Structured Review

Kaneka Corp timp 1
Effects of riociguat on plasma and urinary protein levels of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 <t>(TIMP-1)</t> in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p
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Images

1) Product Images from "Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats"

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021853

Effects of riociguat on plasma and urinary protein levels of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p
Figure Legend Snippet: Effects of riociguat on plasma and urinary protein levels of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p

Techniques Used:

Effects of riociguat on mRNA expression of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the left ventricle and the renal cortex in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p
Figure Legend Snippet: Effects of riociguat on mRNA expression of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the left ventricle and the renal cortex in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p

Techniques Used: Expressing

2) Product Images from "The Expression of the Hepatocyte SLAMF3 (CD229) Receptor Enhances the Hepatitis C Virus Infection"

Article Title: The Expression of the Hepatocyte SLAMF3 (CD229) Receptor Enhances the Hepatitis C Virus Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099601

SLAMF3 over-expression increases the HCV infectivity. Mock and Huh7+ cells were challenged with HCVcc (A, top panel). (A) Supernatants were harvested at 72 h p.i., released viruses quantified by QPCR and equal viral loads from both supernatants were used to infect native Huh-7 cells (A bottom panel). (B) Viral replication was assessed by intracellular viral E2 protein expression measurement by flow cytometry and quantitation of released viral RNA at 72 h p.i. (mean of three independent experiments; error bars: SD *p
Figure Legend Snippet: SLAMF3 over-expression increases the HCV infectivity. Mock and Huh7+ cells were challenged with HCVcc (A, top panel). (A) Supernatants were harvested at 72 h p.i., released viruses quantified by QPCR and equal viral loads from both supernatants were used to infect native Huh-7 cells (A bottom panel). (B) Viral replication was assessed by intracellular viral E2 protein expression measurement by flow cytometry and quantitation of released viral RNA at 72 h p.i. (mean of three independent experiments; error bars: SD *p

Techniques Used: Over Expression, Infection, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Quantitation Assay

3) Product Images from "The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins"

Article Title: The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200403069

Inhibition of S6K restores IRS-1 mRNA. (A) IRS-1 mRNA levels (top) in serum-starved TSC2 +/+ or TSC2 −/− MEFs in untreated cells or after the addition of 20 nM rapamycin for various times. The blot was reprobed for β-actin as a control for mRNA loading (bottom). White line indicates that intervening lanes have been removed. (B) IRS-1 mRNA levels (top) in TSC2 −/− MEFs treated with 20 nM rapamycin alone, or in the presence of 10 μg/ml actinomycin D for the final 10 h of a 24-h treatment. The blot was reprobed for β-actin as a control for mRNA loading (bottom). (C) Quantitative RT-PCR analysis of untreated, rapamycin-treated, and rapamycin plus actinomycin D–treated TSC2 −/− MEFs. Left, β-actin; right, IRS-1. A single graph from triplicate determinations showing identical results is shown. (D) RNAi-mediated inhibition of S6K1 and S6K2. Western blotting of extracts from TSC2 +/+ or TSC2 −/− MEFs transfected with a combination of scrambled siRNAs (C) or S6K1, S6K2, or S6K1 plus S6K2 siRNAs. Top, S6K1; middle, S6K2; bottom, anti-pS6 (Ser240/244). Where indicated, cells were stimulated for 10 min with insulin or treated with 20 nM rapamycin for 1 h. (E) IRS-1 mRNA levels in TSC2 +/+ MEFs or TSC2 −/− MEFs transfected with a combination of S6K1 and S6K2 scrambled siRNAs (C), S6K1, or S6K2 siRNAs, or treated for 24 h with 20 nM rapamycin. Top, IRS-1 mRNA; The blot was also reprobed for β-actin as a control for mRNA loading (bottom). (F) Quantitative RT-PCR analysis of untreated TSC2 +/+ , TSC2 −/− , or TSC2 −/− treated with 20 nM rapamycin or RNAi to S6K1 or S6K2. Left, β-actin; right, IRS-1. A single graph from triplicate determinations showing identical results is shown.
Figure Legend Snippet: Inhibition of S6K restores IRS-1 mRNA. (A) IRS-1 mRNA levels (top) in serum-starved TSC2 +/+ or TSC2 −/− MEFs in untreated cells or after the addition of 20 nM rapamycin for various times. The blot was reprobed for β-actin as a control for mRNA loading (bottom). White line indicates that intervening lanes have been removed. (B) IRS-1 mRNA levels (top) in TSC2 −/− MEFs treated with 20 nM rapamycin alone, or in the presence of 10 μg/ml actinomycin D for the final 10 h of a 24-h treatment. The blot was reprobed for β-actin as a control for mRNA loading (bottom). (C) Quantitative RT-PCR analysis of untreated, rapamycin-treated, and rapamycin plus actinomycin D–treated TSC2 −/− MEFs. Left, β-actin; right, IRS-1. A single graph from triplicate determinations showing identical results is shown. (D) RNAi-mediated inhibition of S6K1 and S6K2. Western blotting of extracts from TSC2 +/+ or TSC2 −/− MEFs transfected with a combination of scrambled siRNAs (C) or S6K1, S6K2, or S6K1 plus S6K2 siRNAs. Top, S6K1; middle, S6K2; bottom, anti-pS6 (Ser240/244). Where indicated, cells were stimulated for 10 min with insulin or treated with 20 nM rapamycin for 1 h. (E) IRS-1 mRNA levels in TSC2 +/+ MEFs or TSC2 −/− MEFs transfected with a combination of S6K1 and S6K2 scrambled siRNAs (C), S6K1, or S6K2 siRNAs, or treated for 24 h with 20 nM rapamycin. Top, IRS-1 mRNA; The blot was also reprobed for β-actin as a control for mRNA loading (bottom). (F) Quantitative RT-PCR analysis of untreated TSC2 +/+ , TSC2 −/− , or TSC2 −/− treated with 20 nM rapamycin or RNAi to S6K1 or S6K2. Left, β-actin; right, IRS-1. A single graph from triplicate determinations showing identical results is shown.

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

4) Product Images from "Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway"

Article Title: Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway

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

doi: 10.1073/pnas.0402135101

IL-6 secretion is inhibited by LMP2A and controls LMP1 expression. ( A ) The ability of LMP2A to down-regulate IL-6 secretion in rEBV-infected HONE-1 and Ad/AH cell lines was determined by using an IL-6 ELISA. The histograms are representative of at least three separate experiments, and the mean ± SE of triplicate determinations is shown. ( B ) Addition of human recombinant IL-6 to the HONE-1 rEBV-infected cell line induces LMP1 expression as demonstrated by immunoblot analysis. ( C ) Addition of human recombinant IL-6 to the C666-1 NPC cell line induces LMP1 expression and phosphorylation of STAT3 as demonstrated by immunoblot analysis. Total STAT3 served as a loading control.
Figure Legend Snippet: IL-6 secretion is inhibited by LMP2A and controls LMP1 expression. ( A ) The ability of LMP2A to down-regulate IL-6 secretion in rEBV-infected HONE-1 and Ad/AH cell lines was determined by using an IL-6 ELISA. The histograms are representative of at least three separate experiments, and the mean ± SE of triplicate determinations is shown. ( B ) Addition of human recombinant IL-6 to the HONE-1 rEBV-infected cell line induces LMP1 expression as demonstrated by immunoblot analysis. ( C ) Addition of human recombinant IL-6 to the C666-1 NPC cell line induces LMP1 expression and phosphorylation of STAT3 as demonstrated by immunoblot analysis. Total STAT3 served as a loading control.

Techniques Used: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Recombinant

Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.
Figure Legend Snippet: Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.

Techniques Used: Expressing, Infection, Transfection, Plasmid Preparation, Negative Control

LMP2A negatively regulates IL-6 secretion by means of inhibition of NF-κB. ( A ) EMSA demonstrates that infection of HONE-1 cells with rEBV inhibits NF-κB activity and that this effect is relieved in cells infected with rEBV-2A. Nuclear extracts isolated from HONE-1 cells or HONE-1 cells infected with rEBVs were analyzed for basal NF-κB activity. Tumor necrosis factor α-stimulated HONE-1 parental cells were used as a positive control. The presence of p65 and p50 subunits in the NF-κB complexes was confirmed by using antibodies to these subunits. ( B ) Blockade of NF-κB activity in rEBV-2A-infected HONE-1 cells with the RAd-IκBα SS/AA virus inhibited IL-6 secretion in a dose-dependent manner as determined by IL-6 ELISA. Data are representative of two separate experiments with triplicate determination, and the mean ± SE were all
Figure Legend Snippet: LMP2A negatively regulates IL-6 secretion by means of inhibition of NF-κB. ( A ) EMSA demonstrates that infection of HONE-1 cells with rEBV inhibits NF-κB activity and that this effect is relieved in cells infected with rEBV-2A. Nuclear extracts isolated from HONE-1 cells or HONE-1 cells infected with rEBVs were analyzed for basal NF-κB activity. Tumor necrosis factor α-stimulated HONE-1 parental cells were used as a positive control. The presence of p65 and p50 subunits in the NF-κB complexes was confirmed by using antibodies to these subunits. ( B ) Blockade of NF-κB activity in rEBV-2A-infected HONE-1 cells with the RAd-IκBα SS/AA virus inhibited IL-6 secretion in a dose-dependent manner as determined by IL-6 ELISA. Data are representative of two separate experiments with triplicate determination, and the mean ± SE were all

Techniques Used: Inhibition, Infection, Activity Assay, Isolation, Positive Control, Enzyme-linked Immunosorbent Assay

5) Product Images from "Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP)"

Article Title: Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP)

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2012.58

Effect of AONs on mouse and human IL-1RAcP pre-mRNA splicing in vitro . ( a ) AONs targeted to mouse IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( b ) Test of AONs on NIH-3T3 cells at 500 nmol/l concentration for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with primers specific for exon 8 and 10. Sequence analysis also confirmed exon 9 skipping. ( c ) RT-PCR results of RNA samples from NIH-3T3 cells transfected with AONs PS300, PS327, 25-mer counterparts of them and PS300L that was designed to increase efficiency of PS300. Triangles show increasing concentrations of 20, 50, 100, 200 and 500 nmol/l for full 2′- O -MePS oligos and additional 10 nmol/l for PS300L (+ = positive control PS300, 100 nmol/l; − = water control, NT = non-transfected cells, Mock tr = only Lipofectamine-2000). ( d ) Quantification of skipping levels of AON concentration from 20 to 500 nmol/l by qPCR analysis. β-Actin was used as the reference gene and each bar represents the mean value of three different experiments ± SEM. ( e ) AONs targeted to human IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( f ) Test of AONs on HEPG2 cells at 500 nmol/l for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with exon 8- and 10-specific primers. The sequence analysis also confirmed exon 9 skipping. AON, antisense oligonucleotide; IL-1RAcP, interleukin-1 receptor accessory protein; qPCR, quantitative PCR; RT-PCR, reverse transcription-PCR.
Figure Legend Snippet: Effect of AONs on mouse and human IL-1RAcP pre-mRNA splicing in vitro . ( a ) AONs targeted to mouse IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( b ) Test of AONs on NIH-3T3 cells at 500 nmol/l concentration for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with primers specific for exon 8 and 10. Sequence analysis also confirmed exon 9 skipping. ( c ) RT-PCR results of RNA samples from NIH-3T3 cells transfected with AONs PS300, PS327, 25-mer counterparts of them and PS300L that was designed to increase efficiency of PS300. Triangles show increasing concentrations of 20, 50, 100, 200 and 500 nmol/l for full 2′- O -MePS oligos and additional 10 nmol/l for PS300L (+ = positive control PS300, 100 nmol/l; − = water control, NT = non-transfected cells, Mock tr = only Lipofectamine-2000). ( d ) Quantification of skipping levels of AON concentration from 20 to 500 nmol/l by qPCR analysis. β-Actin was used as the reference gene and each bar represents the mean value of three different experiments ± SEM. ( e ) AONs targeted to human IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( f ) Test of AONs on HEPG2 cells at 500 nmol/l for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with exon 8- and 10-specific primers. The sequence analysis also confirmed exon 9 skipping. AON, antisense oligonucleotide; IL-1RAcP, interleukin-1 receptor accessory protein; qPCR, quantitative PCR; RT-PCR, reverse transcription-PCR.

Techniques Used: In Vitro, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Transfection, Positive Control, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

6) Product Images from "Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1"

Article Title: Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1

Journal: Journal of medicinal chemistry

doi: 10.1021/acs.jmedchem.8b01529

Overlay of “mode 2” docking poses obtained for stick renderings of MC047 (C, light gray), 11 (C, teal), 25 (C, dark green), and 26 (C, pink) in the active site of APE1 (4QHE). Compounds MC047 , 25 , and 26 all display an interaction between the sulfonamide groups and the magnesium ion (light green sphere), as well as a projection of their halogenated aryl rings toward hot spot region 5, left-hand side.
Figure Legend Snippet: Overlay of “mode 2” docking poses obtained for stick renderings of MC047 (C, light gray), 11 (C, teal), 25 (C, dark green), and 26 (C, pink) in the active site of APE1 (4QHE). Compounds MC047 , 25 , and 26 all display an interaction between the sulfonamide groups and the magnesium ion (light green sphere), as well as a projection of their halogenated aryl rings toward hot spot region 5, left-hand side.

Techniques Used:

). (A) Computational docking of small organic molecules reveals clustering of 50 probes (thin lines, C, cyan, O, red, N blue) into seven consensus sites in or near the active site of APE1 (4QHE) shown as a molecular surface rendering (C, light gray, O, red, N, blue, and S, yellow). DMSO (stick model C, green, O, red, S, yellow) from the crystal structure is shown bound in consensus site 2. Probes at consensus site 1, the most populated site, are in close proximity to the Mg 2+ ion, shown as a surface rendering in green. (B) A secondary site occupied by ethylene glycol in several crystal structures of APE1 is shown with a cluster of 21 probes shown in thin lines. Ethylene glycols bound in this site and in the vicinity are shown as stick renderings (C, magenta, O, red). This is a small pocket on the surface of the molecule. Thus, the major consensus sites for computational docking of probes in the active of APE1 and in another pocket have been validated in our crystallographic analysis.
Figure Legend Snippet: ). (A) Computational docking of small organic molecules reveals clustering of 50 probes (thin lines, C, cyan, O, red, N blue) into seven consensus sites in or near the active site of APE1 (4QHE) shown as a molecular surface rendering (C, light gray, O, red, N, blue, and S, yellow). DMSO (stick model C, green, O, red, S, yellow) from the crystal structure is shown bound in consensus site 2. Probes at consensus site 1, the most populated site, are in close proximity to the Mg 2+ ion, shown as a surface rendering in green. (B) A secondary site occupied by ethylene glycol in several crystal structures of APE1 is shown with a cluster of 21 probes shown in thin lines. Ethylene glycols bound in this site and in the vicinity are shown as stick renderings (C, magenta, O, red). This is a small pocket on the surface of the molecule. Thus, the major consensus sites for computational docking of probes in the active of APE1 and in another pocket have been validated in our crystallographic analysis.

