Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: TaqMan Probes in Human, Mouse, and Rat Gene Expression Assay

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques: Gene Expression, Sequencing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Corin mRNA inversely correlates with Atrial natriuretic peptide (ANP )/Brain natriuretic peptide (BNP ) mRNA in chronic, advanced human systolic heart failure. We examined the relationship of corin mRNA to ANP and BNP RNA (A) in the UPENN database in the nonischemic cardiomyopathy (n=120) and nonischemic (n=80) samples. Data are expressed as log 2 normalized RNA abundance (corin on x ‐axis; BNP on y ‐axis.) Corin mRNA was significantly inversely correlated with either ANP or BNP mRNA. The UK heart collection demonstrated a similar significant inverse relationship between corin mRNA and ANP or BNP mRNA (B). In addition, BNP mRNA was significantly positively correlated with GRP78 mRNA, and corin mRNA was significantly inversely correlated with spliced X‐box protein 1 (XBP1).

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Corin RNA and protein in heart failure compared to nonfailing controls. Using a sample of 100 randomly selected samples of transmural sample hearts from the UPENN collection consisting of left‐ventricular (LV) tissue, we compared Atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP), and corin gene expression compared to nonfailing controls (n=10) using real‐time polymerase chain reaction. We discovered that corin mRNA was increased in 53 of the hearts compared to nonfailing controls ( high corin mRNA group ) and decreased in 47 hearts ( low corin mRNA group ) (A). The low corin mRNA group , when compared to the high corin mRNA group , demonstrated significantly higher expression of ANP (B) and BNP (C). Corin protein in samples from LV apex was significantly lower in the low corin RNA as compared to the high corin RNA group (D), demonstrating that changes in corin mRNA resulted in expected changes in corin protein. * P< 0.05, ** P< 0.01, *** P <0.001. Error bars are SEM.

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Thapsigargin (TG)‐induced ER stress causes and time‐dependent corin mRNA decay in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free DMEM‐M199 (4:1) with and without 0.1 μmol/L TG for 6, 16, 24, and 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and gene expression of the following genes was assessed by RT‐PCR (A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; and (F) ATF4. Compared with non‐TG‐treated controls, cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4, and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P< 0.05, ** P< 0.01. Corin mRNA demonstrated a significant, time‐dependent decrease in the cells treated with TG. We also analyzed XBP splicing using PCR, restriction digestion with Pst1, and gel electrophoresis. Fragments of XBP1 were amplified and resolved in 2% agarose gel. PCR products were not digested by Pst I (G). Lane 1: DNA ladder; Lane 2: TG untreated; Lane 3: TG treated. XBP1 fragment: Spliced=450 bp; unspliced=476 bp. PCR products were digested by Pst I (H). Lane 1: DNA ladder; Lane 2: TG untreated; Lane 3: TG treated. Spliced rat XBP1 fragment was 450 bp, while unspliced rat XBP1 appears as 2 fragments of 186 and 290 bp due to the presence of the Pst I restriction site, which is contained in the intronic segment that is removed by IRE1 endonuclease activity. Error bars represent SEM. ATF6 indicates activating transcription factor‐6; ER, endoplasmic reticulum; IRE1, inositol‐requiring protein 1; RT‐PCR, real‐time polymerase chain reaction; CHOP, C/EBP homology protein; DMEM, Dulbecco's Modified Eagle's medium; XBP1, X‐box protein 1.

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques: Cell Culture, Reverse Transcription, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Modification

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Thapsigargin (TG)‐induced ER stress and time‐dependent corin mRNA decay in adult mouse atrial cardiomyocytes (HL‐1) cells. HL‐1 cells were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free Claycomb medium with and without thapsigargin (TG) 0.1 μmol/L for 6, 16, 24, 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and mRNA abundance of the following genes was assessed by RT‐PCR ( A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; (F) ATF4. Compared with non‐TG‐treated HL‐1 control cells, HL‐1 cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4 and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P< 0.05, ** P< 0.01. Corin mRNA demonstrated a significant, time‐dependent decrease in the TG‐treated cells. Error bars represent standard error of the mean. ATF indicates activating transcription factor; ER, endoplasmic reticulum; HL‐1, adult murine atrial myocytes; RT‐PCR, real‐time polymerase chain reaction; XBP1, X‐box protein 1.

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques: Cell Culture, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: IRE1‐gene overexpression. Neonatal murine cardiomyocytes were plated in 6‐well plates with complete medium 1 day before infection. On the second day, cells were infected with IRE1‐overexpressed lentivirus containing 8 μg/mL polybrene and incubated overnight. After that, the cells were fed with fresh complete medium. Cells were harvested at 72 hours after transduction and the total RNA was extracted. Quantitative polymerase chain reaction was conducted using specific TaqMan mRNA probes: (A) IRE1 alpha; (B) Corin; (C) Spliced XBP1; (D) GRP78; (E) CHOP; (F) ATF4 mRNA were quantified by normalization to 18S ribosomal RNA. IRE1‐overexpression activated IRE1 endoribonuclease activity, resulting in a significant increase in XBP1s and a parallel significant decrease in corin mRNA. Note: Compared with mock group, * P< 0.05; ** P< 0.01; *** P< 0.001. Mock: cells transduced with empty lentivirus vehicle; IRE1‐: cells transduced with IRE1‐overexpressed lentivirus. Error bars represent standard error of the mean. ATF indicates activating transcription factor; CHOP, ; IRE1, inositol‐requiring protein 1; XBP1, X‐box protein 1.

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques: Over Expression, Infection, Incubation, Transduction, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: IRE1 shRNA knockdown attenuates corin mRNA decay. HL‐1 cells were transfected with 2 μg mammalian expression plasmid containing IRE1 shRNA or nontargeted negative control (Negative Ctr), separately. Cells were exposed to thapsigargin 0.1 μmol/L for 12 hours. Thereafter, cellular total RNA was isolated and quantitative polymerase chain reaction was conducted using specific TaqMan probes: (A) IRE1 alpha; (B) Corin; (C) Spliced XBP1; (D) GRP78; (E) CHOP; (F) ATF4 mRNA were quantified by normalization to 18S ribosomal RNA. Cells with IRE1 shRNA knockdown prior to exposure to thapsigargin (12 hours) demonstrated less XBP1 splicing and increased corin mRNA values compared to cells transfected with control plasmid and also exposed to 12 hours thapsigargin. * P< 0.05 and ** P< 0.01. Error bars represent SEM. ATF indicates activating transcription factor; CHOP, C/EBP homology protein; HL‐1, adult murine atrial myocytes; IRE1, inositol‐requiring protein 1; XBP1, X‐box protein 1.

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques: shRNA, Knockdown, Transfection, Expressing, Plasmid Preparation, Negative Control, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Effect of actinomycin D prior to thapsigargin (TG). HL‐1 cells were incubated with or without actinomycin D at a concentration of 1 μg/μL 3 hours prior to administration of TG (0.1 μmol/L) for 12 hours. Cells were harvested and the relative abundance of corin mRNA and XBP1s (normalized to 18S rRNA) was compared to cells not receiving TG. We compared the changes in corin and XBP1s in cells not treated (A) or treated with actinomycin D (B) prior to the administration of TG to the medium. We compared relative mRNA abundance (normalized to 18S ribosomal RNA) of corin and XBP1s 12 hours after TG as compared to baseline. As demonstrated in (B), the decrease in corin mRNA and corresponding increase in XBP1s ( P <0.001), a marker of IRE1 endoribonuclease activity, was similar in cells pretreated with actinomycin D as compared to cells not pretreated with actinomycin D (B) prior to the induction of ER stress with TG. Baseline XBP1s was higher in cells not pretreated with actinomycin D. The error bars represent SEM for the relative mRNA expression. ( P <0.001 for baseline compared to 12 hours). Error bars represent SEM. ER indicates endoplasmic reticulum; HL‐1, adult murine atrial myocytes; IRE1, inositol‐requiring protein 1; XBP1s, spliced X‐box protein 1.

Article Snippet: Assays for ANP and BNP were designed in house, whereas assays for 18S ribosomal RNA, spliced X box protein 1 (XBP1s), corin, and GRP78 were purchased from Life Technologies (catalogue numbers: 4310893E, Hs03929085_g1, Hs00198141_m1, and Hs00946084_g1, respectively).

