Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: circRNA profiles differentiate the normal group from model group. (a) Principal component analysis (PCA) shows the difference between normal and model groups. (b) Scatter plots assess the variation of circRNA expression between two groups. The values plotted on x and y axes are the normalized signal values of each group (log2 scaled). Dots above the top green line and below the bottom green line represent differentially expressed circRNAs. (c, d) Unsupervised hierarchical clustering (c) and volcano plot (d) demonstrate the differential expression of circRNAs during hepatic steatosis. Both downregulated (green) and upregulated (red) circRNAs were visualized in the cluster. In similar, the red points in volcano plot represent the differentially expressed circRNAs with statistical significance. (e, f) Validation of the upregulated (e) and downregulated circRNAs (f) using QPCR. (g) The results of QPCR exhibit well consistence with those of microarray. Pairwise scatter plots reflect the fold changes (log2 transformed) of both microarray (horizontal axis) and the QPCR (vertical axis). The R stands for linear correlation coefficient. The presented results are expressed as means ± SD. ∗∗ P < 0.01.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques: Expressing, Quantitative Proteomics, Biomarker Discovery, Microarray, Transformation Assay

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: Predicted targets of top-20 differentially expressed circRNAs.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques:

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: Enumeration data of differentially expressed circRNAs and target miRNAs/mRNAs.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques:

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: circRNA-miRNA-mRNA regulatory network uncovers the circRNA_021412-miR-1972-LPIN1 signaling underlying circRNAs' effects. (a) The circRNA-miRNA-mRNA network related to transcriptional regulation. Red cycles, violet squares, and blue cycles represent circRNAs, miRNAs, and mRNAs, respectively. The size of each symbol reflects its degree, which is scored by the number of downstream targets. (b) circRNA_021412-miR-1972-LPIN1 signaling within the circRNA-miRNA-mRNA network is recognized to underlie the actions of circRNAs. (c) The circRNA_021412-miR-1972-LPIN1 signaling controls the pathway of lipid degradation via PPAR α -induced ACSLs expression.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques: Expressing

Journal: BioMed Research International

Article Title: Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

doi: 10.1155/2017/5936171

Figure Lengend Snippet: Diagram of circRNA_021412-based regulation of hepatic steatosis. Arrow, blunt-headed line, and red cross represent effects of activation, inhibition, and blocking, respectively.

Article Snippet: To remove linear RNAs and enrich circRNA, total RNA from each sample was treated with Rnase R. circRNA was then transcribed into fluorescent cRNA by random primer according to the Arraystar Super RNA Labeling protocol (Arraystar, Inc., USA) and hybridized onto the Arraystar Human circRNA Arrays V2.0 (Arraystar, Inc., USA) at 65°C for 17 hours.

Techniques: Activation Assay, Inhibition, Blocking Assay

Journal: Frontiers in Oncology

Article Title: CircHADHA-augmented autophagy suppresses tumor growth of colon cancer by regulating autophagy-related gene via miR-361