Techniques Used:

Macrocycle treatment alone and in combination with DNA damaging agent methyl methanesulfonate (MMS) in malignant peripheral nerve sheath tumor cell line ST8814 in alkaline CometAssay. APE1 repair inhibitor candidates (macrocycles) and APE1 repair inhibitor control, APE1 repair inhibitor III (ARiIII; Calbiochem), were tested in ST8814 at a single dose (100 μ M or EC 30 ) and in combination with a single dose of MMS (1.0 mM) for a 1.0 h treatment. Cells were seeded in 6-well tissue culture plates at 130 000 cells/well in DMEM + 10% FBS and grown overnight at 37 °C, 5% CO 2 . Medium was exchanged with Opti-MEM (Gibco) medium containing macrocycle alone or spiked with 1.0 mM MMS. Cells were then incubated for 1.0 h at 37 °C, 5% CO 2 . Medium was exchanged with PBS, and cells were treated with 0.25% trypsin (HyClone), collected, and washed with PBS. Cells were then counted by hemacytometer. DNA damage was evaluated by CometAssay (Trevigen) performed under alkaline conditions. Cells were resuspended in PBS at 1 × 10 5 /mL and then added to premelted and cooled (37 °C) agarose at a 1:10 ratio. Cells were gently mixed, and then 50 μ L of the agarose cell mix was transferred to prewarmed comet slides (37 °C, CometSlide). After solidifying at 4 °C, slides were placed in lysis solution for 60 min and then placed in freshly prepared alkaline unwinding solution (NaOH, pH 13) for 20 min. Slides were then subjected to electrophoresis under alkaline conditions (NaOH, pH 13) at 1 V/cm (300 mA) for 30 min. Slides were washed in H 2 O, placed in 70% ethanol, and allowed to dry at 37 °C for 30 min. Slides were stained with 100 μ L of 1:10 000 diluted SYBR Gold (Invitrogen) in TE pH 7.5 and incubated for 30 min. Slides were then rinsed in H 2 O briefly, and comets were captured by fluorescent microscope (Leica DMIL) and quantified by CometScore Pro (TriTec Corp). DNA damage measured by percent Tail DNA for controls and selected macrocycles is shown in (A) in two independent experiments. Representative images of comet slide DNA damage are shown in (B) for controls and selected macrocycles. There was an average of 20 comet readings/compound.
Figure Legend Snippet: Macrocycle treatment alone and in combination with DNA damaging agent methyl methanesulfonate (MMS) in malignant peripheral nerve sheath tumor cell line ST8814 in alkaline CometAssay. APE1 repair inhibitor candidates (macrocycles) and APE1 repair inhibitor control, APE1 repair inhibitor III (ARiIII; Calbiochem), were tested in ST8814 at a single dose (100 μ M or EC 30 ) and in combination with a single dose of MMS (1.0 mM) for a 1.0 h treatment. Cells were seeded in 6-well tissue culture plates at 130 000 cells/well in DMEM + 10% FBS and grown overnight at 37 °C, 5% CO 2 . Medium was exchanged with Opti-MEM (Gibco) medium containing macrocycle alone or spiked with 1.0 mM MMS. Cells were then incubated for 1.0 h at 37 °C, 5% CO 2 . Medium was exchanged with PBS, and cells were treated with 0.25% trypsin (HyClone), collected, and washed with PBS. Cells were then counted by hemacytometer. DNA damage was evaluated by CometAssay (Trevigen) performed under alkaline conditions. Cells were resuspended in PBS at 1 × 10 5 /mL and then added to premelted and cooled (37 °C) agarose at a 1:10 ratio. Cells were gently mixed, and then 50 μ L of the agarose cell mix was transferred to prewarmed comet slides (37 °C, CometSlide). After solidifying at 4 °C, slides were placed in lysis solution for 60 min and then placed in freshly prepared alkaline unwinding solution (NaOH, pH 13) for 20 min. Slides were then subjected to electrophoresis under alkaline conditions (NaOH, pH 13) at 1 V/cm (300 mA) for 30 min. Slides were washed in H 2 O, placed in 70% ethanol, and allowed to dry at 37 °C for 30 min. Slides were stained with 100 μ L of 1:10 000 diluted SYBR Gold (Invitrogen) in TE pH 7.5 and incubated for 30 min. Slides were then rinsed in H 2 O briefly, and comets were captured by fluorescent microscope (Leica DMIL) and quantified by CometScore Pro (TriTec Corp). DNA damage measured by percent Tail DNA for controls and selected macrocycles is shown in (A) in two independent experiments. Representative images of comet slide DNA damage are shown in (B) for controls and selected macrocycles. There was an average of 20 comet readings/compound.

Techniques Used: Incubation, Lysis, Electrophoresis, Staining, Microscopy

7) Product Images from "Activation of Human and Chicken Toll-Like Receptors by Campylobacter spp. ▿"

Article Title: Activation of Human and Chicken Toll-Like Receptors by Campylobacter spp. ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.00897-09

Induction of IL-1β and IL-8 by live and disrupted C. jejuni . MM6 cells (A) and chicken HD11 cells (B) were stimulated for 2 h with 5 × 10 7 CFU of live or lysed C. jejuni strain 81116 ml −1 . As a positive control, 1 μg of
Figure Legend Snippet: Induction of IL-1β and IL-8 by live and disrupted C. jejuni . MM6 cells (A) and chicken HD11 cells (B) were stimulated for 2 h with 5 × 10 7 CFU of live or lysed C. jejuni strain 81116 ml −1 . As a positive control, 1 μg of

Techniques Used: Positive Control

8) Product Images from "Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway"

Article Title: Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway

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

doi: 10.1073/pnas.0402135101

Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.
Figure Legend Snippet: Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.

Techniques Used: Expressing, Infection, Transfection, Plasmid Preparation, Negative Control

9) Product Images from "Microbial community dynamics in replicate anaerobic digesters exposed sequentially to increasing organic loading rate, acidosis, and process recovery"

Article Title: Microbial community dynamics in replicate anaerobic digesters exposed sequentially to increasing organic loading rate, acidosis, and process recovery

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-015-0309-9

Bacterial and archaeal diversity dynamic over time as assessed by high-throughput 16S rRNA amplicon sequencing. P0–P5 are the sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading; results are presented at the phylum level and the OTU level (top 50) for bacteria and at the species level for archaea; gaps for R1 at sampling periods P3 and P4 were due to the poor quality of extracted DNA during severe acidosis; the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period
Figure Legend Snippet: Bacterial and archaeal diversity dynamic over time as assessed by high-throughput 16S rRNA amplicon sequencing. P0–P5 are the sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading; results are presented at the phylum level and the OTU level (top 50) for bacteria and at the species level for archaea; gaps for R1 at sampling periods P3 and P4 were due to the poor quality of extracted DNA during severe acidosis; the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period

Techniques Used: High Throughput Screening Assay, Amplification, Sequencing, Sampling

Canonical correspondence analysis (CCA) ordination diplot for the archaeal community. Red vectors represent the influence of the process parameters such as pH (pH), organic loading rate (OLR), biogas production (Biogas), total solids (TS), volatile solids (VS), methane (CH 4 ), carbon dioxide (CO 2 ) and hydrogen sulphide (H 2 S) contents in the biogas, alkalinity (Alk.), ammonium–nitrogen (NH 4 –N), total volatile fatty acids (Total VFAs), acetate (Acet.) and propionate (Propio.), contents in the sludge; blue triangles represent archaeal taxa derived from the high-throughput 16S rRNA amplicon sequencing at the species level: unknown Methanomassiliicoccaceae ( U.Mmass. ), unknown Methanospirillaceae ( U.Mspir .), unknown WSA2 ( U.WSA2. ), unknown vadinCA11 ( U.vadin .), Methanobacterium sp. A ( Meth.A .), Methanobacterium sp. B ( Meth.B. ), Methanoculleus sp. ( Mcul .), Methanomassiliicoccus sp. ( Mliico. ), Methanomethylovorans sp. ( Mmethyl. ), Methanosaeta sp. ( Msae. ), Methanosarcina sp. ( Msar. ), unknown pGrfC26 A ( U.pGrf.A ), unknown pGrfC26 sp. B ( U.pGrf.B ). A detailed correlation matrix including all process parameters and the archaeal community diversity is provided as Additional file 2 : Table S4
Figure Legend Snippet: Canonical correspondence analysis (CCA) ordination diplot for the archaeal community. Red vectors represent the influence of the process parameters such as pH (pH), organic loading rate (OLR), biogas production (Biogas), total solids (TS), volatile solids (VS), methane (CH 4 ), carbon dioxide (CO 2 ) and hydrogen sulphide (H 2 S) contents in the biogas, alkalinity (Alk.), ammonium–nitrogen (NH 4 –N), total volatile fatty acids (Total VFAs), acetate (Acet.) and propionate (Propio.), contents in the sludge; blue triangles represent archaeal taxa derived from the high-throughput 16S rRNA amplicon sequencing at the species level: unknown Methanomassiliicoccaceae ( U.Mmass. ), unknown Methanospirillaceae ( U.Mspir .), unknown WSA2 ( U.WSA2. ), unknown vadinCA11 ( U.vadin .), Methanobacterium sp. A ( Meth.A .), Methanobacterium sp. B ( Meth.B. ), Methanoculleus sp. ( Mcul .), Methanomassiliicoccus sp. ( Mliico. ), Methanomethylovorans sp. ( Mmethyl. ), Methanosaeta sp. ( Msae. ), Methanosarcina sp. ( Msar. ), unknown pGrfC26 A ( U.pGrf.A ), unknown pGrfC26 sp. B ( U.pGrf.B ). A detailed correlation matrix including all process parameters and the archaeal community diversity is provided as Additional file 2 : Table S4

Techniques Used: Derivative Assay, High Throughput Screening Assay, Amplification, Sequencing

Bacterial and archaeal community structure dynamic over time as assessed by 16S rRNA gene-based T-RFLP analysis. P0–P5 are the six sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading rate; T-RFs are characterized by their length in base pairs (bp); the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period
Figure Legend Snippet: Bacterial and archaeal community structure dynamic over time as assessed by 16S rRNA gene-based T-RFLP analysis. P0–P5 are the six sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading rate; T-RFs are characterized by their length in base pairs (bp); the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period

Techniques Used: Sampling

10) Product Images from "Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure"

Article Title: Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure

Journal: Molecular and Cellular Biochemistry

doi: 10.1007/s11010-020-03771-1

GW501516 impact on N-cadherin level in T24 cells. Cells were exposed for 24 h to vehicle or PPARβ/δ activator at the indicated concentrations. a RTqPCR analysis of cdh2 mRNA expression. Fold inductions represent comparison with control cells (set at 1). b , c N-cadherin detection was determined by Western blotting analysis from total protein extracts with GC-4 antibody. β-actin was used as an internal loading control. The density of each band in immunoblots was measured by using ImageJ and N-cadherin levels were determined as arbitrary units in control and treated cells. The amount of N-cadherin protein was normalized to that of β-actin. Data are expressed as means ± SEM of three independent experiments performed in triplicate. * P
Figure Legend Snippet: GW501516 impact on N-cadherin level in T24 cells. Cells were exposed for 24 h to vehicle or PPARβ/δ activator at the indicated concentrations. a RTqPCR analysis of cdh2 mRNA expression. Fold inductions represent comparison with control cells (set at 1). b , c N-cadherin detection was determined by Western blotting analysis from total protein extracts with GC-4 antibody. β-actin was used as an internal loading control. The density of each band in immunoblots was measured by using ImageJ and N-cadherin levels were determined as arbitrary units in control and treated cells. The amount of N-cadherin protein was normalized to that of β-actin. Data are expressed as means ± SEM of three independent experiments performed in triplicate. * P

Techniques Used: Expressing, Western Blot

11) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.379966

Comparison of I-CreI:C1234_Me+2b crystal structures obtained in the presence of Mg 2+ ( left panel, green, PDB code 4AQX ) or Ca 2+ ( right panel, green, PDB code 4AQX ) with their respective unmethylated structures obtained in the presence of Mg 2+ ( left panel,
Figure Legend Snippet: Comparison of I-CreI:C1234_Me+2b crystal structures obtained in the presence of Mg 2+ ( left panel, green, PDB code 4AQX ) or Ca 2+ ( right panel, green, PDB code 4AQX ) with their respective unmethylated structures obtained in the presence of Mg 2+ ( left panel,

Techniques Used:

Influence of CpG methylation on the nuclease activity of I-CreI in vitro . A constant amount of C1234 duplex (50 n m ) was incubated with an excess of I-CreI (1.5 μ m , final concentration) in the reaction buffer (10 m m Tris-HCl, 150 m m NaCl, pH 8)
Figure Legend Snippet: Influence of CpG methylation on the nuclease activity of I-CreI in vitro . A constant amount of C1234 duplex (50 n m ) was incubated with an excess of I-CreI (1.5 μ m , final concentration) in the reaction buffer (10 m m Tris-HCl, 150 m m NaCl, pH 8)

Techniques Used: CpG Methylation Assay, Activity Assay, In Vitro, Incubation, Concentration Assay

Influence of CpG methylation on the DNA binding affinity of I-CreI in vitro ; spectrofluorometric titration of FAM-labeled C1234 by I-CreI wild type. 25 n m unmethylated or methylated C1234 duplex was incubated with increasing concentrations of I-CreI (from
Figure Legend Snippet: Influence of CpG methylation on the DNA binding affinity of I-CreI in vitro ; spectrofluorometric titration of FAM-labeled C1234 by I-CreI wild type. 25 n m unmethylated or methylated C1234 duplex was incubated with increasing concentrations of I-CreI (from

Techniques Used: CpG Methylation Assay, Binding Assay, In Vitro, Titration, Labeling, Methylation, Incubation

Nucleotide sequence of C1234, the cognate target of I-CreI. The central tetrabase is displayed in gray ; overhangs induced by I-CreI endonuclease activity are indicated by dashed lines , and base numbering and positions of methylated cytosines are indicated
Figure Legend Snippet: Nucleotide sequence of C1234, the cognate target of I-CreI. The central tetrabase is displayed in gray ; overhangs induced by I-CreI endonuclease activity are indicated by dashed lines , and base numbering and positions of methylated cytosines are indicated

Techniques Used: Sequencing, Activity Assay, Methylation

Influence of CpG Methylation on the DNA Binding Affinity of I-CreI
Figure Legend Snippet: Influence of CpG Methylation on the DNA Binding Affinity of I-CreI

Techniques Used: CpG Methylation Assay, Binding Assay

12) Product Images from "Microbial community dynamics in replicate anaerobic digesters exposed sequentially to increasing organic loading rate, acidosis, and process recovery"

Article Title: Microbial community dynamics in replicate anaerobic digesters exposed sequentially to increasing organic loading rate, acidosis, and process recovery

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-015-0309-9

Bacterial and archaeal diversity dynamic over time as assessed by high-throughput 16S rRNA amplicon sequencing. P0–P5 are the sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading; results are presented at the phylum level and the OTU level (top 50) for bacteria and at the species level for archaea; gaps for R1 at sampling periods P3 and P4 were due to the poor quality of extracted DNA during severe acidosis; the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period
Figure Legend Snippet: Bacterial and archaeal diversity dynamic over time as assessed by high-throughput 16S rRNA amplicon sequencing. P0–P5 are the sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading; results are presented at the phylum level and the OTU level (top 50) for bacteria and at the species level for archaea; gaps for R1 at sampling periods P3 and P4 were due to the poor quality of extracted DNA during severe acidosis; the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period

Techniques Used: High Throughput Screening Assay, Amplification, Sequencing, Sampling

Canonical correspondence analysis (CCA) ordination diplot for the archaeal community. Red vectors represent the influence of the process parameters such as pH (pH), organic loading rate (OLR), biogas production (Biogas), total solids (TS), volatile solids (VS), methane (CH 4 ), carbon dioxide (CO 2 ) and hydrogen sulphide (H 2 S) contents in the biogas, alkalinity (Alk.), ammonium–nitrogen (NH 4 –N), total volatile fatty acids (Total VFAs), acetate (Acet.) and propionate (Propio.), contents in the sludge; blue triangles represent archaeal taxa derived from the high-throughput 16S rRNA amplicon sequencing at the species level: unknown Methanomassiliicoccaceae ( U.Mmass. ), unknown Methanospirillaceae ( U.Mspir .), unknown WSA2 ( U.WSA2. ), unknown vadinCA11 ( U.vadin .), Methanobacterium sp. A ( Meth.A .), Methanobacterium sp. B ( Meth.B. ), Methanoculleus sp. ( Mcul .), Methanomassiliicoccus sp. ( Mliico. ), Methanomethylovorans sp. ( Mmethyl. ), Methanosaeta sp. ( Msae. ), Methanosarcina sp. ( Msar. ), unknown pGrfC26 A ( U.pGrf.A ), unknown pGrfC26 sp. B ( U.pGrf.B ). A detailed correlation matrix including all process parameters and the archaeal community diversity is provided as Additional file 2 : Table S4
Figure Legend Snippet: Canonical correspondence analysis (CCA) ordination diplot for the archaeal community. Red vectors represent the influence of the process parameters such as pH (pH), organic loading rate (OLR), biogas production (Biogas), total solids (TS), volatile solids (VS), methane (CH 4 ), carbon dioxide (CO 2 ) and hydrogen sulphide (H 2 S) contents in the biogas, alkalinity (Alk.), ammonium–nitrogen (NH 4 –N), total volatile fatty acids (Total VFAs), acetate (Acet.) and propionate (Propio.), contents in the sludge; blue triangles represent archaeal taxa derived from the high-throughput 16S rRNA amplicon sequencing at the species level: unknown Methanomassiliicoccaceae ( U.Mmass. ), unknown Methanospirillaceae ( U.Mspir .), unknown WSA2 ( U.WSA2. ), unknown vadinCA11 ( U.vadin .), Methanobacterium sp. A ( Meth.A .), Methanobacterium sp. B ( Meth.B. ), Methanoculleus sp. ( Mcul .), Methanomassiliicoccus sp. ( Mliico. ), Methanomethylovorans sp. ( Mmethyl. ), Methanosaeta sp. ( Msae. ), Methanosarcina sp. ( Msar. ), unknown pGrfC26 A ( U.pGrf.A ), unknown pGrfC26 sp. B ( U.pGrf.B ). A detailed correlation matrix including all process parameters and the archaeal community diversity is provided as Additional file 2 : Table S4

Techniques Used: Derivative Assay, High Throughput Screening Assay, Amplification, Sequencing

Bacterial and archaeal community structure dynamic over time as assessed by 16S rRNA gene-based T-RFLP analysis. P0–P5 are the six sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading rate; T-RFs are characterized by their length in base pairs (bp); the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period
Figure Legend Snippet: Bacterial and archaeal community structure dynamic over time as assessed by 16S rRNA gene-based T-RFLP analysis. P0–P5 are the six sampling periods chosen for microbial community monitoring; CR is the cautiously fed reactor; R1, R2 and R3 are the reactors exposed to increasing organic loading rate; T-RFs are characterized by their length in base pairs (bp); the white arrow represents the initiation of the hydraulic retention time decrease; the black arrow represents the onset of the starving period

Techniques Used: Sampling

13) Product Images from "DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria"

Article Title: DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003541

GcrA interacts with RNA polymerase. (A) Immunoblots using RNAP β subunit antibodies on several samples. On the left cellular lysates of E. coli cells and C. crescentus cells, both positives to the antibodies. On the right, samples of C. crescentus CB15N cell lysate applied to the column containing His 6 -GcrA and subsequently washed with salt (up to 1 M) and imidazole in order to remove His 6 -GcrA and putative interactors. This procedure was done also with an empty column (“No GcrA” lane). The nickel column loaded with His 6 -GcrA was used to detect the association of GcrA to RNA polymerase. (B) E. coli RNA polymerase core enzyme is able to bind the GcrA-DNA complex (promoter ctrA P1), as visualized by a slower migration rate as the amount of RNA polymerase increased. On the left the same RNA polymerase conditions were tested without GcrA.
Figure Legend Snippet: GcrA interacts with RNA polymerase. (A) Immunoblots using RNAP β subunit antibodies on several samples. On the left cellular lysates of E. coli cells and C. crescentus cells, both positives to the antibodies. On the right, samples of C. crescentus CB15N cell lysate applied to the column containing His 6 -GcrA and subsequently washed with salt (up to 1 M) and imidazole in order to remove His 6 -GcrA and putative interactors. This procedure was done also with an empty column (“No GcrA” lane). The nickel column loaded with His 6 -GcrA was used to detect the association of GcrA to RNA polymerase. (B) E. coli RNA polymerase core enzyme is able to bind the GcrA-DNA complex (promoter ctrA P1), as visualized by a slower migration rate as the amount of RNA polymerase increased. On the left the same RNA polymerase conditions were tested without GcrA.

Techniques Used: Western Blot, Nickel Column, Migration

Electrophoresis mobility shift assay (EMSA) of 5 ChIP–Seq regions with His 6 -GcrA. EMSA results using increasing concentrations of purified GcrA using probes (red line) design in regions with high number of reads in ChIp-Seq results. From the top to the bottom: 1) the sequence with the maximum number of reads ChipSeq results, corresponding to the coding sequence of CCNA_00697; 2) the intergenic sequence between CCNA_00278 and CCNA_00279; 3) The promoter of mipZ ; 4) The promoter ctrA P1 of ctrA ; 5) A negative control, corresponding to the intergenic between CCNA_01926 and CCNA_01927. On the right, P. = Probe signal and C. = complex signal. Dissociation constants (Kd) of the positive probes are shown in Figure S9 .
Figure Legend Snippet: Electrophoresis mobility shift assay (EMSA) of 5 ChIP–Seq regions with His 6 -GcrA. EMSA results using increasing concentrations of purified GcrA using probes (red line) design in regions with high number of reads in ChIp-Seq results. From the top to the bottom: 1) the sequence with the maximum number of reads ChipSeq results, corresponding to the coding sequence of CCNA_00697; 2) the intergenic sequence between CCNA_00278 and CCNA_00279; 3) The promoter of mipZ ; 4) The promoter ctrA P1 of ctrA ; 5) A negative control, corresponding to the intergenic between CCNA_01926 and CCNA_01927. On the right, P. = Probe signal and C. = complex signal. Dissociation constants (Kd) of the positive probes are shown in Figure S9 .

Techniques Used: Electrophoresis, Mobility Shift, Chromatin Immunoprecipitation, Purification, Sequencing, Negative Control

CcrM and GcrA dependence of selected promoters. (A) Immunoblots showing that the Δ ccrM mutation does not affect the steady-state levels of the master cell cycle regulators, GcrA, CtrA and DnaA using polyclonal antibodies to these proteins. (B) GcrA-depletion impairs P mipZ -, P podJ - , P flaY - and P pleC - lacZ activity, while affecting P CCNA_00697 - lacZ to a lesser extent. β-Galactosidase activities were measured in the WT or the GcrA depletion strain harbouring the transcriptional reporters after growth in PYE supplemented with xylose (0.3%) or glucose (0.2%) for 5 hours and tetracycline to select for the reporter plasmid. (C) Mutation of the CcrM recognition sites (GAnTC→GCnTC) cripples P mipZ -, P podJ - , P flaY - and P pleC - lacZ activity, but not P CCNA_00697 - lacZ. WT cells harbouring the reporter plasmids were grown PYE and tetracycline to select for the reporter plasmid.
Figure Legend Snippet: CcrM and GcrA dependence of selected promoters. (A) Immunoblots showing that the Δ ccrM mutation does not affect the steady-state levels of the master cell cycle regulators, GcrA, CtrA and DnaA using polyclonal antibodies to these proteins. (B) GcrA-depletion impairs P mipZ -, P podJ - , P flaY - and P pleC - lacZ activity, while affecting P CCNA_00697 - lacZ to a lesser extent. β-Galactosidase activities were measured in the WT or the GcrA depletion strain harbouring the transcriptional reporters after growth in PYE supplemented with xylose (0.3%) or glucose (0.2%) for 5 hours and tetracycline to select for the reporter plasmid. (C) Mutation of the CcrM recognition sites (GAnTC→GCnTC) cripples P mipZ -, P podJ - , P flaY - and P pleC - lacZ activity, but not P CCNA_00697 - lacZ. WT cells harbouring the reporter plasmids were grown PYE and tetracycline to select for the reporter plasmid.

Techniques Used: Western Blot, Mutagenesis, Activity Assay, Plasmid Preparation

GcrA DNA binding depends on CcrM methylation state. Three regions (CCNA_0697, ctrA , mipZ ) containing methylation sites of CcrM were tested in a competition experiment. Competitor DNA identical to the probe with various methylation states was mixed with biotinylated probes and GcrA in order to evaluate competition. For CCNA_00697 we used 0.375 µM of competitors, while for ctrA and mipZ promoters we used 1.25 µM. Dissociation constants (Kd) of the methylated probes of ctrA and mipZ probes are shown in Figure S9 .
Figure Legend Snippet: GcrA DNA binding depends on CcrM methylation state. Three regions (CCNA_0697, ctrA , mipZ ) containing methylation sites of CcrM were tested in a competition experiment. Competitor DNA identical to the probe with various methylation states was mixed with biotinylated probes and GcrA in order to evaluate competition. For CCNA_00697 we used 0.375 µM of competitors, while for ctrA and mipZ promoters we used 1.25 µM. Dissociation constants (Kd) of the methylated probes of ctrA and mipZ probes are shown in Figure S9 .

Techniques Used: Binding Assay, Methylation

Binding of GcrA to DNA in different GAnTC methylation states and conformation al changes of GcrA induced by DNA. (A) DNase I footprinting of the ctrA P1 promoter by GcrA. The promoter region was tested in different methylation states with increasing concentrations of His 6 -GcrA. The nucleotide sequence of the ctrA P1 promoter with the start site and the CcrM methylation site (green)vis shown on top. In orange, the putative region, protected by GcrA in the fully methylated state. In green the putative region protected in the hemi-methylated strand plus. (B) Proteolytic digestion using V8 in presence of DNA. In particular 4 kinds of DNA corresponding to the ctrA P1 promoter were tested as reported in the bottom part.
Figure Legend Snippet: Binding of GcrA to DNA in different GAnTC methylation states and conformation al changes of GcrA induced by DNA. (A) DNase I footprinting of the ctrA P1 promoter by GcrA. The promoter region was tested in different methylation states with increasing concentrations of His 6 -GcrA. The nucleotide sequence of the ctrA P1 promoter with the start site and the CcrM methylation site (green)vis shown on top. In orange, the putative region, protected by GcrA in the fully methylated state. In green the putative region protected in the hemi-methylated strand plus. (B) Proteolytic digestion using V8 in presence of DNA. In particular 4 kinds of DNA corresponding to the ctrA P1 promoter were tested as reported in the bottom part.

Techniques Used: Binding Assay, Methylation, Footprinting, Sequencing

14) Product Images from "Stability of telomeric G-quadruplexes"

Article Title: Stability of telomeric G-quadruplexes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1292

( A and B ) Thermal melting (cooling and heating) followed by absorbance at 295 nm of (A) Asc20 and (B) Bom17 in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( C and D ) CD spectra at 4°C of (C) FAsc20T and (D) FBom17T in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( E and F ) Thermal melting (heating) followed by FRET (excitation at 470 nm, emission at 520 nm) of (E) FAsc20T and (F) FBom17T in NaCl (circles) and KCl (triangles), at 0.2 µM oligonucleotide strand concentration.
Figure Legend Snippet: ( A and B ) Thermal melting (cooling and heating) followed by absorbance at 295 nm of (A) Asc20 and (B) Bom17 in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( C and D ) CD spectra at 4°C of (C) FAsc20T and (D) FBom17T in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( E and F ) Thermal melting (heating) followed by FRET (excitation at 470 nm, emission at 520 nm) of (E) FAsc20T and (F) FBom17T in NaCl (circles) and KCl (triangles), at 0.2 µM oligonucleotide strand concentration.

Techniques Used: Concentration Assay

15) Product Images from "Role of the DNA Base Excision Repair Protein, APE1 in Cisplatin, Oxaliplatin, or Carboplatin Induced Sensory Neuropathy"

Article Title: Role of the DNA Base Excision Repair Protein, APE1 in Cisplatin, Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0106485

Apoptosis induced by cisplatin and oxaliplatin, but not carboplatin, is increased by reducing the expression of APE1 in sensory neuronal cultures. Neuronal cultures were treated with siRNAs on days 3–5 in culture then exposed to various concentrations of platins for 72 hours starting on day nine in culture. Cell apoptosis was detected by Annexin-V and PI staining and FACS analyses after cells were grown for 12 days. The left panels show representative fluorescence-activated cell sorting (FACS) for cells treated with various concentrations of cisplatin (A), oxaliplatin (B), or carboplatin (C) and scrambled siRNA (SCsiRNA) or APE1siRNA as indicated. The panels on the right show the quantification of data from five independent harvests. Each column represents the mean ± SEM of the percent of apoptotic cells from cultures treated with SCsiRNA (lightly shaded) or with APE1siRNA (heavy shaded) and various concentrations of cisplatin (A); oxaliplatin (B) or carboplatin (C) as indicated. An asterisk indicates significant difference in survival in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using Student's t -test.
Figure Legend Snippet: Apoptosis induced by cisplatin and oxaliplatin, but not carboplatin, is increased by reducing the expression of APE1 in sensory neuronal cultures. Neuronal cultures were treated with siRNAs on days 3–5 in culture then exposed to various concentrations of platins for 72 hours starting on day nine in culture. Cell apoptosis was detected by Annexin-V and PI staining and FACS analyses after cells were grown for 12 days. The left panels show representative fluorescence-activated cell sorting (FACS) for cells treated with various concentrations of cisplatin (A), oxaliplatin (B), or carboplatin (C) and scrambled siRNA (SCsiRNA) or APE1siRNA as indicated. The panels on the right show the quantification of data from five independent harvests. Each column represents the mean ± SEM of the percent of apoptotic cells from cultures treated with SCsiRNA (lightly shaded) or with APE1siRNA (heavy shaded) and various concentrations of cisplatin (A); oxaliplatin (B) or carboplatin (C) as indicated. An asterisk indicates significant difference in survival in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using Student's t -test.