Techniques: Incubation, Concentration Assay, Marker, Activity Assay, Expressing

Journal: bioRxiv

Article Title: A circular engineered sortase for interrogating histone H3 in chromatin

doi: 10.1101/2024.09.10.612318

Figure Lengend Snippet: ( A ) Workflow for isolating tandem mass tagged histone H3 tails with cW11 sortase. ( B ) SDS-PAGE of 14-hour sortase reaction in a nuclear acid extract. ( C ) Tris-Tricine PAGE of histone H3(1-34) synthetic standards (1: 140 ng; 2: 420 ng) and TCA precipitated H3(1-32)-TMT from the acid extract reaction; bands between 25 and 37 kDa are histone H1. ( D ) Structure of the TMT-labelled oligoglycine peptide illustrating sortase reactive GGG, charge carrying K and carboxamide, and TMT labelled aminoalanine. ( E ) Unique proteoforms detected in each 6plex sample and shared between the two samples. ( F ) Quantifiable proteoforms detected in each 6plex sample and shared between the two samples. Volcano plot of significant (p<0.05) log2-fold change and overall abundance of H3 proteoforms in HEK293T cells following treatment with ( G ) LSD1/HDAC1/CoREST complex-specific inhibitor Corin, or ( H ) pan class one HDAC inhibitor MS275.

Article Snippet: For treatment with DMSO vehicle, MS275 (LC Laboratories, Cat# E-3866, Lot: ENT-102) or Corin (Med Chem Express, Cat# HY-111048/CS0034060, Lot: 41808), a single 15 cm plate of HEK293T cells was grown to near confluence and split.

Techniques: SDS Page

Journal: PLoS ONE

Article Title: Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

doi: 10.1371/journal.pone.0135378

Figure Lengend Snippet: Anti-LRP4 immunoreactivity in 101 Italian myasthenic patients (MG) and controls.

Article Snippet: Briefly, cells (1 x 10 5 per test) were resuspended in 50 μl PBS/BSA 0,1% (FACS buffer) and incubated with primary antibodies: sera from MG patients and HBD (dilution 1:5), or a commercially available rabbit anti-LRP4 antiserum (Atlas Antibodies, Stockholm, Sweden; dilution 1:150); the latter antibody, that served as positive control, is specific for LRP4 and does not cross-react with other members of the LRP protein family.

Techniques:

Journal: PLoS ONE

Article Title: Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

doi: 10.1371/journal.pone.0135378

Figure Lengend Snippet: Clinical correlates with and without anti-LRP4 antibodies in double seronegative myasthenic patients.

Article Snippet: Briefly, cells (1 x 10 5 per test) were resuspended in 50 μl PBS/BSA 0,1% (FACS buffer) and incubated with primary antibodies: sera from MG patients and HBD (dilution 1:5), or a commercially available rabbit anti-LRP4 antiserum (Atlas Antibodies, Stockholm, Sweden; dilution 1:150); the latter antibody, that served as positive control, is specific for LRP4 and does not cross-react with other members of the LRP protein family.

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: TaqMan Probes in Human, Mouse, and Rat Gene Expression Assay

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Gene Expression, Sequencing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Corin mRNA inversely correlates with Atrial natriuretic peptide (ANP )/Brain natriuretic peptide (BNP ) mRNA in chronic, advanced human systolic heart failure. We examined the relationship of corin mRNA to ANP and BNP RNA (A) in the UPENN database in the nonischemic cardiomyopathy (n=120) and nonischemic (n=80) samples. Data are expressed as log 2 normalized RNA abundance (corin on x ‐axis; BNP on y ‐axis.) Corin mRNA was significantly inversely correlated with either ANP or BNP mRNA. The UK heart collection demonstrated a similar significant inverse relationship between corin mRNA and ANP or BNP mRNA (B). In addition, BNP mRNA was significantly positively correlated with GRP78 mRNA, and corin mRNA was significantly inversely correlated with spliced X‐box protein 1 (XBP1).