doi: 10.3389/fonc.2022.937209

Figure Lengend Snippet: Plasma circRNA expression pattern in healthy individuals and colon polyp and colon cancer patients. (A) The endoscopic diagnosis of healthy individuals (left panel) and colon polyp (central panel) and colon cancer (right panel) patients. (B) The schematic diagram of Arraystar assay performance in plasma circRNAs. (C) The differential expression of plasma circRNAs in polyp patients ( N = 5) and healthy individuals ( N = 5). The expression variation of circRNAs was assessed by the scatter plot between polyp patients and healthy individuals (left panel). The green lines represented fold change lines. The circRNAs above the top green line and below the bottom green line indicated more than 1.5-fold change of circRNAs between the two compared samples. The differential expression of circRNAs was analyzed by volcano plots between polyp patients and healthy individuals (right panel). Red points represented the differentially expressed circRNAs with statistical significance (1.5-fold upregulation and downregulation, P < 0.05). (D) The differential expression of plasma circRNAs in colon cancer ( N = 5) and colon polyp patients ( N = 5). The expression variation of circRNAs was assessed by the scatter plot between colon cancer and colon polyp patients (left panel). The green lines represented fold change lines. The circRNAs above the top green line and below the bottom green line indicated more than 1.5-fold change of circRNAs between the two compared samples. The differential expression of circRNAs was analyzed by volcano plots between colon cancer and colon polyp patients (right panel). Red points represented the differentially expressed circRNAs with statistical significance (1.5-fold upregulation and downregulation, P < 0.05). (E) The differential expression of plasma circRNAs in colon cancer patients ( N = 5) and healthy individuals ( N = 5). The expression variation of circRNAs was assessed by the scatter plot between colon cancer patients and healthy individuals (left panel). The green lines represented fold change lines. The circRNAs above the top green line and below the bottom green line indicated more than 1.5-fold change of circRNAs between the two compared samples. The differential expression of circRNAs was analyzed by volcano plots between cancer patients and healthy individuals (right panel). Red points represented the differentially expressed circRNAs with statistical significance (1.5-fold upregulation and downregulation, P < 0.05). (F–H) The composition of types in detectable circRNAs. (F) The composition of circRNA types in polyp and colon cancer patients. (I) The Venn analysis between upregulated circRNAs from colon cancer patients compared with colon polyp patients and downregulated circRNAs from colon polyp patients compared with healthy individuals. (J) The Venn analysis between downregulated circRNAs from colon cancer patients compared with colon polyp patients and upregulated circRNAs from colon polyp patients compared with healthy individuals. (K) The potential circRNA candidates were validated by performing real-time PCR in plasma from healthy individuals ( N = 15) and colon polyp ( N = 15) and colon cancer ( N = 15) patients. (L) The schematic diagram of circHADHA dynamic alteration in healthy individuals and colon polyp and colon cancer patients. **** P < 0.0001; ns, no significant difference.

Article Snippet: We collected plasma from healthy individuals and colon polyp and colon cancer patients confirmed by endoscopic diagnosis ( ) and analyzed 2,162 human circRNAs by Arraystar ( ).

Techniques: Clinical Proteomics, Expressing, Biomarker Discovery, Quantitative Proteomics, Real-time Polymerase Chain Reaction

Journal: EBioMedicine

Article Title: Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis

doi: 10.1016/j.ebiom.2017.12.007

Figure Lengend Snippet: The experimental scheme of the study. The discovery cohort was composed of two TB patients and two age- and gender-matched healthy controls: one young male case-control pair and one senior female case-control pair. PBMC circRNA expression in the discovery cohort was profiled by both rRNA-depleted RNA-seq and a circRNA expression microarray. We aggregated circRNAs into pathway-level mechanisms using KEGG pathway definitions. Pathways with significant circRNA dysregulation were prioritized by performing a paired Wilcoxon signed-rank test between paired control and TB samples. A molecular signature was developed based on the dysregulated circRNAs within the prioritized pathways. This circRNA signature was further validated by qRT-PCR in a validation cohort with 11 healthy controls and 10 TB patients.

Article Snippet: The CapitalBio Technology Human CircRNA Array v2 (4 × 180K) was used to quantify circRNA expression.

Techniques: Control, Expressing, RNA Sequencing, Microarray, Quantitative RT-PCR, Biomarker Discovery

Journal: EBioMedicine

Article Title: Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis

doi: 10.1016/j.ebiom.2017.12.007

Figure Lengend Snippet: Landscape of PBMC circRNA expression. (a) A Venn diagram of the parental genes with circRNA transcripts among PBMCs, platelets, RBCs, and whole blood. (b) Distribution of log 2 -transformed TPM values of circular transcripts in PBMCs, platelets, RBCs, and whole blood. (c) The fraction of RNA-seq reads from coding linear, non-coding linear, and circular transcripts. (d) The cumulative distribution of TPM values of circular transcripts. A significantly increased circRNA TPM was observed in the TB patients compared with the healthy controls. The P -values were computed by the Kolmogorov-Smirnov test.

Article Snippet: The CapitalBio Technology Human CircRNA Array v2 (4 × 180K) was used to quantify circRNA expression.

Techniques: Expressing, Transformation Assay, RNA Sequencing

Journal: EBioMedicine

Article Title: Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis

doi: 10.1016/j.ebiom.2017.12.007

Figure Lengend Snippet: The KEGG pathways enriched by upregulated circRNAs. A paired Wilcoxon test was applied to identify the KEGG pathways enriched by dysregulated circRNAs in young and senior TB patients. (a) The correlation in Z -scores (computed by a paired Wilcoxon test) between young and senior case-control pairs. Each dot represents one KEGG pathway. The red points stand for the pathways with circular transcripts commonly upregulated in both young and senior TB patients. (b) Paired comparison of circRNA expression in the five prioritized KEGG pathways. (c) Comparison of the fraction of circRNA reads in the five pathways between healthy controls and TB patients.

Article Snippet: The CapitalBio Technology Human CircRNA Array v2 (4 × 180K) was used to quantify circRNA expression.

Techniques: Control, Comparison, Expressing

Journal: EBioMedicine

Article Title: Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis

doi: 10.1016/j.ebiom.2017.12.007

Figure Lengend Snippet: Technical validation of the five prioritized KEGG pathways using a circRNA expression microarray. Total RNA was treated with RNase R to remove linear transcripts and then subject to microarray hybridization.

Article Snippet: The CapitalBio Technology Human CircRNA Array v2 (4 × 180K) was used to quantify circRNA expression.

Techniques: Biomarker Discovery, Expressing, Microarray, Hybridization

Journal: EBioMedicine

Article Title: Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis

doi: 10.1016/j.ebiom.2017.12.007

Figure Lengend Snippet: The 7-circRNA signature.

Article Snippet: The CapitalBio Technology Human CircRNA Array v2 (4 × 180K) was used to quantify circRNA expression.

Techniques:

Journal: EBioMedicine

Article Title: Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis

doi: 10.1016/j.ebiom.2017.12.007

Figure Lengend Snippet: The 7-circRNA signature. (a) Expression heatmap of the circRNAs within the 7-circRNA signature in the discovery cohort. The expression measured by both RNA-seq and circRNA microarray is displayed. (b) Comparison of the expression levels of the 7-circRNA signature between the healthy controls and TB patients in the validation cohort. The error bars represent the standard error of the mean. (c) Principal component analysis of the 7-circRNA signature. PC1: the first principal component; PC2: the second principal component. (d) Comparison of the circRNA-based TB index between the healthy controls and TB patients in the validation cohort. (e) The ROC curve of the TB index in distinguishing TB patients from healthy controls. The light blue area indicates the confidence interval of the ROC curve.

Article Snippet: The CapitalBio Technology Human CircRNA Array v2 (4 × 180K) was used to quantify circRNA expression.

Techniques: Expressing, RNA Sequencing, Microarray, Comparison, Biomarker Discovery

Journal: Molecular Medicine Reports

Article Title: CircRNA expression profiles in human visceral preadipocytes and adipocytes

doi: 10.3892/mmr.2019.10886

Figure Lengend Snippet: Comparison of circRNA expression profiles between HPA-v and adipocytes. (A) Box plots revealed the distribution of circRNAs in the six samples after normalization. (B) Volcano plots revealed the differentially expressed circRNAs. Green and red dots represent significantly down- and upregulated circRNAs in adipocytes compared with HPA-v, respectively (fold change ≥5.0, P<0.01). (C) Hierarchical clustering was performed to reveal the differentially expressed circRNAs between HPA-v and adipocytes. (D) Expression patterns of select differentially expressed circRNAs in HPA-v and adipocytes were determined by qPCR. (E) The heatmap revealed the selected differentially expressed circRNAs in HPA-v and adipocytes. *P<0.05. circRNA, circular RNA; HPA-v, human preadipocytes from visceral fat tissue; AD, adipocytes; n.d., not detected.