Techniques Used: Expressing, Staining, FACS, Fluorescence

Reducing the expression of APE1 augments the ability of cisplatin and oxaliplatin, but not carboplatin to reduce cell viability in sensory neuronal cultures. Neuronal cultures were treated with siRNAs on days 3–5 in culture then exposed to various concentrations of platins for 72 hours starting on day nine in culture. Cell viability as measured by trypan blue exclusion was determined on day 12 in culture from three independent harvests. Each column represents the mean ± SEM of percent survival of cells from cultures treated with scrambled siRNA (SCsiRNA; lightly shaded columns) or with APE1si RNA (heavy shaded columns), then exposed to various concentrations of cisplatin (panel A), oxaliplatin (panel B), or carboplatin (panel C) as indicated. An asterisk indicates significant difference in survival in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using ANOVA and Tukey's post hoc test.
Figure Legend Snippet: Reducing the expression of APE1 augments the ability of cisplatin and oxaliplatin, but not carboplatin to reduce cell viability in sensory neuronal cultures. Neuronal cultures were treated with siRNAs on days 3–5 in culture then exposed to various concentrations of platins for 72 hours starting on day nine in culture. Cell viability as measured by trypan blue exclusion was determined on day 12 in culture from three independent harvests. Each column represents the mean ± SEM of percent survival of cells from cultures treated with scrambled siRNA (SCsiRNA; lightly shaded columns) or with APE1si RNA (heavy shaded columns), then exposed to various concentrations of cisplatin (panel A), oxaliplatin (panel B), or carboplatin (panel C) as indicated. An asterisk indicates significant difference in survival in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using ANOVA and Tukey's post hoc test.

Techniques Used: Expressing

Platinum-induced phosphorylation of H2AX in sensory neuronal cultures is increased by reducing APE1 expression. The top panels show representative Western blots of phospho-H2AX (P-H2AX) and actin from cultures prior to and after 8, 24 and 48 hours of exposure to 300 µM oxaliplatin (A) or 500 µM carboplatin (B). Cultures were exposed to SCsiRNA or APE1siRNA as indicated. The bottom panels represent the densitometry of P-H2AX expression normalized to actin from three independent experiments. The columns represent the mean ± SEM from cultures treated with SCsiRNA (lightly shaded columns) or APE1siRNA (heavy shaded columns) prior to or after exposure to 300 µM oxaliplatin (A) or 500 µM carboplatin (B). An asterisk indicates a statistically significant increase in P-H2AX density in cells treated with APE1siRNA compared to those treated with SCsiRNA. Cisplatin data can be found in our previous publication [24] .
Figure Legend Snippet: Platinum-induced phosphorylation of H2AX in sensory neuronal cultures is increased by reducing APE1 expression. The top panels show representative Western blots of phospho-H2AX (P-H2AX) and actin from cultures prior to and after 8, 24 and 48 hours of exposure to 300 µM oxaliplatin (A) or 500 µM carboplatin (B). Cultures were exposed to SCsiRNA or APE1siRNA as indicated. The bottom panels represent the densitometry of P-H2AX expression normalized to actin from three independent experiments. The columns represent the mean ± SEM from cultures treated with SCsiRNA (lightly shaded columns) or APE1siRNA (heavy shaded columns) prior to or after exposure to 300 µM oxaliplatin (A) or 500 µM carboplatin (B). An asterisk indicates a statistically significant increase in P-H2AX density in cells treated with APE1siRNA compared to those treated with SCsiRNA. Cisplatin data can be found in our previous publication [24] .

Techniques Used: Expressing, Western Blot

Reducing APE1 expression enhances the ability of oxaliplatin but not carboplatin to reduce capsaicin-evoked release of CGRP from sensory neurons in culture. Each column represents the mean ± SEM of CGRP release in fmol/well/min for untreated sensory neurons in culture (controls) or cultures treated with SCsiRNA or APE1siRNA as indicated. Cells were exposed to 30 µM oxaliplatin (A) or 300 µM carboplatin (B) for 24 hours prior to release experiments. For release, wells of cells from three independent harvests were exposed for 10 min to HEPES alone (basal; open columns), or HEPES in the presence of 30 nM capsaicin (solid columns) as indicated. An asterisk indicates a significant difference in capsaicin-stimulated release compared to untreated cells, whereas a cross indicates a significant difference in cultures treated with APE1siRNA versus those treated with SCsiRNA using Student's t -test. Cisplatin analyses can be found in our previous publication [24] .
Figure Legend Snippet: Reducing APE1 expression enhances the ability of oxaliplatin but not carboplatin to reduce capsaicin-evoked release of CGRP from sensory neurons in culture. Each column represents the mean ± SEM of CGRP release in fmol/well/min for untreated sensory neurons in culture (controls) or cultures treated with SCsiRNA or APE1siRNA as indicated. Cells were exposed to 30 µM oxaliplatin (A) or 300 µM carboplatin (B) for 24 hours prior to release experiments. For release, wells of cells from three independent harvests were exposed for 10 min to HEPES alone (basal; open columns), or HEPES in the presence of 30 nM capsaicin (solid columns) as indicated. An asterisk indicates a significant difference in capsaicin-stimulated release compared to untreated cells, whereas a cross indicates a significant difference in cultures treated with APE1siRNA versus those treated with SCsiRNA using Student's t -test. Cisplatin analyses can be found in our previous publication [24] .

Techniques Used: Expressing

Platinum-induced oxidative DNA damage measured by 8-oxoG DNA adduct immunocytochemistry is increased by reducing APE1 expression in cisplatin and oxaliplatin treated sensory neuronal cultures. The top panels (A) show representative immunohistochemical staining for 8-oxoG DNA adducts in control (media), scrambled (SCsiRNA) or APE1 knockdown (APEsiRNA) from cultures after 24 hrs treatment with cisplatin (50 µM), oxaliplatin (300 µM) or carboplatin (500 µM). The bottom panels (B) represent the quantitation of the 8-oxoG positive staining cells as described in ”Methods.” The columns represent the mean ± SEM from cultures treated with media along (lightly shaded columns), SCsiRNA (medium shaded columns) or APE1siRNA (heavy shaded columns). An asterisk indicates a statistically significant increase in 8-oxoG in cells treated with APE1siRNA compared to those treated with SCsiRNA.
Figure Legend Snippet: Platinum-induced oxidative DNA damage measured by 8-oxoG DNA adduct immunocytochemistry is increased by reducing APE1 expression in cisplatin and oxaliplatin treated sensory neuronal cultures. The top panels (A) show representative immunohistochemical staining for 8-oxoG DNA adducts in control (media), scrambled (SCsiRNA) or APE1 knockdown (APEsiRNA) from cultures after 24 hrs treatment with cisplatin (50 µM), oxaliplatin (300 µM) or carboplatin (500 µM). The bottom panels (B) represent the quantitation of the 8-oxoG positive staining cells as described in ”Methods.” The columns represent the mean ± SEM from cultures treated with media along (lightly shaded columns), SCsiRNA (medium shaded columns) or APE1siRNA (heavy shaded columns). An asterisk indicates a statistically significant increase in 8-oxoG in cells treated with APE1siRNA compared to those treated with SCsiRNA.

Techniques Used: Immunocytochemistry, Expressing, Immunohistochemistry, Staining, Quantitation Assay

Production of reactive oxygen species (ROS) by cisplatin and oxaliplatin, but not carboplatin is increased by reducing the expression of APE1 in sensory neuronal cultures. Neuronal cultures were exposed to siRNAs on days 3-5 in culture then exposed to various concentrations of platins for 24 hours starting on day 11 in culture. ROS generation was measured by Carboxy-H2DCFDA and FACS analysis. The left panels show representative FACS for cells treated with various concentrations of cisplatin (A), oxaliplatin (B) or carboplatin (C) and scramble siRNA (SCsiRNA) or APE1siRNA as indicated. The panels on the right show the quantification of data from 4-6 independent harvests. Each column represents the mean ± SEM of the percent of ROS positive cells from cultures treated with SCsiRNA (lightly shaded) or with APE1siRNA (heavy shaded) and treated with various concentrations of cisplatin (A); oxaliplatin (B) or carboplatin (C) as indicated. An asterisk indicates significant difference in the number of ROS positive cells in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using Student's t -test.
Figure Legend Snippet: Production of reactive oxygen species (ROS) by cisplatin and oxaliplatin, but not carboplatin is increased by reducing the expression of APE1 in sensory neuronal cultures. Neuronal cultures were exposed to siRNAs on days 3-5 in culture then exposed to various concentrations of platins for 24 hours starting on day 11 in culture. ROS generation was measured by Carboxy-H2DCFDA and FACS analysis. The left panels show representative FACS for cells treated with various concentrations of cisplatin (A), oxaliplatin (B) or carboplatin (C) and scramble siRNA (SCsiRNA) or APE1siRNA as indicated. The panels on the right show the quantification of data from 4-6 independent harvests. Each column represents the mean ± SEM of the percent of ROS positive cells from cultures treated with SCsiRNA (lightly shaded) or with APE1siRNA (heavy shaded) and treated with various concentrations of cisplatin (A); oxaliplatin (B) or carboplatin (C) as indicated. An asterisk indicates significant difference in the number of ROS positive cells in the absence or presence of drug treatment, whereas a cross indicates significant difference in cultures treated with SCsiRNA versus APE1siRNA using Student's t -test.

Techniques Used: Expressing, FACS

16) Product Images from "Inhibition of hepatitis C virus replication by semi-synthetic derivatives of glycopeptide antibiotics"

Article Title: Inhibition of hepatitis C virus replication by semi-synthetic derivatives of glycopeptide antibiotics

Journal: Journal of Antimicrobial Chemotherapy

doi: 10.1093/jac/dkr104

Clearance–rebound in the Huh 9-13 replicon assay. Replicon-containing cells were treated for two or four consecutive passages with LCTA-949 (black bars), white bars or VX-950 (top panels), 2′- C -methylcytidine (middle panels), 4′-azidocytidine (bottom panels) or the combination of these selective HCV inhibitors with LCTA-949 (grey bars; each compound was at a concentration of 4× EC 50 ). In the clearance phase (C), cells were incubated in the compound's presence in the absence of G418. After this clearance phase, cells were further cultured twice in fresh medium containing 1 mg/mL G418 without antiviral agents (rebound phase, R). Intracellular HCV RNA in the surviving cells was quantified by qRT–PCR. UTC, untreated controls (Cx, clearance phase at passage x; Rx, rebound phase at passage x; Cx-Rx, rebound following clearance x).
Figure Legend Snippet: Clearance–rebound in the Huh 9-13 replicon assay. Replicon-containing cells were treated for two or four consecutive passages with LCTA-949 (black bars), white bars or VX-950 (top panels), 2′- C -methylcytidine (middle panels), 4′-azidocytidine (bottom panels) or the combination of these selective HCV inhibitors with LCTA-949 (grey bars; each compound was at a concentration of 4× EC 50 ). In the clearance phase (C), cells were incubated in the compound's presence in the absence of G418. After this clearance phase, cells were further cultured twice in fresh medium containing 1 mg/mL G418 without antiviral agents (rebound phase, R). Intracellular HCV RNA in the surviving cells was quantified by qRT–PCR. UTC, untreated controls (Cx, clearance phase at passage x; Rx, rebound phase at passage x; Cx-Rx, rebound following clearance x).

Techniques Used: Concentration Assay, Incubation, Cell Culture, Quantitative RT-PCR

17) Product Images from "A MicroRNA-Transcription Factor Blueprint for Early Atrial Arrhythmogenic Remodeling"

Article Title: A MicroRNA-Transcription Factor Blueprint for Early Atrial Arrhythmogenic Remodeling

Journal: BioMed Research International

doi: 10.1155/2015/263151

MicroRNAs which can be potentially responsible for the downregulation of Pitx2 , Tbx5 , and Myocd expression in atrial myocardium after pacing. (a) miRNA:mRNA alignments. Shown are the schematics of porcine Pitx2 3′-UTR sequence targeted by miR-21, porcine Tbx5 3′-UTR sequence targeted by miR-10a and miR-10b, and human Myocd 3′-UTR sequence targeted by miR-1. The porcine Tbx5 3′-UTR was identified within the pig genomic chromosome-14 sequence (NC_010456.4), starting from nt 40259322. (b) Overall relative levels of Pitx2c (grey boxes) and Mef2c (white boxes) transcripts in HL-1 cells transfected with 50 nM miR-21 mimic, anti-miR-21 inhibitor, and FAM-labeled pre-miR negative control (Con). The results of qRT-PCR analysis are shown. Data from 3 replicates of each transfection were pooled and averaged. ∗ p ≤ 0.05. (c) Control and transfected cells were pooled (from triplicate wells in each setup), lysed, electrophoresed, and immunoblotted with antibodies against PITX2A,B,C. MW values (kDa) of the bands detected are shown. (d) Membrane stained with Amido Black 10B. (e) The histogram demonstrating inverse expression correlations of selected miRNA/target gene pairs in paced pigs as revealed by qRT-PCR (miR-21, miR-10b, and miR-1) and Western blot (PITX2C, TBX5, and MYOCD). See text for details.
Figure Legend Snippet: MicroRNAs which can be potentially responsible for the downregulation of Pitx2 , Tbx5 , and Myocd expression in atrial myocardium after pacing. (a) miRNA:mRNA alignments. Shown are the schematics of porcine Pitx2 3′-UTR sequence targeted by miR-21, porcine Tbx5 3′-UTR sequence targeted by miR-10a and miR-10b, and human Myocd 3′-UTR sequence targeted by miR-1. The porcine Tbx5 3′-UTR was identified within the pig genomic chromosome-14 sequence (NC_010456.4), starting from nt 40259322. (b) Overall relative levels of Pitx2c (grey boxes) and Mef2c (white boxes) transcripts in HL-1 cells transfected with 50 nM miR-21 mimic, anti-miR-21 inhibitor, and FAM-labeled pre-miR negative control (Con). The results of qRT-PCR analysis are shown. Data from 3 replicates of each transfection were pooled and averaged. ∗ p ≤ 0.05. (c) Control and transfected cells were pooled (from triplicate wells in each setup), lysed, electrophoresed, and immunoblotted with antibodies against PITX2A,B,C. MW values (kDa) of the bands detected are shown. (d) Membrane stained with Amido Black 10B. (e) The histogram demonstrating inverse expression correlations of selected miRNA/target gene pairs in paced pigs as revealed by qRT-PCR (miR-21, miR-10b, and miR-1) and Western blot (PITX2C, TBX5, and MYOCD). See text for details.

