Solutions ... TaqMan® Universal Master Mix, No AmpErase® UNG (Applied Biosystems).

    CNV Assay:

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: Paragraph title: CNV assay ... Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems).

    RNA Extraction:

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: The mirVana miRNA Isolation Kit (Ambion, USA) was used for total RNA extraction from cultured cells. .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions.

    Plasmid Preparation:

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women
    Article Snippet: The reaction contained 100 ng of DNA, 900 nM of each primer, 250 nM probe, and the TaqMan Universal Master Mix II (Applied Biosystems). .. To calculate the sensitivity of the test and the viral copy number, we generated a complete standard curve with the plasmid DNA from the MMTV C3H strain by preparing serial dilutions (10-1 – 10-7 ) for the positive control reaction with 1 ng of template.

    Article Title: Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR
    Article Snippet: Real-time PCR was performed in 25-μl reaction mixtures containing 12.5 μl of TaqMan universal master mix (Applied Biosystems), 300 nM concentrations of each primer, 250 nM WT probe, 200 nM vaccine probe, and 5 μl of sample DNA. .. VZV DNA quantification was done by comparing the cycle threshold ( CT ) value (PCR cycle at which the reporter fluorescence reaches 20 standard deviations above background emissions) of the samples to the CT versus plasmid quantity standard curve.

    Software:

    Article Title: MicroRNA-1 inhibits tumorigenicity of esophageal squamous cell carcinoma and enhances sensitivity to gefitinib
    Article Snippet: Cycle threshold (Ct) values were determined using the SDS version 2.4 software (Applied Biosystems; Thermo Fisher Scientific, Inc.). .. The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-1 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: Each probe was designed based on a genomic sequence (Homo_sapiens.GRCh37/hg19) using Applied Biosystems proprietary software. .. Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems).

    Article Title: Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR
    Article Snippet: Primer and probe design was performed by using Primer Express software, version 3.0 (Applied Biosystems), and sequence homogeneity was confirmed by comparison to all available sequences on the GenBank database by using BLAST ( ). .. Real-time PCR was performed in 25-μl reaction mixtures containing 12.5 μl of TaqMan universal master mix (Applied Biosystems), 300 nM concentrations of each primer, 250 nM WT probe, 200 nM vaccine probe, and 5 μl of sample DNA.

    SYBR Green Assay:

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape
    Article Snippet: The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block. .. All protocols were performed following the manufacturer’s instructions. qPCR reactions for TurboGFP were performed using QuantiTect SYBR Green PCR kit (204143) and run using the Stratagene Mx300P qPCR system (Agilent Genomics).

    Negative Control:

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women
    Article Snippet: The reaction contained 100 ng of DNA, 900 nM of each primer, 250 nM probe, and the TaqMan Universal Master Mix II (Applied Biosystems). .. As a negative control, nuclease-free sterile water was reacted with the primers in the absence of a template, and the MMTV env gene from the C3H strain (GenBank AF228552) was used as a positive control.

    Laser Capture Microdissection:

    Article Title: miR-182 Regulates Slit2-Mediated Axon Guidance by Modulating the Local Translation of a Specific mRNA
    Article Snippet: TaqMan qPCR for miR-182 Total RNA collected following LCM (described earlier) was retro-transcribed using the TaqMan MiRNA Reverse Transcription Kit. .. The cDNA obtained was used for the TaqMan Micro RNA assay using xtr-miR-182-5p and U6 snRNA-specific primers and probes and the TaqMan Universal Master Mix II (MMIX II) no AmpErase Uracil N-Glycosylase (UNG) (all Thermo Fisher).

    Activation Assay:

    Article Title: Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR
    Article Snippet: Real-time PCR was performed in 25-μl reaction mixtures containing 12.5 μl of TaqMan universal master mix (Applied Biosystems), 300 nM concentrations of each primer, 250 nM WT probe, 200 nM vaccine probe, and 5 μl of sample DNA. .. Thermocycling was performed on the Prism 7900HT (Applied Biosystems) and consisted of 2 min at 50°C, 10 min at 95°C for AmpliTaq Gold activation, and 40 cycles of 95°C for 15 s and 60°C for 1 min.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher real time pcr system
    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time <t>PCR</t> and <t>TaqMan</t> ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.
    Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2976 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time pcr system/product/Thermo Fisher
    Average 99 stars, based on 2976 article reviews
    Price from $9.99 to $1999.99
    real time pcr system - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher steponeplus real time system
    p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems <t>StepOnePlus</t> Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p
    Steponeplus Real Time System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/steponeplus real time system/product/Thermo Fisher
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    steponeplus real time system - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher applied biosystemstm thermo fisher scientific steponeplustm real time pcr system
    p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems <t>StepOnePlus</t> Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p
    Applied Biosystemstm Thermo Fisher Scientific Steponeplustm Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/applied biosystemstm thermo fisher scientific steponeplustm real time pcr system/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    applied biosystemstm thermo fisher scientific steponeplustm real time pcr system - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    Image Search Results


    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time PCR and TaqMan ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.

    Journal: Molecular Medicine Reports

    Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

    doi: 10.3892/mmr.2018.9141

    Figure Lengend Snippet: Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time PCR and TaqMan ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.

    Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

    Techniques: Genotyping Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Fluorescence, Software, Polymerase Chain Reaction

    Detection of the BRCA1 ex9-12del mutation by real-time PCR and TaqMan ® probe. Red amplification curves represent the fluorescent signal given by the FAM ™ dye from the TaqMan ® probe detecting the variant allele in the four positive samples, which had a mean Ct of 27.46. No amplification curve is observed for the negative samples neither for negative controls (horizontal red lines). The ‘Y’ axis values represent the fluorescent signal normalized of the FAM ™ dye with the ROX, whereas the ‘X’ axis represent number of cycles. PCR. PCR, polymerase chain reaction; ROX, passive reference dye.

    Journal: Molecular Medicine Reports

    Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

    doi: 10.3892/mmr.2018.9141

    Figure Lengend Snippet: Detection of the BRCA1 ex9-12del mutation by real-time PCR and TaqMan ® probe. Red amplification curves represent the fluorescent signal given by the FAM ™ dye from the TaqMan ® probe detecting the variant allele in the four positive samples, which had a mean Ct of 27.46. No amplification curve is observed for the negative samples neither for negative controls (horizontal red lines). The ‘Y’ axis values represent the fluorescent signal normalized of the FAM ™ dye with the ROX, whereas the ‘X’ axis represent number of cycles. PCR. PCR, polymerase chain reaction; ROX, passive reference dye.

    Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

    Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Amplification, Variant Assay, Polymerase Chain Reaction

    p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo

    doi: 10.4049/jimmunol.1502399

    Figure Lengend Snippet: p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

    Article Snippet: QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System (Thermo fisher) using pre-designed TaqMan® Gene Expression Assays (Life Technologies) (18S RNA Mm03928990 and p21 Mm04205640) according to manufacturer's instructions.

    Techniques: Isolation, Cell Culture, Recombinant, FACS, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing