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NeuroMab kchip2
<t>KChIP2</t> represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
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1) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

2) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

3) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

4) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

5) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

6) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

7) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

8) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

9) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

10) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

11) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

12) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

13) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

14) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

15) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

16) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

17) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

18) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

19) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

20) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

21) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

22) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

23) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

24) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

25) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

26) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

27) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

28) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

29) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

30) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

31) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

32) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

33) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

34) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

35) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

36) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

37) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

38) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

39) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p
Figure Legend Snippet: Preservation of the KChIP2/miR-34b/c axis in human heart failure. ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p

Techniques Used: Preserving, RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization

In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p
Figure Legend Snippet: In vitro cardiac disease signaling links KChIP2 loss with miR-34 elevation. ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Functional Assay

miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p
Figure Legend Snippet: miR-34 regulation linked to changes in KChIP2 expression. ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Microarray, Hybridization, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Staining

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