Techniques Used: Expressing, Sequencing, Transfection, Labeling, Negative Control, Quantitative RT-PCR, Staining, Western Blot

18) Product Images from "Preparation of antibody-immobilized gelatin nanospheres incorporating a molecular beacon to visualize the biological function of macrophages"

Article Title: Preparation of antibody-immobilized gelatin nanospheres incorporating a molecular beacon to visualize the biological function of macrophages

Journal: Regenerative Therapy

doi: 10.1016/j.reth.2019.12.009

(A) The relative miRNA 155–5p expression of RAW264 before (M0) and after the stimulation with pro-inflammatory (M1) or anti-inflammatory agents (M2) (B) The representative fluorescent images of RAW264 before (a) and after the stimulation with pro-inflammatory (b) or anti-inflammatory agents (c). The RAW264 were cultured for 3 h with MB-gelatin NS (30 μg/ml) 24 h after the stimulation. The nucleus are stained in blue, while the MB interacted with miRNA 155–5p emits in green. The arrows indicate the cells which emit the fluorescence derived from MB. The size of scale bar is 50 μm (C) Percentage of MB-derived fluorescent cells which are not stimulated (M0) or stimulated with pro-inflammatory (M1) or anti-inflammatory agents (M2). The RAW264 were cultured for 3 h with MB-gelatin NS (30 μg/ml) 24 h after the stimulation. *, p 
Figure Legend Snippet: (A) The relative miRNA 155–5p expression of RAW264 before (M0) and after the stimulation with pro-inflammatory (M1) or anti-inflammatory agents (M2) (B) The representative fluorescent images of RAW264 before (a) and after the stimulation with pro-inflammatory (b) or anti-inflammatory agents (c). The RAW264 were cultured for 3 h with MB-gelatin NS (30 μg/ml) 24 h after the stimulation. The nucleus are stained in blue, while the MB interacted with miRNA 155–5p emits in green. The arrows indicate the cells which emit the fluorescence derived from MB. The size of scale bar is 50 μm (C) Percentage of MB-derived fluorescent cells which are not stimulated (M0) or stimulated with pro-inflammatory (M1) or anti-inflammatory agents (M2). The RAW264 were cultured for 3 h with MB-gelatin NS (30 μg/ml) 24 h after the stimulation. *, p 

Techniques Used: Expressing, Cell Culture, Staining, Fluorescence, Derivative Assay

19) Product Images from "Exposure to a Complex Cocktail of Environmental Endocrine-Disrupting Compounds Disturbs the Kisspeptin/GPR54 System in Ovine Hypothalamus and Pituitary Gland"

Article Title: Exposure to a Complex Cocktail of Environmental Endocrine-Disrupting Compounds Disturbs the Kisspeptin/GPR54 System in Ovine Hypothalamus and Pituitary Gland

Journal: Environmental Health Perspectives

doi: 10.1289/ehp.0900699

Maternal ER α mRNA expression (mean ± SE) in the hypothalamus ( A ) and the pituitary ( B ) of T sheep were not significantly affected by exposure to sewage sludge relative to control sheep.
Figure Legend Snippet: Maternal ER α mRNA expression (mean ± SE) in the hypothalamus ( A ) and the pituitary ( B ) of T sheep were not significantly affected by exposure to sewage sludge relative to control sheep.

Techniques Used: Expressing

20) Product Images from "Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway"

Article Title: Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway

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

doi: 10.1073/pnas.0402135101

Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.
Figure Legend Snippet: Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.

Techniques Used: Expressing, Infection, Transfection, Plasmid Preparation, Negative Control

21) Product Images from "Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure"

Article Title: Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure

Journal: Molecular and Cellular Biochemistry

doi: 10.1007/s11010-020-03771-1

GW501516 impact on N-cadherin level in T24 cells. Cells were exposed for 24 h to vehicle or PPARβ/δ activator at the indicated concentrations. a RTqPCR analysis of cdh2 mRNA expression. Fold inductions represent comparison with control cells (set at 1). b , c N-cadherin detection was determined by Western blotting analysis from total protein extracts with GC-4 antibody. β-actin was used as an internal loading control. The density of each band in immunoblots was measured by using ImageJ and N-cadherin levels were determined as arbitrary units in control and treated cells. The amount of N-cadherin protein was normalized to that of β-actin. Data are expressed as means ± SEM of three independent experiments performed in triplicate. * P
Figure Legend Snippet: GW501516 impact on N-cadherin level in T24 cells. Cells were exposed for 24 h to vehicle or PPARβ/δ activator at the indicated concentrations. a RTqPCR analysis of cdh2 mRNA expression. Fold inductions represent comparison with control cells (set at 1). b , c N-cadherin detection was determined by Western blotting analysis from total protein extracts with GC-4 antibody. β-actin was used as an internal loading control. The density of each band in immunoblots was measured by using ImageJ and N-cadherin levels were determined as arbitrary units in control and treated cells. The amount of N-cadherin protein was normalized to that of β-actin. Data are expressed as means ± SEM of three independent experiments performed in triplicate. * P

Techniques Used: Expressing, Western Blot

22) Product Images from "Stability of telomeric G-quadruplexes"

Article Title: Stability of telomeric G-quadruplexes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1292

( A and B ) Thermal melting (cooling and heating) followed by absorbance at 295 nm of (A) Asc20 and (B) Bom17 in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( C and D ) CD spectra at 4°C of (C) FAsc20T and (D) FBom17T in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( E and F ) Thermal melting (heating) followed by FRET (excitation at 470 nm, emission at 520 nm) of (E) FAsc20T and (F) FBom17T in NaCl (circles) and KCl (triangles), at 0.2 µM oligonucleotide strand concentration.
Figure Legend Snippet: ( A and B ) Thermal melting (cooling and heating) followed by absorbance at 295 nm of (A) Asc20 and (B) Bom17 in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( C and D ) CD spectra at 4°C of (C) FAsc20T and (D) FBom17T in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( E and F ) Thermal melting (heating) followed by FRET (excitation at 470 nm, emission at 520 nm) of (E) FAsc20T and (F) FBom17T in NaCl (circles) and KCl (triangles), at 0.2 µM oligonucleotide strand concentration.

Techniques Used: Concentration Assay

23) Product Images from "Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats"

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021853

Effects of riociguat on plasma and urinary protein levels of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p
Figure Legend Snippet: Effects of riociguat on plasma and urinary protein levels of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p

Techniques Used:

Effects of riociguat on mRNA expression of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the left ventricle and the renal cortex in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p
Figure Legend Snippet: Effects of riociguat on mRNA expression of osteopontin (OPN), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the left ventricle and the renal cortex in the vehicle (n = 7) - and riociguat-treated (3 or 10 mg/kg/d, n = 11 per group) Dahl/ss rats maintained on a high-salt diet. Healthy, age-matched animals were used as controls (n = 10). Data are mean±SEM; *p

Techniques Used: Expressing

24) Product Images from "Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway"

Article Title: Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway

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

doi: 10.1073/pnas.0402135101

IL-6 secretion is inhibited by LMP2A and controls LMP1 expression. ( A ) The ability of LMP2A to down-regulate IL-6 secretion in rEBV-infected HONE-1 and Ad/AH cell lines was determined by using an IL-6 ELISA. The histograms are representative of at least three separate experiments, and the mean ± SE of triplicate determinations is shown. ( B ) Addition of human recombinant IL-6 to the HONE-1 rEBV-infected cell line induces LMP1 expression as demonstrated by immunoblot analysis. ( C ) Addition of human recombinant IL-6 to the C666-1 NPC cell line induces LMP1 expression and phosphorylation of STAT3 as demonstrated by immunoblot analysis. Total STAT3 served as a loading control.
Figure Legend Snippet: IL-6 secretion is inhibited by LMP2A and controls LMP1 expression. ( A ) The ability of LMP2A to down-regulate IL-6 secretion in rEBV-infected HONE-1 and Ad/AH cell lines was determined by using an IL-6 ELISA. The histograms are representative of at least three separate experiments, and the mean ± SE of triplicate determinations is shown. ( B ) Addition of human recombinant IL-6 to the HONE-1 rEBV-infected cell line induces LMP1 expression as demonstrated by immunoblot analysis. ( C ) Addition of human recombinant IL-6 to the C666-1 NPC cell line induces LMP1 expression and phosphorylation of STAT3 as demonstrated by immunoblot analysis. Total STAT3 served as a loading control.

Techniques Used: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Recombinant

Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.
Figure Legend Snippet: Confirmation that LMP2A regulates LMP1 expression. ( A ) Transient delivery of LMP2A represses LMP1 expression in rEBV-2A-infected HONE-1 cells. HONE-1 cells infected with rEBV-2A were transiently transfected with a vector (pSG-LMP2A) expressing HA-tagged LMP2A or with a control vector (pSG5), and, after 48 h, the protein expression was assessed by immunoblotting with antibodies to HA for detection of tagged LMP2A, LMP1, phospho-STAT3, and total STAT3. LMP2A expression resulted in decreased expression of both LMP1 and phospho-STAT3. pSG5 control transfected cells were used as a negative control. ( B ) HONE-1 rEBV-infected cells were transiently transfected with siRNA targeted to LMP2A or with a control scrambled siRNA. This treatment resulted in a 50% reduction in the levels of LMP2A relative to control ( Upper ) and a 6-fold increase in LMP1 relative to control ( Lower ) at 48 h posttransfection. Data shown are representative of two separate experiments with triplicate determination in each experiment, and data are expressed as the mean ± SE.

Techniques Used: Expressing, Infection, Transfection, Plasmid Preparation, Negative Control

LMP2A negatively regulates IL-6 secretion by means of inhibition of NF-κB. ( A ) EMSA demonstrates that infection of HONE-1 cells with rEBV inhibits NF-κB activity and that this effect is relieved in cells infected with rEBV-2A. Nuclear extracts isolated from HONE-1 cells or HONE-1 cells infected with rEBVs were analyzed for basal NF-κB activity. Tumor necrosis factor α-stimulated HONE-1 parental cells were used as a positive control. The presence of p65 and p50 subunits in the NF-κB complexes was confirmed by using antibodies to these subunits. ( B ) Blockade of NF-κB activity in rEBV-2A-infected HONE-1 cells with the RAd-IκBα SS/AA virus inhibited IL-6 secretion in a dose-dependent manner as determined by IL-6 ELISA. Data are representative of two separate experiments with triplicate determination, and the mean ± SE were all
Figure Legend Snippet: LMP2A negatively regulates IL-6 secretion by means of inhibition of NF-κB. ( A ) EMSA demonstrates that infection of HONE-1 cells with rEBV inhibits NF-κB activity and that this effect is relieved in cells infected with rEBV-2A. Nuclear extracts isolated from HONE-1 cells or HONE-1 cells infected with rEBVs were analyzed for basal NF-κB activity. Tumor necrosis factor α-stimulated HONE-1 parental cells were used as a positive control. The presence of p65 and p50 subunits in the NF-κB complexes was confirmed by using antibodies to these subunits. ( B ) Blockade of NF-κB activity in rEBV-2A-infected HONE-1 cells with the RAd-IκBα SS/AA virus inhibited IL-6 secretion in a dose-dependent manner as determined by IL-6 ELISA. Data are representative of two separate experiments with triplicate determination, and the mean ± SE were all

Techniques Used: Inhibition, Infection, Activity Assay, Isolation, Positive Control, Enzyme-linked Immunosorbent Assay

25) Product Images from "Stability of telomeric G-quadruplexes"

Article Title: Stability of telomeric G-quadruplexes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1292

( A and B ) Thermal melting (cooling and heating) followed by absorbance at 295 nm of (A) Asc20 and (B) Bom17 in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( C and D ) CD spectra at 4°C of (C) FAsc20T and (D) FBom17T in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( E and F ) Thermal melting (heating) followed by FRET (excitation at 470 nm, emission at 520 nm) of (E) FAsc20T and (F) FBom17T in NaCl (circles) and KCl (triangles), at 0.2 µM oligonucleotide strand concentration.
Figure Legend Snippet: ( A and B ) Thermal melting (cooling and heating) followed by absorbance at 295 nm of (A) Asc20 and (B) Bom17 in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( C and D ) CD spectra at 4°C of (C) FAsc20T and (D) FBom17T in NaCl (circles) and KCl (triangles), at 3 µM oligonucleotide strand concentration. ( E and F ) Thermal melting (heating) followed by FRET (excitation at 470 nm, emission at 520 nm) of (E) FAsc20T and (F) FBom17T in NaCl (circles) and KCl (triangles), at 0.2 µM oligonucleotide strand concentration.

Techniques Used: Concentration Assay

26) Product Images from "Inhibition of hepatitis C virus replication by semi-synthetic derivatives of glycopeptide antibiotics"

Article Title: Inhibition of hepatitis C virus replication by semi-synthetic derivatives of glycopeptide antibiotics

Journal: Journal of Antimicrobial Chemotherapy

doi: 10.1093/jac/dkr104

Clearance–rebound in the Huh 9-13 replicon assay. Replicon-containing cells were treated for two or four consecutive passages with LCTA-949 (black bars), white bars or VX-950 (top panels), 2′- C -methylcytidine (middle panels), 4′-azidocytidine (bottom panels) or the combination of these selective HCV inhibitors with LCTA-949 (grey bars; each compound was at a concentration of 4× EC 50 ). In the clearance phase (C), cells were incubated in the compound's presence in the absence of G418. After this clearance phase, cells were further cultured twice in fresh medium containing 1 mg/mL G418 without antiviral agents (rebound phase, R). Intracellular HCV RNA in the surviving cells was quantified by qRT–PCR. UTC, untreated controls (Cx, clearance phase at passage x; Rx, rebound phase at passage x; Cx-Rx, rebound following clearance x).
Figure Legend Snippet: Clearance–rebound in the Huh 9-13 replicon assay. Replicon-containing cells were treated for two or four consecutive passages with LCTA-949 (black bars), white bars or VX-950 (top panels), 2′- C -methylcytidine (middle panels), 4′-azidocytidine (bottom panels) or the combination of these selective HCV inhibitors with LCTA-949 (grey bars; each compound was at a concentration of 4× EC 50 ). In the clearance phase (C), cells were incubated in the compound's presence in the absence of G418. After this clearance phase, cells were further cultured twice in fresh medium containing 1 mg/mL G418 without antiviral agents (rebound phase, R). Intracellular HCV RNA in the surviving cells was quantified by qRT–PCR. UTC, untreated controls (Cx, clearance phase at passage x; Rx, rebound phase at passage x; Cx-Rx, rebound following clearance x).

Techniques Used: Concentration Assay, Incubation, Cell Culture, Quantitative RT-PCR

27) Product Images from "Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression"

