Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

doi: 10.1016/j.jcmgh.2018.10.011

Figure Lengend Snippet: Transcriptomic profiling of CAID patient dermal fibroblasts. ( A ) Volcano plot of the differentially expressed genes at early (p8) and late stage (p14). Log 2 fold change: blue, <-1; red, >1. ( B ) Number of up-regulated and down-regulated genes identified at early (p8) and late passage (p14). ( C ) Cluster of genes associated with cell cycle identified at p8 that became larger at p14. ( D ) mRNA expression of candidate genes at early (p8) and late passage (p14) showing up-regulation of SGO1 , SGO2 , BUB1 , PLK1 , ESCO2 , DUSP2 , and BIRC5 and down-regulation of KCND2 , KCNJ2 , and KCNJ8 . Error bars signify SD. For each group, N = 3 independent biological replicates. P values were corrected for multiple testing using the Benjamini–Hochberg method (q value). FC, fold change; MUT, K23E; WT, wild-type.

Article Snippet: Five times excess of BUB1 (H00000699-Q01; Novus Biologicals, Littleton, CO), and TAGLN (NBP1-45267; Novus Biologicals) human recombinant peptides was added to the primary antibody by weight.

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

doi: 10.1016/j.jcmgh.2018.10.011

Figure Lengend Snippet: Candidates validation and expression profile of cohesinopathies genes. ( A ) Reverse transcription quantitative PCR (qPCR) validations of SGO1, SGO2, BUB1, PLK1, DUSP2, and KCND2 expression at early (p8) and late (p14) passage. Error bars signify SD. For each condition, the experiment was performed on N = 3 independent biological replicates in technical replicates. Significance was calculated using 1-way analysis of variance with the Bonferroni post-test ( • P < .1, * P < .05, and ** P < .01). ( B ) mRNA expression levels of genes involved in cohesinopathies at early (p8) and late (p14) passage. AFF4, CHOPS syndrome; DDX11, Warsaw breakage syndrome; NIPBL, RAD21, and SMC3, Cornelia de Lange syndrome. Error bars signify SD. P values were corrected for multiple testing using the Benjamini–Hochberg method (q value). For each group, N = 3 independent biological replicates. MUT, K23E; WT, wild-type.

Article Snippet: Five times excess of BUB1 (H00000699-Q01; Novus Biologicals, Littleton, CO), and TAGLN (NBP1-45267; Novus Biologicals) human recombinant peptides was added to the primary antibody by weight.

Techniques: Biomarker Discovery, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

doi: 10.1016/j.jcmgh.2018.10.011

Figure Lengend Snippet: Proteomic profiling of CAID patient dermal fibroblasts . ( A ) Venn diagram of proteins differentially expressed in CAID patient/control duos at early (p10) and late passage (p14). ( B ) Protein network of differentially expressed proteins in CAID patients at early (p10) and late (p14) passage. N = 3 independent biological replicates were analyzed for each condition. P values were corrected for multiple testing using the Benjamini–Hochberg method (q value). ( C ) Western blot validation of TPM1, TAGLN, and BUB1 overexpression. ( D ) Densitometric quantification of BUB1, TAGLN, and TPM1 overexpression by Western blot. Error bars signify SD. For each group, N = 3 independent biological replicates (2 independent experiments). Significance was calculated by 1-way analysis of variance with the Bonferroni post-test (* P < .05, ** P < .01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MUT, K23E; WT, wild-type.

Article Snippet: Five times excess of BUB1 (H00000699-Q01; Novus Biologicals, Littleton, CO), and TAGLN (NBP1-45267; Novus Biologicals) human recombinant peptides was added to the primary antibody by weight.

Techniques: Control, Western Blot, Biomarker Discovery, Over Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

doi: 10.1016/j.jcmgh.2018.10.011

Figure Lengend Snippet: Gastrointestinal histology of CAID patients. Biopsy specimens (5 μm) from controls and individuals with CAID. All biopsy specimens are oriented to show the villi on top and the muscularis externa at the bottom. ( A ) Negative controls show that no signal was obtained in the absence of the primary antibody. ( B–D ) DAB staining was performed for SGO1, BUB1, and TAGLN. ( B ) SGO1 and ( C ) BUB1 show higher expression in the villi and muscularis externa of CAID patient intestines vs controls. ( D ) TAGLN shows higher expression in muscularis mucosa and muscularis externa in CAID patient intestines vs controls (N = 3, 2 shown). Experiment was performed 3 times independently on 3 biological replicates for each condition. The Scale bar is 1000 μm except MUT/MUT1 SGO1, BUB1, TAGLN, and MUT/MUT 2 TAGLN where Scale bar : 2000 μm. Images were acquired using a Zeiss AxioScan Z1 slide scanner and processed with Zen software. MUT, K23E; WT, wild-type.

Article Snippet: Five times excess of BUB1 (H00000699-Q01; Novus Biologicals, Littleton, CO), and TAGLN (NBP1-45267; Novus Biologicals) human recombinant peptides was added to the primary antibody by weight.

Techniques: Staining, Expressing, Software

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

doi: 10.1016/j.jcmgh.2018.10.011

Figure Lengend Snippet: Gastrointestinal fluorescent histology of CAID patients . Biopsy specimens (5 μm) from controls and individuals with CAID. All biopsy specimens are oriented to show the villi on top and the muscularis externa at the bottom. ( A ) Immunizing peptide blocking experiment showing the specificity of BUB1 and TAGLN antibodies. ( B ) Negative controls showing that no signal was obtained in the villi (SGO1 and BUB1), the muscularis mucosa (TAGLN), and the muscularis externa (SGO1, BUB1, and TAGLN) in the absence of the primary antibody. ( C and D ) Fluorescence staining was performed for SGO1, BUB1, and TAGLN. ( C ) SGO1 and BUB1 expression were higher in the villi and muscularis externa of CAID patient intestines vs controls. In controls, SGO1 expression was mainly nuclear whereas in CAID patients, SGO1 was delocalized to the cytoplasm. ( D ) TAGLN expression was higher in the muscularis mucosa and the muscularis externa in CAID patient intestines vs controls. V, villi; ME, muscularis externa; MM, muscularis mucosa (N = 3, 2 shown). Experiment was performed 3 times independently on 3 biological replicates for each condition. Scale bar : 100 μm. Images were acquired at 40× using a Leica DMi8 inverted fluorescence microscope and processed with Leica LAS X software. DAPI, 4′,6-diamidino-2-phenylindole. MUT, K23E; WT, wild-type.

Article Snippet: Five times excess of BUB1 (H00000699-Q01; Novus Biologicals, Littleton, CO), and TAGLN (NBP1-45267; Novus Biologicals) human recombinant peptides was added to the primary antibody by weight.

Techniques: Blocking Assay, Fluorescence, Staining, Expressing, Microscopy, Software

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

doi: 10.1016/j.jcmgh.2018.10.011

Figure Lengend Snippet: Primers List for Reverse-Transcription PCR Validations

Article Snippet: Five times excess of BUB1 (H00000699-Q01; Novus Biologicals, Littleton, CO), and TAGLN (NBP1-45267; Novus Biologicals) human recombinant peptides was added to the primary antibody by weight.

Techniques: Reverse Transcription, Sequencing