Article Snippet: To investigate whether circRNAs are associated with lipid deposition, a human circRNA microarray (version 2.0; Agilent Technologies, Inc.) was used to assess circRNA expression profiles in HPA-v and adipocytes.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: CircRNA expression profiles in human visceral preadipocytes and adipocytes

doi: 10.3892/mmr.2019.10886

Figure Lengend Snippet: Top 10 up- and downregulated circRNAs in adipocytes.

Article Snippet: To investigate whether circRNAs are associated with lipid deposition, a human circRNA microarray (version 2.0; Agilent Technologies, Inc.) was used to assess circRNA expression profiles in HPA-v and adipocytes.

Techniques:

Journal: Molecular Medicine Reports

Article Title: CircRNA expression profiles in human visceral preadipocytes and adipocytes

doi: 10.3892/mmr.2019.10886

Figure Lengend Snippet: The top 15 significantly enriched pathways associated with the differentially expressed circRNA parental genes according to KEGG analysis. circRNAs, circular RNAs; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: To investigate whether circRNAs are associated with lipid deposition, a human circRNA microarray (version 2.0; Agilent Technologies, Inc.) was used to assess circRNA expression profiles in HPA-v and adipocytes.

Techniques:

Journal: Molecular Medicine Reports

Article Title: CircRNA expression profiles in human visceral preadipocytes and adipocytes

doi: 10.3892/mmr.2019.10886

Figure Lengend Snippet: miRNAs with >2 miRNA response elements targeting the top 10 upregulated and downregulated circRNAs.

Article Snippet: To investigate whether circRNAs are associated with lipid deposition, a human circRNA microarray (version 2.0; Agilent Technologies, Inc.) was used to assess circRNA expression profiles in HPA-v and adipocytes.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: PRAP1 is an intrinsically disordered protein conserved in placental mammals. ( A ) Amino acid sequence of human PRAP1 with signal peptide and secreted portion of the protein labeled. ( B ) Analysis of the PRAP1 amino acid sequence using the Predictor of Natural Disordered Regions (PONDR) software. The predicted ordered and disordered regions are plotted for each residue. ( C ) Size exclusion chromatogram of purified recombinant PRAP1 protein compared with a molecular weight standard: (a) thyroglobulin (670,000 daltons), (b) γ-globulin (158,000 daltons), (c) ovalbumin (44,000 daltons), (d) myoglobin (17,000 daltons), and (e) vitamin B12 (1350 daltons). ( D ) Circular dichroism spectra of recombinant PRAP1. ( E ) Analysis of the human PRAP1 amino acid sequence using the comparative genomics feature of Ensembl software. The number of PRAP1 orthologs identified in each taxonomic clade are indicated.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Sequencing, Labeling, Software, Residue, Purification, Recombinant, Molecular Weight, Circular Dichroism

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Generation and validation of PRAP1 recombinant protein, PRAP1 antisera, and Prap1 -/- mice. ( A ) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Coomassie staining of recombinant 6xHis-PRAP1 expressed in E coli and purified by a Ni-NTA affinity chromatography column followed by a size exclusion column. ( B ) A human colonic epithelial cell line (SK-CO15) was transfected to overexpress human (H) and mouse (M) PRAP1. Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera. Whole-body knockout mice were procured from MMRRC-UC Davis and backcrossed to obtain a fully congenic C57BL/6 background. Scale bar : 100 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout, SI, small intestine; WT, wild-type; UT, uterine tissue.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Biomarker Discovery, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Purification, Affinity Column, Transfection, Generated, Western Blot, Immunofluorescence, Knock-Out

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: PRAP1 is highly expressed by the epithelium of the gastrointestinal tract in mice and human beings. ( A ) Quantification of Prap1 transcript measured via quantitative PCR in the indicated tissues from 8-week-old wild-type C57BL/6 mice (n = 3 mice). ( B ) Western blot analysis for the detection of PRAP1 protein abundance in the indicated tissues dissected from 8-week-old wild-type C57BL/6 mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Images are representative of 3 mice per tissue collected. ( C ) Immunofluorescence for the detection of PRAP1 (green) in the duodenum of 8-week-old wild-type C57BL/6 mice or Prap1 -/- mice. Images are representative of the analysis of 5 mice per tissue collected. ( D ) Immunofluorescence staining of PRAP1 (green) in the duodenum of 8-week-old wild-type mice at 60× magnification. ( E and F ) Immunohistochemistry staining for the detection of PRAP1 (brown) in the human ileum ( E ) and colon ( F ). Image is representative of 3 subjects. Prox, proximal.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Quantitative Proteomics, Control, Immunofluorescence, Staining, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Prap1 -/- mice have an altered microbiota in the small intestine. ( A ) The body weight of wild-type and Prap1 -/- littermates at 10 weeks old. ( B ) H&E staining of wild-type and Prap1 -/- small intestine. Images are representative of 3 mice per group. ( C ) Quantification of villi and crypt length in the small intestine of wild-type and Prap1 -/- mice. Significance was determined using an unpaired t test (n = 3 mice). ∗ P < .05. ( D and E ) Quantitative PCR analysis of Pcna ( D ) and Bax ( E ) expression in whole tissue from the small intestine of wild-type and Prap1 -/- mice. Expression levels are relative to Gapdh . Significance was determined using an unpaired t test. ∗ P < .05 (n = 6 mice). ( F ) Pie chart comparison of the average Bacteriodetes:Firmicutes phyla ratio in the small intestine of wild-type and Prap1 -/- littermates measured via 16S ribosomal RNA sequencing (n ≥ 12 mice). ( G ) Relative abundance of Firmicutes and Bacteroidetes in the small intestine of wild-type and Prap1 -/- littermates. Significance was determined using an unpaired t test (n ≥ 12 mice). ∗ P < .05. All data are graphed as the means ± SEM. Bax RA, Bax relative abundance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; Pcna RA, Pcna relative abundance; WT, wild-type.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing, Comparison, RNA Sequencing, Knock-Out