Article Title: Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression

Journal: Nature Communications

doi: 10.1038/ncomms10846

Characterization of monocyte and macrophage biology in a unique QKI haploinsufficient patient. ( a ) Schematic of chromosomal translocation event in the qkI haploinsufficient patient (Pat- QKI +/− ), reducing QKI expression to ∼50% that of her sister control (Sib- QKI +/+ ). ( b ) Top: UCSC Genome Browser display of reference genome QKI locus with standard and chimeric reads for the patient and sibling. The reduced expression levels and altered 3′-untranslated region (UTR) composition in the patient RNA as compared with a sibling control is noteworthy. Patient shows increased intronic RNA extending to the point where chimeric reads map at the breakpoint to chr5. Middle: chromosome diagrams showing normal chromosomes 5 and 6 with the red line, indicating the location of the breakage fusion point. Bottom: sequence across the fusion point. The chromosomal origin of the AG dinucleotide is ambiguous. ( c ) Photomicrographs of sibling and patient macrophages, cultured in GM-CSF or M-CSF for 7 days, respectively. ( d , e ) Assessment of QKI isoform mRNA and protein expression in 4-day GM-CSF-stimulated macrophages in the sibling and patient. ( f ) Hierarchical clustering (Euclidean algorithm) of key monocyte differentiation genes depicting changes in RNA-seq-derived mRNA abundance where dark blue=low expression, whereas light blue=high expression (* and/or # indicates ≥1.5-fold expression change in monocytes or macrophages, respectively). ( g ) Venn diagrams with numbers of differentially expressed genes (minimally ±1.5-fold; patient/sibling expression) for unstimulated (top) and GM-CSF stimulated macrophages (bottom). An expression cutoff (Pat+Sib expression≥1CPM) was applied. ( h ) The most differentially expressed genes, harbouring a QRE are depicted. ( i ) Genome-wide scatterplot of mRNA abundance ( y axis: Log 10 CPM) versus the log 2 FC ( x axis: Patient/sibling CPM) after an expression cutoff (Pat+Sib expression ≥1 CPM) in monocytes (left) and GM-CSF-stimulated macrophages (right). Blue dots indicate QRE-containing transcripts minimally ±1.5-fold differentially expressed. Grey dots do not fulfill these criteria. ( j ) CDF ( y axis) for QKI target (QRE containing: blue line) and non-target (non-QRE containing: cyan line) mRNAs ( x axis: log 2 FC) in monocytes (left) and macrophages (right). Left shift indicates lower expression of QKI target genes, whereas a right shift indicates higher expression of QKI targets in the patient samples. Distributions were compared using a Wilcoxon rank-sum test.
Figure Legend Snippet: Characterization of monocyte and macrophage biology in a unique QKI haploinsufficient patient. ( a ) Schematic of chromosomal translocation event in the qkI haploinsufficient patient (Pat- QKI +/− ), reducing QKI expression to ∼50% that of her sister control (Sib- QKI +/+ ). ( b ) Top: UCSC Genome Browser display of reference genome QKI locus with standard and chimeric reads for the patient and sibling. The reduced expression levels and altered 3′-untranslated region (UTR) composition in the patient RNA as compared with a sibling control is noteworthy. Patient shows increased intronic RNA extending to the point where chimeric reads map at the breakpoint to chr5. Middle: chromosome diagrams showing normal chromosomes 5 and 6 with the red line, indicating the location of the breakage fusion point. Bottom: sequence across the fusion point. The chromosomal origin of the AG dinucleotide is ambiguous. ( c ) Photomicrographs of sibling and patient macrophages, cultured in GM-CSF or M-CSF for 7 days, respectively. ( d , e ) Assessment of QKI isoform mRNA and protein expression in 4-day GM-CSF-stimulated macrophages in the sibling and patient. ( f ) Hierarchical clustering (Euclidean algorithm) of key monocyte differentiation genes depicting changes in RNA-seq-derived mRNA abundance where dark blue=low expression, whereas light blue=high expression (* and/or # indicates ≥1.5-fold expression change in monocytes or macrophages, respectively). ( g ) Venn diagrams with numbers of differentially expressed genes (minimally ±1.5-fold; patient/sibling expression) for unstimulated (top) and GM-CSF stimulated macrophages (bottom). An expression cutoff (Pat+Sib expression≥1CPM) was applied. ( h ) The most differentially expressed genes, harbouring a QRE are depicted. ( i ) Genome-wide scatterplot of mRNA abundance ( y axis: Log 10 CPM) versus the log 2 FC ( x axis: Patient/sibling CPM) after an expression cutoff (Pat+Sib expression ≥1 CPM) in monocytes (left) and GM-CSF-stimulated macrophages (right). Blue dots indicate QRE-containing transcripts minimally ±1.5-fold differentially expressed. Grey dots do not fulfill these criteria. ( j ) CDF ( y axis) for QKI target (QRE containing: blue line) and non-target (non-QRE containing: cyan line) mRNAs ( x axis: log 2 FC) in monocytes (left) and macrophages (right). Left shift indicates lower expression of QKI target genes, whereas a right shift indicates higher expression of QKI targets in the patient samples. Distributions were compared using a Wilcoxon rank-sum test.

Techniques Used: Translocation Assay, Expressing, Sequencing, Cell Culture, RNA Sequencing Assay, Derivative Assay, Genome Wide

QKI influences pre-mRNA splicing in naive PB monocytes and macrophages. ( a ) SpliceTrap assessment of the proximal ACUAA RNA motif enrichment in 50-bp windows upstream and downstream of alternatively spliced cassette exons (as compared with a background set of exons; grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment over the genomic locus are depicted. ( b ) Sashimi plots illustrate RNA-seq read coverage for selected alternative splicing events in Pat- QKI +/− versus Sib- QKI +/+ PB monocytes (orange) and macrophages (blue). Splicing events (se) are highlighted by inverted brackets. The location of ACUAA motifs and QKI PAR-CLIPs are provided below. Splicing events were defined based on the genomic organization of RefSeq transcripts (bottom tracks). Full event details are provided in Supplementary Data 3 . ( c ) PCR validation of alternatively spliced cassette exons in Sib- QKI +/+ and Pat- QKI +/ − PB-derived monocytes and macrophages. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). ( d ) Phase-contrast and fluorescence-microscopy photographs (scale bar, 50 μm) of primary human, PB macrophages of healthy controls that have been treated with FAM-labelled GapmeRs, to reduce QKI expression. ( e ) Quantitative RT–PCR (qRT–PCR) of QKI mRNA isoform expression in GapmeR-treated macrophages ( n =3). Data expressed as mean±s.e.m.; Student's t -test, with ** P
Figure Legend Snippet: QKI influences pre-mRNA splicing in naive PB monocytes and macrophages. ( a ) SpliceTrap assessment of the proximal ACUAA RNA motif enrichment in 50-bp windows upstream and downstream of alternatively spliced cassette exons (as compared with a background set of exons; grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment over the genomic locus are depicted. ( b ) Sashimi plots illustrate RNA-seq read coverage for selected alternative splicing events in Pat- QKI +/− versus Sib- QKI +/+ PB monocytes (orange) and macrophages (blue). Splicing events (se) are highlighted by inverted brackets. The location of ACUAA motifs and QKI PAR-CLIPs are provided below. Splicing events were defined based on the genomic organization of RefSeq transcripts (bottom tracks). Full event details are provided in Supplementary Data 3 . ( c ) PCR validation of alternatively spliced cassette exons in Sib- QKI +/+ and Pat- QKI +/ − PB-derived monocytes and macrophages. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). ( d ) Phase-contrast and fluorescence-microscopy photographs (scale bar, 50 μm) of primary human, PB macrophages of healthy controls that have been treated with FAM-labelled GapmeRs, to reduce QKI expression. ( e ) Quantitative RT–PCR (qRT–PCR) of QKI mRNA isoform expression in GapmeR-treated macrophages ( n =3). Data expressed as mean±s.e.m.; Student's t -test, with ** P

Techniques Used: RNA Sequencing Assay, Polymerase Chain Reaction, Derivative Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR

QKI expression levels influence pre-mRNA splicing during THP-1-based monocyte-like to macrophage-like cell differentiation. ( a ) Schematic depicting detectable alternative splicing events with the splicing-sensitive microarray platform and number of inclusion (incl.; top lines) or exclusion (excl.; bottom lines) events observed in unstimulated THP-1 ‘monocytes' (left) and 3-day PMA-stimulated THP-1 ‘macrophages' ( n =3, q ≤0.05). ( b ) Scatterplots of skip ( y axis) and include ( x axis) probe set intensity for selected alternative splicing events in sh-Cont (blue boxes) versus sh-QKI (orange circles) in unstimulated and 3 days PMA-stimulated THP-1 ‘monocytes' and ‘macrophages', respectively. Regression coefficients (constrained to pass the origin) are depicted as solid lines. The log 2 difference in the slopes (termed separation score; ss) are provided to the right of the plots for each event, with for example, an ss of −1.72, indicating a 3.3-fold more inclusion of ADD3 exon 13 in sh-QKI versus sh-Cont THP-1 ‘monocytes'. Full event details are provided in Supplementary Data 6 . CE, cassette exon; Alt 5′ or 3′, alternative 5′ or 3′ splice site; RI, retained intron. ( c ) SpliceTrap assessment of average proximal ACUAA RNA motif enrichment in 50 bp windows upstream and downstream of alternatively spliced cassette exons as compared with a background set of exons (grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment are depicted. ( d ) PCR validation of alternatively spliced cassette exons in sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages'. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). All experiments depict biological n =3. ( e ) PCR validation of three splicing events in wt and qk v mouse-derived primary monocytes and 7 days M-CSF-stimulated macrophages. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). Depicted is a representative PCR for at least a biological n ≥3.
Figure Legend Snippet: QKI expression levels influence pre-mRNA splicing during THP-1-based monocyte-like to macrophage-like cell differentiation. ( a ) Schematic depicting detectable alternative splicing events with the splicing-sensitive microarray platform and number of inclusion (incl.; top lines) or exclusion (excl.; bottom lines) events observed in unstimulated THP-1 ‘monocytes' (left) and 3-day PMA-stimulated THP-1 ‘macrophages' ( n =3, q ≤0.05). ( b ) Scatterplots of skip ( y axis) and include ( x axis) probe set intensity for selected alternative splicing events in sh-Cont (blue boxes) versus sh-QKI (orange circles) in unstimulated and 3 days PMA-stimulated THP-1 ‘monocytes' and ‘macrophages', respectively. Regression coefficients (constrained to pass the origin) are depicted as solid lines. The log 2 difference in the slopes (termed separation score; ss) are provided to the right of the plots for each event, with for example, an ss of −1.72, indicating a 3.3-fold more inclusion of ADD3 exon 13 in sh-QKI versus sh-Cont THP-1 ‘monocytes'. Full event details are provided in Supplementary Data 6 . CE, cassette exon; Alt 5′ or 3′, alternative 5′ or 3′ splice site; RI, retained intron. ( c ) SpliceTrap assessment of average proximal ACUAA RNA motif enrichment in 50 bp windows upstream and downstream of alternatively spliced cassette exons as compared with a background set of exons (grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment are depicted. ( d ) PCR validation of alternatively spliced cassette exons in sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages'. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). All experiments depict biological n =3. ( e ) PCR validation of three splicing events in wt and qk v mouse-derived primary monocytes and 7 days M-CSF-stimulated macrophages. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). Depicted is a representative PCR for at least a biological n ≥3.

Techniques Used: Expressing, Cell Differentiation, Microarray, Polymerase Chain Reaction, Derivative Assay

28) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.379966

Influence of CpG methylation on the nuclease activity of ADCY9m activity in vivo . A, determination of TM frequencies induced by ADCY9m and CAPNS1m in 293H cells. 293H cells were transfected with 5 μg of meganuclease-expressing vector or empty
Figure Legend Snippet: Influence of CpG methylation on the nuclease activity of ADCY9m activity in vivo . A, determination of TM frequencies induced by ADCY9m and CAPNS1m in 293H cells. 293H cells were transfected with 5 μg of meganuclease-expressing vector or empty

Techniques Used: CpG Methylation Assay, Activity Assay, In Vivo, Transfection, Expressing, Plasmid Preparation

Effect of valine 73 to alanine substitution on the nuclease activity of ADCY9m toward extrachromosomal unmethylated ADCY9t in yeast ( A ) and in mammalian cells ( B ). Effect of valine 73 to alanine substitution on the frequency of TM induced by ADCY9m in
Figure Legend Snippet: Effect of valine 73 to alanine substitution on the nuclease activity of ADCY9m toward extrachromosomal unmethylated ADCY9t in yeast ( A ) and in mammalian cells ( B ). Effect of valine 73 to alanine substitution on the frequency of TM induced by ADCY9m in

Techniques Used: Activity Assay

Effect of 5-aza-dC treatment on HGT induced by ADCY9m. 293H cells were pretreated for 2 days with or without 0.2 μ m 5-aza-dC and then co-transfected with or without 5 μg of meganuclease expressing vector and 2 μg of DNA matrix
Figure Legend Snippet: Effect of 5-aza-dC treatment on HGT induced by ADCY9m. 293H cells were pretreated for 2 days with or without 0.2 μ m 5-aza-dC and then co-transfected with or without 5 μg of meganuclease expressing vector and 2 μg of DNA matrix

Techniques Used: Transfection, Expressing, Plasmid Preparation

29) Product Images from "Small molecule activation of apurinic/apyrimidinic endonuclease 1 reduces DNA damage induced by cisplatin in cultured sensory neurons"

Article Title: Small molecule activation of apurinic/apyrimidinic endonuclease 1 reduces DNA damage induced by cisplatin in cultured sensory neurons

Journal: DNA repair

doi: 10.1016/j.dnarep.2016.03.009

3.1 Activation of APE1 activity by nicorandil
Figure Legend Snippet: 3.1 Activation of APE1 activity by nicorandil

Techniques Used: Activation Assay, Activity Assay

Nicorandil stimulates both wild-type and R177A APE1 endonuclease activity. APE1 endonuclease activity in the absence of nicorandil was measured for wild-type and R177A APE1. Enzyme concentrations of 0.2 nM and 0.0125 nM were used for wild-type and R177A
Figure Legend Snippet: Nicorandil stimulates both wild-type and R177A APE1 endonuclease activity. APE1 endonuclease activity in the absence of nicorandil was measured for wild-type and R177A APE1. Enzyme concentrations of 0.2 nM and 0.0125 nM were used for wild-type and R177A

Techniques Used: Activity Assay

Nicorandil and/or daidzin do not inhibit APE1 redox signaling activity in a cell-based reporter transactivation or redox EMSA assays. Pa02c cells were cotransfected for 24 hours (37°C, 5% CO 2 ) with AP-1 Luciferase Firefly vector and control pRL
Figure Legend Snippet: Nicorandil and/or daidzin do not inhibit APE1 redox signaling activity in a cell-based reporter transactivation or redox EMSA assays. Pa02c cells were cotransfected for 24 hours (37°C, 5% CO 2 ) with AP-1 Luciferase Firefly vector and control pRL

Techniques Used: Activity Assay, Luciferase, Plasmid Preparation

Overexpression of R177A APE1 in cisplatin-treated sensory neurons has no effect on DNA damage; levels of cell death or neurotransmitter release were comparable to control samples. (A) The top panels show representative Western blots of APE1, HA, phospho-H2AX
Figure Legend Snippet: Overexpression of R177A APE1 in cisplatin-treated sensory neurons has no effect on DNA damage; levels of cell death or neurotransmitter release were comparable to control samples. (A) The top panels show representative Western blots of APE1, HA, phospho-H2AX

Techniques Used: Over Expression, Western Blot

Effect of nicorandil on APE1 activity. (A) Chemical structures for nicorandil (top) and the denitrated metabolite, N-(2-hydroxyethyl)nicotinamide (bottom) are shown. Increasing concentrations of nicorandil stimulate wild-type (B) and R177A APE1 (C) endonuclease
Figure Legend Snippet: Effect of nicorandil on APE1 activity. (A) Chemical structures for nicorandil (top) and the denitrated metabolite, N-(2-hydroxyethyl)nicotinamide (bottom) are shown. Increasing concentrations of nicorandil stimulate wild-type (B) and R177A APE1 (C) endonuclease

Techniques Used: Activity Assay

30) Product Images from "DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria"

Article Title: DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003541

Electrophoresis mobility shift assay (EMSA) of 5 ChIP–Seq regions with His 6 -GcrA. EMSA results using increasing concentrations of purified GcrA using probes (red line) design in regions with high number of reads in ChIp-Seq results. From the top to the bottom: 1) the sequence with the maximum number of reads ChipSeq results, corresponding to the coding sequence of CCNA_00697; 2) the intergenic sequence between CCNA_00278 and CCNA_00279; 3) The promoter of mipZ ; 4) The promoter ctrA P1 of ctrA ; 5) A negative control, corresponding to the intergenic between CCNA_01926 and CCNA_01927. On the right, P. = Probe signal and C. = complex signal. Dissociation constants (Kd) of the positive probes are shown in Figure S9 .
Figure Legend Snippet: Electrophoresis mobility shift assay (EMSA) of 5 ChIP–Seq regions with His 6 -GcrA. EMSA results using increasing concentrations of purified GcrA using probes (red line) design in regions with high number of reads in ChIp-Seq results. From the top to the bottom: 1) the sequence with the maximum number of reads ChipSeq results, corresponding to the coding sequence of CCNA_00697; 2) the intergenic sequence between CCNA_00278 and CCNA_00279; 3) The promoter of mipZ ; 4) The promoter ctrA P1 of ctrA ; 5) A negative control, corresponding to the intergenic between CCNA_01926 and CCNA_01927. On the right, P. = Probe signal and C. = complex signal. Dissociation constants (Kd) of the positive probes are shown in Figure S9 .