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Corin RNA and protein in heart failure compared to nonfailing controls. Using a sample of 100 randomly selected samples of transmural sample hearts from the UPENN collection consisting of left‐ventricular (LV) tissue, we compared Atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP), and corin gene expression compared to nonfailing controls (n=10) using real‐time polymerase chain reaction. We discovered that corin mRNA was increased in 53 of the hearts compared to nonfailing controls ( high corin mRNA group ) and decreased in 47 hearts ( low corin mRNA group ) (A). The low corin mRNA group , when compared to the high corin mRNA group , demonstrated significantly higher expression of ANP (B) and BNP (C). Corin protein in samples from LV apex was significantly lower in the low corin RNA as compared to the high corin RNA group (D), demonstrating that changes in corin mRNA resulted in expected changes in corin protein. * P< 0.05, ** P< 0.01, *** P <0.001. Error bars are SEM.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Thapsigargin (TG)‐induced ER stress causes and time‐dependent corin mRNA decay in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free DMEM‐M199 (4:1) with and without 0.1 μmol/L TG for 6, 16, 24, and 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and gene expression of the following genes was assessed by RT‐PCR (A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; and (F) ATF4. Compared with non‐TG‐treated controls, cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4, and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P< 0.05, ** P< 0.01. Corin mRNA demonstrated a significant, time‐dependent decrease in the cells treated with TG. We also analyzed XBP splicing using PCR, restriction digestion with Pst1, and gel electrophoresis. Fragments of XBP1 were amplified and resolved in 2% agarose gel. PCR products were not digested by Pst I (G). Lane 1: DNA ladder; Lane 2: TG untreated; Lane 3: TG treated. XBP1 fragment: Spliced=450 bp; unspliced=476 bp. PCR products were digested by Pst I (H). Lane 1: DNA ladder; Lane 2: TG untreated; Lane 3: TG treated. Spliced rat XBP1 fragment was 450 bp, while unspliced rat XBP1 appears as 2 fragments of 186 and 290 bp due to the presence of the Pst I restriction site, which is contained in the intronic segment that is removed by IRE1 endonuclease activity. Error bars represent SEM. ATF6 indicates activating transcription factor‐6; ER, endoplasmic reticulum; IRE1, inositol‐requiring protein 1; RT‐PCR, real‐time polymerase chain reaction; CHOP, C/EBP homology protein; DMEM, Dulbecco's Modified Eagle's medium; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Reverse Transcription, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Modification

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Thapsigargin (TG)‐induced ER stress and time‐dependent corin mRNA decay in adult mouse atrial cardiomyocytes (HL‐1) cells. HL‐1 cells were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free Claycomb medium with and without thapsigargin (TG) 0.1 μmol/L for 6, 16, 24, 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and mRNA abundance of the following genes was assessed by RT‐PCR ( A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; (F) ATF4. Compared with non‐TG‐treated HL‐1 control cells, HL‐1 cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4 and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P< 0.05, ** P< 0.01. Corin mRNA demonstrated a significant, time‐dependent decrease in the TG‐treated cells. Error bars represent standard error of the mean. ATF indicates activating transcription factor; ER, endoplasmic reticulum; HL‐1, adult murine atrial myocytes; RT‐PCR, real‐time polymerase chain reaction; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: IRE1‐gene overexpression. Neonatal murine cardiomyocytes were plated in 6‐well plates with complete medium 1 day before infection. On the second day, cells were infected with IRE1‐overexpressed lentivirus containing 8 μg/mL polybrene and incubated overnight. After that, the cells were fed with fresh complete medium. Cells were harvested at 72 hours after transduction and the total RNA was extracted. Quantitative polymerase chain reaction was conducted using specific TaqMan mRNA probes: (A) IRE1 alpha; (B) Corin; (C) Spliced XBP1; (D) GRP78; (E) CHOP; (F) ATF4 mRNA were quantified by normalization to 18S ribosomal RNA. IRE1‐overexpression activated IRE1 endoribonuclease activity, resulting in a significant increase in XBP1s and a parallel significant decrease in corin mRNA. Note: Compared with mock group, * P< 0.05; ** P< 0.01; *** P< 0.001. Mock: cells transduced with empty lentivirus vehicle; IRE1‐: cells transduced with IRE1‐overexpressed lentivirus. Error bars represent standard error of the mean. ATF indicates activating transcription factor; CHOP, ; IRE1, inositol‐requiring protein 1; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Over Expression, Infection, Incubation, Transduction, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: IRE1 shRNA knockdown attenuates corin mRNA decay. HL‐1 cells were transfected with 2 μg mammalian expression plasmid containing IRE1 shRNA or nontargeted negative control (Negative Ctr), separately. Cells were exposed to thapsigargin 0.1 μmol/L for 12 hours. Thereafter, cellular total RNA was isolated and quantitative polymerase chain reaction was conducted using specific TaqMan probes: (A) IRE1 alpha; (B) Corin; (C) Spliced XBP1; (D) GRP78; (E) CHOP; (F) ATF4 mRNA were quantified by normalization to 18S ribosomal RNA. Cells with IRE1 shRNA knockdown prior to exposure to thapsigargin (12 hours) demonstrated less XBP1 splicing and increased corin mRNA values compared to cells transfected with control plasmid and also exposed to 12 hours thapsigargin. * P< 0.05 and ** P< 0.01. Error bars represent SEM. ATF indicates activating transcription factor; CHOP, C/EBP homology protein; HL‐1, adult murine atrial myocytes; IRE1, inositol‐requiring protein 1; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: shRNA, Knockdown, Transfection, Expressing, Plasmid Preparation, Negative Control, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Effect of actinomycin D prior to thapsigargin (TG). HL‐1 cells were incubated with or without actinomycin D at a concentration of 1 μg/μL 3 hours prior to administration of TG (0.1 μmol/L) for 12 hours. Cells were harvested and the relative abundance of corin mRNA and XBP1s (normalized to 18S rRNA) was compared to cells not receiving TG. We compared the changes in corin and XBP1s in cells not treated (A) or treated with actinomycin D (B) prior to the administration of TG to the medium. We compared relative mRNA abundance (normalized to 18S ribosomal RNA) of corin and XBP1s 12 hours after TG as compared to baseline. As demonstrated in (B), the decrease in corin mRNA and corresponding increase in XBP1s ( P <0.001), a marker of IRE1 endoribonuclease activity, was similar in cells pretreated with actinomycin D as compared to cells not pretreated with actinomycin D (B) prior to the induction of ER stress with TG. Baseline XBP1s was higher in cells not pretreated with actinomycin D. The error bars represent SEM for the relative mRNA expression. ( P <0.001 for baseline compared to 12 hours). Error bars represent SEM. ER indicates endoplasmic reticulum; HL‐1, adult murine atrial myocytes; IRE1, inositol‐requiring protein 1; XBP1s, spliced X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Concentration Assay, Marker, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Single cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation

doi: 10.1101/2022.09.15.507987

Figure Lengend Snippet: ( A ) qPCR analysis of canonical WNT pathway and non-canonical WNT pathway at day 16. *p < 0.05 vs. D11; ††p < 0.01, †††p < 0.001 vs. 1.5 µM CHIR99021 (n = 6). ( B ) qPCR analysis of EN1 at day 16. ***p < 0.001 vs. D11; ††p < 0.01 vs. 1.5 µM CHIR99021 (n = 6). ( C ) Immunostaining of LMX1A + /EN1 + cells. Scale bar, 100 µm. ( D ) Quantification of LMX1A + /EN1 + cells. *p < 0.05 vs. 1.5 µM CHIR99021 (n = 3). ( E ) qPCR analysis of rostral and lateral midbrain markers at day 16. **p < 0.01, ***p < 0.001 vs. D11; †p < 0.05 vs. 1.5 µM CHIR99021 (n = 6). ( F ) Violin plots of CORIN, LMX1A and NGN2 generated from scRNA-seq data of developing human ventral midbrain. ( G ) Immunostaining of CORIN + /LMX1A + cells and NGN2 + /LMX1A + cells. Scale bar, 100 µm. ( H and I ) Quantification of CORIN + /LMX1A + cells ( H ) and NGN2 + /LMX1A + cells ( I ). *p < 0.05, **p < 0.01 vs. 1.5 µM CHIR99021 (n = 3).

Article Snippet: The primary antibodies were used as follows: ALDH1A1 (rabbit, 1:1,000, Abcam, ab23375), COL1A1 (sheep, 1:200, R&D Systems, AF6220), CORIN (rat, 1:1,000, R&D Systems, MAB2209), DCX (goat, 1:500, SantaCruz, sc-8066), EN1 (mouse 1:50, DSHB, 4G11), FOXA2 (goat, 1:500, R&D Systems, AF2400), GIRK2 (rabbit, 1:400, Alomone, APC006), LMX1A (rabbit, 1:4,000, Millipore, AB10533), LMO3 (goat, 1:200, SantaCruz, sc-82647), MAP2 (mouse 1:1,000, Sigma, M4403), NGN2 (goat, 1:200, SantaCruz, sc-19233), NURR1 (rabbit, 1:500, SantaCruz, sc-990), OTX2 (goat, 1:1,000, R&D Systems, AF1979), pH3 (rabbit, 1:500, Millipore, 06-570), PITX3 (goat, 1:500, SantaCruz, sc-19307), PDGFRa (rabbit, 1:100, Cell Signaling, 5241), SOX2 (rabbit, 1:500, Millipore, AB5603), TH (rabbit, 1:1,000, Millipore, AB152), TH (mouse, 1:500, ImmunoStar, 22941), and TH (sheep, 1:500, Novus, NB300).

Techniques: Immunostaining, Generated