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Prap1 -/- mice have increased inflammation but no significant intestinal barrier defect. ( A ) A multiplex enzyme-linked immunosorbent assay (ELISA) was used for the detection of 10 proinflammatory cytokines in sera of 10-week-old wild-type and Prap1 -/- littermates. Data are shown as a heat map, with red indicating a higher than average concentration. Each column represents 1 mouse. ( B ) Graphic representation of significantly different cytokine levels shown in panel A , including IL2, IL4, and IL12p70. Statistical significance was determined using an unpaired t test (n = 5 mice). ∗ P < .05. ( C ) Quantification of IL12β transcript levels measured via quantitative PCR in the colon of 10-week-old wild-type and Prap1 -/- mice relative to the abundance of β-actin. Statistical significance was determined using an unpaired t test (n = 5 mice). ∗ P < .05. ( D ) Quantification of IgA levels in fecal pellets collected from 10-week-old wild-type and Prap1 -/- mice, measured via ELISA. Statistical significance was determined using an unpaired t test (n = 5 mice). ( E ) Quantification of FITC dextran in the sera of unchallenged 10-week-old wild-type and Prap1 -/- mice 4 hours after oral gavage with 4 kilodaltons FITC dextran. Statistical significance was determined using an unpaired t test (n = 5 mice). IFN, interferon; KC, keratinocyte chemoattractant; RA, relative abundance; TNF, tumor necrosis factor.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Prap1 -/- mice are more susceptible to radiologic challenge and have increased apoptosis in the intestinal epithelium. ( A ) Percentage body weight loss of wild-type C57BL/6 and littermate Prap1 -/- mice after 10 Gy TBI. Statistical analysis represents a comparison of wild-type vs Prap1 -/- on each respective day using 2-way analysis of variance, Bonferroni multiple comparisons test (n = 4 mice). ∗ P < .05, ∗∗ P < .01. ( B ) Survival of wild-type, Prap1 +/- or Prap1 -/- mice after 10 Gy TBI. Statistical significance was determined using the log-rank test (n ≥ 11 mice). ∗∗ P < .01. ( C ) Representative images of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells (green) within the small intestine of 8-week-old wild-type and Prap1 -/- littermates 6 hours after receiving 10 Gy TBI. ( D ) Representative images of cleaved caspase-3–positive cells (green) within the small intestine of 8-week-old wild-type and Prap1 -/- littermates 72 hours after receiving 10 Gy TBI. ( E ) Quantification of TUNEL-positive cells in panel C . Significance was determined using an unpaired t test (n ≥ 11 mice). ∗∗ P < .01. ( F ) Quantification of cleaved caspase-3–positive cells in panel F . Significance was determined using an unpaired t test (n ≥ 6 mice). ∗∗ P < .01. ( G ) Quantification of Bax transcript via quantitative PCR on whole tissue from the small intestine of wild-type and Prap1 -/- littermates 96 hours after 10 Gy TBI. Significance was determined using an unpaired t test (n = 6 mice). ∗ P < .05. ( H ) Quantification of Pcna transcript via quantitative PCR on whole tissue from the small intestine of wild-type and Prap1 -/- littermates 96 hours after 10 Gy TBI. Significance was determined using an unpaired t test (n = 6 mice). ( I ) Quantification of serum FITC dextran in wild-type and Prap1 -/- littermates after oral gavage with 4 kilodaltons FITC dextran 72 hours after 10 Gy TBI. Significance was determined using an unpaired t test (n ≥ 3 mice). All data are graphed as the means ± SEM. ( J ) Quantification of Prap1 transcript via quantitative PCR on whole tissue from the small intestine of wild-type mice at different time points after 10 Gy TBI. Significance was determined using 1-way analysis of variance, Tukey multiple comparisons test (n = 6 mice). ∗∗ P < .005. Bax RA, Bax relative abundance; DAPI, 4′,6-diamidino-2-phenylindole; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; Pcna RA, Pcna relative abundance; WT, wild-type.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Comparison, End Labeling, TUNEL Assay, Real-time Polymerase Chain Reaction, Knock-Out

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: PRAP1 protects enteroids from irradiation-induced apoptosis by limiting p21 expression. ( A ) The percentage of viability of wild-type or Prap1 -/- enteroids 48 hours after 2 Gy. Cell viability was measured using the addition of MTT and the percentage of viability was calculated using the cell viability measured before irradiation. Significance was determined using an unpaired t test (n ≥ 7 wells). ∗ P < .05. ( B ) Representative immunofluorescence images for the detection of cleaved caspase-3 in wild-type and Prap1 -/- enteroids 24 hours after 2 Gy. Examples of cleaved caspase-3–positive enteroids are indicated by a white arrowhead . Scale bar : 1000 μm. ( C ) Quantification of cleaved caspase-3–positive enteroids in panel B . Each data point represents the percentage of cleaved caspase-3–positive enteroids in a well. Data were pooled from 4 independent experiments. Significance was determined via unpaired t test (n = 4 mice per group). ∗∗ P < .01. ( D–F ) Quantification of Prap1 ( D ), p21 ( E ), and p18 ( F ) transcript via quantitative PCR in wild-type and Prap1 -/- enteroids 24 hours after 0 Gy and 1 Gy. Significance was determined via an unpaired t test. ∗ P < .05. Each data point represents enteroids harvested from a unique mouse (n = 3 mice per group). ( G ) Protein levels were determined via Western blot from epithelial cells transfected with an empty cytomegalovirus expression vector (pCMV) pCMV or a cytomegalovirus expression vector encoding PRAP1 (pCMV-PRAP1) 48 hours after 8 Gy. ( H and I ) Quantification of p21 ( H ) and p18 ( I ) protein levels in panel G determined via signal intensity relative to GAPDH. Significance was determined using an unpaired t test (n = 4). ∗ P > .05. All data are graphed as means ± SEM. Casp3, caspase-3; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; pCMV, empty cytomegalovirus expression vector; WT, wild-type

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Irradiation, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Knock-Out