Techniques Used: Electrophoresis, Mobility Shift, Chromatin Immunoprecipitation, Purification, Sequencing, Negative Control

Binding of GcrA to DNA in different GAnTC methylation states and conformation al changes of GcrA induced by DNA. (A) DNase I footprinting of the ctrA P1 promoter by GcrA. The promoter region was tested in different methylation states with increasing concentrations of His 6 -GcrA. The nucleotide sequence of the ctrA P1 promoter with the start site and the CcrM methylation site (green)vis shown on top. In orange, the putative region, protected by GcrA in the fully methylated state. In green the putative region protected in the hemi-methylated strand plus. (B) Proteolytic digestion using V8 in presence of DNA. In particular 4 kinds of DNA corresponding to the ctrA P1 promoter were tested as reported in the bottom part.
Figure Legend Snippet: Binding of GcrA to DNA in different GAnTC methylation states and conformation al changes of GcrA induced by DNA. (A) DNase I footprinting of the ctrA P1 promoter by GcrA. The promoter region was tested in different methylation states with increasing concentrations of His 6 -GcrA. The nucleotide sequence of the ctrA P1 promoter with the start site and the CcrM methylation site (green)vis shown on top. In orange, the putative region, protected by GcrA in the fully methylated state. In green the putative region protected in the hemi-methylated strand plus. (B) Proteolytic digestion using V8 in presence of DNA. In particular 4 kinds of DNA corresponding to the ctrA P1 promoter were tested as reported in the bottom part.

Techniques Used: Binding Assay, Methylation, Footprinting, Sequencing

31) Product Images from "Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1"

Article Title: Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1

Journal: Journal of medicinal chemistry

doi: 10.1021/acs.jmedchem.8b01529

Overlay of “mode 2” docking poses obtained for stick renderings of MC047 (C, light gray), 11 (C, teal), 25 (C, dark green), and 26 (C, pink) in the active site of APE1 (4QHE). Compounds MC047 , 25 , and 26 all display an interaction between the sulfonamide groups and the magnesium ion (light green sphere), as well as a projection of their halogenated aryl rings toward hot spot region 5, left-hand side.
Figure Legend Snippet: Overlay of “mode 2” docking poses obtained for stick renderings of MC047 (C, light gray), 11 (C, teal), 25 (C, dark green), and 26 (C, pink) in the active site of APE1 (4QHE). Compounds MC047 , 25 , and 26 all display an interaction between the sulfonamide groups and the magnesium ion (light green sphere), as well as a projection of their halogenated aryl rings toward hot spot region 5, left-hand side.

Techniques Used:

). (A) Computational docking of small organic molecules reveals clustering of 50 probes (thin lines, C, cyan, O, red, N blue) into seven consensus sites in or near the active site of APE1 (4QHE) shown as a molecular surface rendering (C, light gray, O, red, N, blue, and S, yellow). DMSO (stick model C, green, O, red, S, yellow) from the crystal structure is shown bound in consensus site 2. Probes at consensus site 1, the most populated site, are in close proximity to the Mg 2+ ion, shown as a surface rendering in green. (B) A secondary site occupied by ethylene glycol in several crystal structures of APE1 is shown with a cluster of 21 probes shown in thin lines. Ethylene glycols bound in this site and in the vicinity are shown as stick renderings (C, magenta, O, red). This is a small pocket on the surface of the molecule. Thus, the major consensus sites for computational docking of probes in the active of APE1 and in another pocket have been validated in our crystallographic analysis.
Figure Legend Snippet: ). (A) Computational docking of small organic molecules reveals clustering of 50 probes (thin lines, C, cyan, O, red, N blue) into seven consensus sites in or near the active site of APE1 (4QHE) shown as a molecular surface rendering (C, light gray, O, red, N, blue, and S, yellow). DMSO (stick model C, green, O, red, S, yellow) from the crystal structure is shown bound in consensus site 2. Probes at consensus site 1, the most populated site, are in close proximity to the Mg 2+ ion, shown as a surface rendering in green. (B) A secondary site occupied by ethylene glycol in several crystal structures of APE1 is shown with a cluster of 21 probes shown in thin lines. Ethylene glycols bound in this site and in the vicinity are shown as stick renderings (C, magenta, O, red). This is a small pocket on the surface of the molecule. Thus, the major consensus sites for computational docking of probes in the active of APE1 and in another pocket have been validated in our crystallographic analysis.

Techniques Used:

Macrocycle treatment alone and in combination with DNA damaging agent methyl methanesulfonate (MMS) in malignant peripheral nerve sheath tumor cell line ST8814 in alkaline CometAssay. APE1 repair inhibitor candidates (macrocycles) and APE1 repair inhibitor control, APE1 repair inhibitor III (ARiIII; Calbiochem), were tested in ST8814 at a single dose (100 μ M or EC 30 ) and in combination with a single dose of MMS (1.0 mM) for a 1.0 h treatment. Cells were seeded in 6-well tissue culture plates at 130 000 cells/well in DMEM + 10% FBS and grown overnight at 37 °C, 5% CO 2 . Medium was exchanged with Opti-MEM (Gibco) medium containing macrocycle alone or spiked with 1.0 mM MMS. Cells were then incubated for 1.0 h at 37 °C, 5% CO 2 . Medium was exchanged with PBS, and cells were treated with 0.25% trypsin (HyClone), collected, and washed with PBS. Cells were then counted by hemacytometer. DNA damage was evaluated by CometAssay (Trevigen) performed under alkaline conditions. Cells were resuspended in PBS at 1 × 10 5 /mL and then added to premelted and cooled (37 °C) agarose at a 1:10 ratio. Cells were gently mixed, and then 50 μ L of the agarose cell mix was transferred to prewarmed comet slides (37 °C, CometSlide). After solidifying at 4 °C, slides were placed in lysis solution for 60 min and then placed in freshly prepared alkaline unwinding solution (NaOH, pH 13) for 20 min. Slides were then subjected to electrophoresis under alkaline conditions (NaOH, pH 13) at 1 V/cm (300 mA) for 30 min. Slides were washed in H 2 O, placed in 70% ethanol, and allowed to dry at 37 °C for 30 min. Slides were stained with 100 μ L of 1:10 000 diluted SYBR Gold (Invitrogen) in TE pH 7.5 and incubated for 30 min. Slides were then rinsed in H 2 O briefly, and comets were captured by fluorescent microscope (Leica DMIL) and quantified by CometScore Pro (TriTec Corp). DNA damage measured by percent Tail DNA for controls and selected macrocycles is shown in (A) in two independent experiments. Representative images of comet slide DNA damage are shown in (B) for controls and selected macrocycles. There was an average of 20 comet readings/compound.
Figure Legend Snippet: Macrocycle treatment alone and in combination with DNA damaging agent methyl methanesulfonate (MMS) in malignant peripheral nerve sheath tumor cell line ST8814 in alkaline CometAssay. APE1 repair inhibitor candidates (macrocycles) and APE1 repair inhibitor control, APE1 repair inhibitor III (ARiIII; Calbiochem), were tested in ST8814 at a single dose (100 μ M or EC 30 ) and in combination with a single dose of MMS (1.0 mM) for a 1.0 h treatment. Cells were seeded in 6-well tissue culture plates at 130 000 cells/well in DMEM + 10% FBS and grown overnight at 37 °C, 5% CO 2 . Medium was exchanged with Opti-MEM (Gibco) medium containing macrocycle alone or spiked with 1.0 mM MMS. Cells were then incubated for 1.0 h at 37 °C, 5% CO 2 . Medium was exchanged with PBS, and cells were treated with 0.25% trypsin (HyClone), collected, and washed with PBS. Cells were then counted by hemacytometer. DNA damage was evaluated by CometAssay (Trevigen) performed under alkaline conditions. Cells were resuspended in PBS at 1 × 10 5 /mL and then added to premelted and cooled (37 °C) agarose at a 1:10 ratio. Cells were gently mixed, and then 50 μ L of the agarose cell mix was transferred to prewarmed comet slides (37 °C, CometSlide). After solidifying at 4 °C, slides were placed in lysis solution for 60 min and then placed in freshly prepared alkaline unwinding solution (NaOH, pH 13) for 20 min. Slides were then subjected to electrophoresis under alkaline conditions (NaOH, pH 13) at 1 V/cm (300 mA) for 30 min. Slides were washed in H 2 O, placed in 70% ethanol, and allowed to dry at 37 °C for 30 min. Slides were stained with 100 μ L of 1:10 000 diluted SYBR Gold (Invitrogen) in TE pH 7.5 and incubated for 30 min. Slides were then rinsed in H 2 O briefly, and comets were captured by fluorescent microscope (Leica DMIL) and quantified by CometScore Pro (TriTec Corp). DNA damage measured by percent Tail DNA for controls and selected macrocycles is shown in (A) in two independent experiments. Representative images of comet slide DNA damage are shown in (B) for controls and selected macrocycles. There was an average of 20 comet readings/compound.

Techniques Used: Incubation, Lysis, Electrophoresis, Staining, Microscopy

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Amplification:

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats
Article Snippet: .. Real-time quantitative polymerase chain reactions (RTPCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA) were performed to determine relative gene expression of OPN, TIMP-1, PAI-1. cDNA samples were amplified with a PCR mix containing Taq polymerase (qPCR MasterMix Plus, Ref. RT-QP2x-03-075+; Eurogentec, Seraing, Belgium) and primer sets with 6-FAM and TAMRA labeled probes (Operon Biotechnologies, Cologne, Germany) in 96-well or 384-well microtiter plates. ..

Synthesized:

Article Title: Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1
Article Snippet: .. The APE1 repair activity assay was performed in a 96-well plate assay using purified full-length APE1 enzyme and an AP site mimic consisting of two annealed oligonucleotides (5′−6-FAM-GCCCCC*-GGGGACGTACGATATCCCGCTCC-3′ and 3′-Q-CGGGGGCCCCCTGCATGCTATAGGGCGAGG-5′) custom synthesized by Eurogentec Ltd. (Belgium). ..

Article Title: Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP)
Article Snippet: .. AONs with phosphorothioate backbones and 2′-O -methyl ribose modifications (2′-O -MePS) were synthesized by Prosensa (Leiden, Netherlands) and AONs with 2′-O -MePS with additional LNA modifications were synthesized by Eurogentec (Seraing, Belgium). .. Transfection efficiency was assessed using a control AON with 5′-fluorescein group (6-FAM).

Labeling:

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats
Article Snippet: .. Real-time quantitative polymerase chain reactions (RTPCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA) were performed to determine relative gene expression of OPN, TIMP-1, PAI-1. cDNA samples were amplified with a PCR mix containing Taq polymerase (qPCR MasterMix Plus, Ref. RT-QP2x-03-075+; Eurogentec, Seraing, Belgium) and primer sets with 6-FAM and TAMRA labeled probes (Operon Biotechnologies, Cologne, Germany) in 96-well or 384-well microtiter plates. ..

Article Title: The Expression of the Hepatocyte SLAMF3 (CD229) Receptor Enhances the Hepatitis C Virus Infection
Article Snippet: .. The following primers were used for QPCR to quatify HCV transcripts: forward primer (300 nM) HCV-S1 (nucleotides (nt): −215 to −197; 5′-TCCCGGGAGAGCCATAGTG-3′ ), 600 nM reverse primer HCV-AS2 (nt: −158 to −140; 5′-TCCAAGAAAGGACCCRGT-3′), and 250 nM TaqMan minor groove binding (MGB) probe labeled with 6-carboxyfluorescein (nt: −195 to −181; 5′-FAM-TCTGCGGAACCGGTG-MGB-3′) as previously described (Eurogentec, Angers, France) . .. For SLAMF3 transcript quantification, the primers were as follows: forward 5′-TGG GAC TAA GAG CCT CTG GAA A-3′, reverse 5′-CCAGATGACGTTCTCAATCTC-3′ and an MGB probe with 6-FAM ( 5′-CCCCAACAGTGGTGTC-3′ ).

Purification:

Article Title: Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1
Article Snippet: .. The APE1 repair activity assay was performed in a 96-well plate assay using purified full-length APE1 enzyme and an AP site mimic consisting of two annealed oligonucleotides (5′−6-FAM-GCCCCC*-GGGGACGTACGATATCCCGCTCC-3′ and 3′-Q-CGGGGGCCCCCTGCATGCTATAGGGCGAGG-5′) custom synthesized by Eurogentec Ltd. (Belgium). ..

SYBR Green Assay:

Article Title: Detrimental effects of rat mesenchymal stromal cell pre-treatment in a model of acute kidney rejection
Article Snippet: .. The PCRs for tumor necrosis factor α (TNFα), interferon γ (IFNγ), interleukin-6 (IL-6), CD25, and MHC class II were performed using TaqMan chemistry (TaqMan® Universal PCR Mastermix; Applied Biosystems), and for chemokine ligand (CCL) 21, IL-1β, and β-actin using SYBR® Green qPCR MasterMix Plus dTTP for SYBR® Assay ROX (Eurogentec, Seraing, Belgium). .. Primers and probes were synthesized by Metabion (Martinsried, Germany) with sequences given in Table .

Sequencing:

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats
Article Snippet: .. Real-time quantitative polymerase chain reactions (RTPCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA) were performed to determine relative gene expression of OPN, TIMP-1, PAI-1. cDNA samples were amplified with a PCR mix containing Taq polymerase (qPCR MasterMix Plus, Ref. RT-QP2x-03-075+; Eurogentec, Seraing, Belgium) and primer sets with 6-FAM and TAMRA labeled probes (Operon Biotechnologies, Cologne, Germany) in 96-well or 384-well microtiter plates. ..

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan
Article Snippet: .. Both TDP1 and TOP1 siRNA sequence pools were bought as Dharmacon ON-TARGET plus smartpools (Fisher Scientific, Loughborough, UK) whilst the BLAST validated scrambled siRNA sequence (5′-UUCUUCGAACGUGUCACGU-3′) and individual TDP1 siRNA sequences (TDP1si-05: 5′-GGAGUUAAGCCAAAGUAUA-3′, TDP1si-06: 5′-UCAGUUACUUGAUGGCUUA-3′, TDP1si-07: 5′-GACCAUAUCUAGUAGUGAU-3′, TDP1si-08: 5′-CUAGACAGUUUCAAAGUGA-3′) were obtained from Eurogentec (Southampton, UK). .. DNA single strand breaks were measured by alkaline comet assay (ACA) ( ).

Article Title: Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-?B transcription factor pathway
Article Snippet: .. Small Interfering RNAs (siRNAs) Directed to LMP2A. siRNA sequence directed to LMP2A (5′-CUCCCAAUAUCCAUCUGCU-3′) and an irrelevant/control siRNA (5′-GGGUAGAUAGACUCUCGCU-3′) were designed and manufactured by Eurogentec (Brussels). .. Sequences were labeled with a fluorescent dye (5′-fluorescein, 6-FAM) to monitor transfection efficiency.

Activity Assay:

Article Title: Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1
Article Snippet: .. The APE1 repair activity assay was performed in a 96-well plate assay using purified full-length APE1 enzyme and an AP site mimic consisting of two annealed oligonucleotides (5′−6-FAM-GCCCCC*-GGGGACGTACGATATCCCGCTCC-3′ and 3′-Q-CGGGGGCCCCCTGCATGCTATAGGGCGAGG-5′) custom synthesized by Eurogentec Ltd. (Belgium). ..

Expressing:

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats
Article Snippet: .. Real-time quantitative polymerase chain reactions (RTPCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA) were performed to determine relative gene expression of OPN, TIMP-1, PAI-1. cDNA samples were amplified with a PCR mix containing Taq polymerase (qPCR MasterMix Plus, Ref. RT-QP2x-03-075+; Eurogentec, Seraing, Belgium) and primer sets with 6-FAM and TAMRA labeled probes (Operon Biotechnologies, Cologne, Germany) in 96-well or 384-well microtiter plates. ..

Polymerase Chain Reaction:

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats
Article Snippet: .. Real-time quantitative polymerase chain reactions (RTPCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA) were performed to determine relative gene expression of OPN, TIMP-1, PAI-1. cDNA samples were amplified with a PCR mix containing Taq polymerase (qPCR MasterMix Plus, Ref. RT-QP2x-03-075+; Eurogentec, Seraing, Belgium) and primer sets with 6-FAM and TAMRA labeled probes (Operon Biotechnologies, Cologne, Germany) in 96-well or 384-well microtiter plates. ..

Article Title: Detrimental effects of rat mesenchymal stromal cell pre-treatment in a model of acute kidney rejection
Article Snippet: .. The PCRs for tumor necrosis factor α (TNFα), interferon γ (IFNγ), interleukin-6 (IL-6), CD25, and MHC class II were performed using TaqMan chemistry (TaqMan® Universal PCR Mastermix; Applied Biosystems), and for chemokine ligand (CCL) 21, IL-1β, and β-actin using SYBR® Green qPCR MasterMix Plus dTTP for SYBR® Assay ROX (Eurogentec, Seraing, Belgium). .. Primers and probes were synthesized by Metabion (Martinsried, Germany) with sequences given in Table .

Article Title: The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins
Article Snippet: .. Reactions were performed using the PCR Master Mix for probe assays (Eurogentech) with β-actin LUX™ primers at a final concentration of 200 nM, the IRS-1 primers at a final concentration of 300 nM, and the probe at 100 nM. ..

Binding Assay:

Article Title: The Expression of the Hepatocyte SLAMF3 (CD229) Receptor Enhances the Hepatitis C Virus Infection
Article Snippet: .. The following primers were used for QPCR to quatify HCV transcripts: forward primer (300 nM) HCV-S1 (nucleotides (nt): −215 to −197; 5′-TCCCGGGAGAGCCATAGTG-3′ ), 600 nM reverse primer HCV-AS2 (nt: −158 to −140; 5′-TCCAAGAAAGGACCCRGT-3′), and 250 nM TaqMan minor groove binding (MGB) probe labeled with 6-carboxyfluorescein (nt: −195 to −181; 5′-FAM-TCTGCGGAACCGGTG-MGB-3′) as previously described (Eurogentec, Angers, France) . .. For SLAMF3 transcript quantification, the primers were as follows: forward 5′-TGG GAC TAA GAG CCT CTG GAA A-3′, reverse 5′-CCAGATGACGTTCTCAATCTC-3′ and an MGB probe with 6-FAM ( 5′-CCCCAACAGTGGTGTC-3′ ).

Concentration Assay:

Article Title: The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins
Article Snippet: .. Reactions were performed using the PCR Master Mix for probe assays (Eurogentech) with β-actin LUX™ primers at a final concentration of 200 nM, the IRS-1 primers at a final concentration of 300 nM, and the probe at 100 nM. ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats
Article Snippet: .. Real-time quantitative polymerase chain reactions (RTPCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA) were performed to determine relative gene expression of OPN, TIMP-1, PAI-1. cDNA samples were amplified with a PCR mix containing Taq polymerase (qPCR MasterMix Plus, Ref. RT-QP2x-03-075+; Eurogentec, Seraing, Belgium) and primer sets with 6-FAM and TAMRA labeled probes (Operon Biotechnologies, Cologne, Germany) in 96-well or 384-well microtiter plates. ..

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    Kaneka Corp ubiquinol
    Simvastatin treatment in HepG2 cells. (a) Respective microRNA expression are shown after varying doses of (i) simvastatin treatment only and (ii) co-treatment of varying doses of simvastatin and 5 µg/ml <t>ubiquinol</t> or 10 µg/ml
    Ubiquinol, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ubiquinol/product/Kaneka Corp
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ubiquinol - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    92
    Kaneka Corp coq10
    Correlation of cholesterol-adjusted <t>CoQ10</t> concentration and redox status in 53 male volunteers before (T0) and after 14 days (T14) of supplementation with CoQ10 (150 mg ubiquinol/day).
    Coq10, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/coq10/product/Kaneka Corp
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    coq10 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Simvastatin treatment in HepG2 cells. (a) Respective microRNA expression are shown after varying doses of (i) simvastatin treatment only and (ii) co-treatment of varying doses of simvastatin and 5 µg/ml ubiquinol or 10 µg/ml

    Journal: Experimental Biology and Medicine

    Article Title: MicroRNAs as biomarkers of hepatotoxicity in a randomized placebo-controlled study of simvastatin and ubiquinol supplementation

    doi: 10.1177/1535370215605588

    Figure Lengend Snippet: Simvastatin treatment in HepG2 cells. (a) Respective microRNA expression are shown after varying doses of (i) simvastatin treatment only and (ii) co-treatment of varying doses of simvastatin and 5 µg/ml ubiquinol or 10 µg/ml

    Article Snippet: Capsules of ubiquinol and placebo of ubiquinol were obtained from Kaneka Corporation (Japan).

    Techniques: Expressing

    Proposed model of miRNA regulation by simvastation and possible protective role of ubiquinol. (a, b) Possible targets of miRNAs regulated in the liver. (c) miRNAs regulated in macrophages and monocytes. Targets of miRNA were hypothesized by published

    Journal: Experimental Biology and Medicine

    Article Title: MicroRNAs as biomarkers of hepatotoxicity in a randomized placebo-controlled study of simvastatin and ubiquinol supplementation

    doi: 10.1177/1535370215605588

    Figure Lengend Snippet: Proposed model of miRNA regulation by simvastation and possible protective role of ubiquinol. (a, b) Possible targets of miRNAs regulated in the liver. (c) miRNAs regulated in macrophages and monocytes. Targets of miRNA were hypothesized by published

    Article Snippet: Capsules of ubiquinol and placebo of ubiquinol were obtained from Kaneka Corporation (Japan).

    Techniques:

    Bibliosphere network of ubiquinol-sensitive genes regulated in liver tissues of C57BL6J mice. Based on co-citations with transcription factors and other genes in the network (GFG level B4), 3 ubiquinol-inducible genes were connected with each other by BibliospherePathwayEdition Software. According to this, the uploaded genes seem to play a key role in PPAR-α signalling pathways. IN, input gene; TF, transcription factor; M, gene product is part of a metabolic pathway; ST, gene product is part of a Genomatix signal transduction pathway.

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Ubiquinol affects the expression of genes involved in PPAR? signalling and lipid metabolism without changes in methylation of CpG promoter islands in the liver of mice

    doi: 10.3164/jcbn.11-19

    Figure Lengend Snippet: Bibliosphere network of ubiquinol-sensitive genes regulated in liver tissues of C57BL6J mice. Based on co-citations with transcription factors and other genes in the network (GFG level B4), 3 ubiquinol-inducible genes were connected with each other by BibliospherePathwayEdition Software. According to this, the uploaded genes seem to play a key role in PPAR-α signalling pathways. IN, input gene; TF, transcription factor; M, gene product is part of a metabolic pathway; ST, gene product is part of a Genomatix signal transduction pathway.

    Article Snippet: Ubiquinol (KANEKA, QHTM ) was added to a standard laboratory mouse diet (powdered CE-2, CLEA, Osaka, Japan) at a concentration of 0.2% with corn oil (1%, v/w).

    Techniques: Mouse Assay, Software, Transduction

    Verification of selected ”top 20” genes from microarray experiments by qRT-PCR. For verification of microarray data, two ”top 20” up-regulated genes (GPX3, NELF) were further selected for qRT-PCR experiments. Finally, GPX3 (A) and NELF (B) genes were up-regulated about 2.0-fold ( p = 0.0196) and 1.3-fold ( p = 0.026) in the ubiquinol-treated (+Q 10 H 2 ) animals, respectively, when related to control mice (−Q 10 H 2 ).

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Ubiquinol affects the expression of genes involved in PPAR? signalling and lipid metabolism without changes in methylation of CpG promoter islands in the liver of mice

    doi: 10.3164/jcbn.11-19

    Figure Lengend Snippet: Verification of selected ”top 20” genes from microarray experiments by qRT-PCR. For verification of microarray data, two ”top 20” up-regulated genes (GPX3, NELF) were further selected for qRT-PCR experiments. Finally, GPX3 (A) and NELF (B) genes were up-regulated about 2.0-fold ( p = 0.0196) and 1.3-fold ( p = 0.026) in the ubiquinol-treated (+Q 10 H 2 ) animals, respectively, when related to control mice (−Q 10 H 2 ).

    Article Snippet: Ubiquinol (KANEKA, QHTM ) was added to a standard laboratory mouse diet (powdered CE-2, CLEA, Osaka, Japan) at a concentration of 0.2% with corn oil (1%, v/w).

    Techniques: Microarray, Quantitative RT-PCR, Mouse Assay

    Body weight development of 10-week old C57BL6J mice during acclimation period and one-week supplementation period with ubiquinol (250 mg/kg/d) or a respective control diet. Body weights of Q 10 H 2 -treated and control animals increased slightly during acclimation period (from day 1 [−6] to day 7 [0]). Strong increases in body weights were obtained during grouping period (from day 0 to day 1) in both groups. No significant changes of body weights have been found during supplementation period from day 1 until sacrifice of mice (day 8) both in the ubquinol-treated (+Q 10 H 2 ) and control group (−Q 10 H 2 ).

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Ubiquinol affects the expression of genes involved in PPAR? signalling and lipid metabolism without changes in methylation of CpG promoter islands in the liver of mice

    doi: 10.3164/jcbn.11-19

    Figure Lengend Snippet: Body weight development of 10-week old C57BL6J mice during acclimation period and one-week supplementation period with ubiquinol (250 mg/kg/d) or a respective control diet. Body weights of Q 10 H 2 -treated and control animals increased slightly during acclimation period (from day 1 [−6] to day 7 [0]). Strong increases in body weights were obtained during grouping period (from day 0 to day 1) in both groups. No significant changes of body weights have been found during supplementation period from day 1 until sacrifice of mice (day 8) both in the ubquinol-treated (+Q 10 H 2 ) and control group (−Q 10 H 2 ).

    Article Snippet: Ubiquinol (KANEKA, QHTM ) was added to a standard laboratory mouse diet (powdered CE-2, CLEA, Osaka, Japan) at a concentration of 0.2% with corn oil (1%, v/w).

    Techniques: Mouse Assay

    Increase in ubiquinone and ubiquinol content within the epidermis after treatment with Q10‐containing formulas. Following a 14‐day treatment with formula 1 and formula 2 ubiquinone (A) and ubiquinol (B) levels were determined within the epidermis obtained from treated forearm skin compared with untreated control skin. Results are depicted as mean ± SEM (20–66 years; n = 73). Significant differences are marked with an asterisk [* P

    Journal: Biofactors (Oxford, England)

    Article Title: Topical treatment with coenzyme Q10‐containing formulas improves skin's Q10 level and provides antioxidative effects

    doi: 10.1002/biof.1239

    Figure Lengend Snippet: Increase in ubiquinone and ubiquinol content within the epidermis after treatment with Q10‐containing formulas. Following a 14‐day treatment with formula 1 and formula 2 ubiquinone (A) and ubiquinol (B) levels were determined within the epidermis obtained from treated forearm skin compared with untreated control skin. Results are depicted as mean ± SEM (20–66 years; n = 73). Significant differences are marked with an asterisk [* P

    Article Snippet: Mass spectrometric detection was carried out as follows: ubiquinone (Sigma‐Aldrich): 880.5 amu [M + NH4 ]+ → 197 amu (Quantifier); ubiquinol (Kaneka Nutrients): 882.7 amu [M + NH4 ]+ → 197 amu (Quantifier); 882.7 amu [M + NH4 ]+ → 81 amu (Qualifer); cholesterol (Sigma‐Aldrich): 369.4 amu [M + H‐H2 O]+ → 91 amu (Quantifier); 369.4 amu [M + NH4 ]+ → 81 amu (Qualifer).

    Techniques:

    Correlation of cholesterol-adjusted CoQ10 concentration and redox status in 53 male volunteers before (T0) and after 14 days (T14) of supplementation with CoQ10 (150 mg ubiquinol/day).

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Coenzyme Q10 serum concentration and redox status in European adults: influence of age, sex, and lipoprotein concentration

    doi: 10.3164/jcbn.15-73

    Figure Lengend Snippet: Correlation of cholesterol-adjusted CoQ10 concentration and redox status in 53 male volunteers before (T0) and after 14 days (T14) of supplementation with CoQ10 (150 mg ubiquinol/day).

    Article Snippet: Intervention study Subject characteristics and study design for the intervention study have been described previously. Fifty-three healthy male volunteers aged between 21 and 48 years received 150 mg/day of the reduced form of CoQ10 (Q10 H2 , ubiquinol; Kaneka Corporation, Japan) with each principal meal for 14 days.

    Techniques: Concentration Assay

    Cholesterol-adjusted CoQ10 concentration correlated to redox status of CoQ10 (Spearman’s, p ≤1E-14) in 860 subjects ranging in age from 18 to 82 years: compartmented into quadrants corresponding to mean values of all data points (12.9% redox status, 179 µmol CoQ10/mol cholesterol).

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Coenzyme Q10 serum concentration and redox status in European adults: influence of age, sex, and lipoprotein concentration

    doi: 10.3164/jcbn.15-73

    Figure Lengend Snippet: Cholesterol-adjusted CoQ10 concentration correlated to redox status of CoQ10 (Spearman’s, p ≤1E-14) in 860 subjects ranging in age from 18 to 82 years: compartmented into quadrants corresponding to mean values of all data points (12.9% redox status, 179 µmol CoQ10/mol cholesterol).

    Article Snippet: Intervention study Subject characteristics and study design for the intervention study have been described previously. Fifty-three healthy male volunteers aged between 21 and 48 years received 150 mg/day of the reduced form of CoQ10 (Q10 H2 , ubiquinol; Kaneka Corporation, Japan) with each principal meal for 14 days.

    Techniques: Concentration Assay

    CoQ10 and cholesterol concentrations of 860 subjects split into age-groups (5-year-steps). Mean values are connected by trend lines of moving average.

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Coenzyme Q10 serum concentration and redox status in European adults: influence of age, sex, and lipoprotein concentration

    doi: 10.3164/jcbn.15-73

    Figure Lengend Snippet: CoQ10 and cholesterol concentrations of 860 subjects split into age-groups (5-year-steps). Mean values are connected by trend lines of moving average.

    Article Snippet: Intervention study Subject characteristics and study design for the intervention study have been described previously. Fifty-three healthy male volunteers aged between 21 and 48 years received 150 mg/day of the reduced form of CoQ10 (Q10 H2 , ubiquinol; Kaneka Corporation, Japan) with each principal meal for 14 days.

    Techniques: