Journal: Human Molecular Genetics

Article Title: Inherited thrombocytopenia associated with mutation of UDP-galactose-4-epimerase ( GALE )

doi: 10.1093/hmg/ddy334

Figure Lengend Snippet: Inherited thrombocytopenia in family AH; GALE conservation, structure and mutations. (A) Six individuals in four-generation family AH presented with severe thrombocytopenia (black symbols). Current ages in years of individuals are listed below symbols. V indicates variant allele GALE p.R51W (c.C151T); N indicates reference allele. Asterisks indicate individuals evaluated by whole-exome sequencing. (B) Bone marrow aspirate treated with Wright-Giemsa stain from affected family member IV.1. Arrow indicates a megakaryocyte with hypogranular cytoplasm and hypolobated nuclei. (C) Protein sequence alignment from multiple species of the GALE region including Arg51 (red text) showing evolutionary conservation. Gold indicates complete conservation through S. cerevisiae. (D) GALE crystal structure (PDB ID:1HZJ), viewed in PyMOL (72). Left: GALE homodimer (green and gold), highlighting Arg51 (magenta), Asn340 (orange), UDP-N-acetylgalactosamine (blue), NAD+ cofactor (red), C-terminal end of GALE (yellow). Right: close-up view of Arg51 near the C-terminal end of GALE. Dotted yellow line indicates the 3-Å distance between Arg51 and Asn340. (E) Galactosemia-associated mutations of GALE reported in the literature. GALE domains are the substrate-binding site (gold), NAD+-binding site (orange) and catalytic sites (red dots at residues 132, 157, 307). Asterisks indicate mutations reported as homozygotes; others were reported as compound heterozygotes. GALE p.V94M (red text) is associated with severe generalized galactosemia. GALE p.R51W is shown in bold.

Article Snippet: TaqMan probes for GALE (Hs00166181_m1) and control GAPDH were used.

Techniques: Variant Assay, Sequencing, Giemsa Stain, Binding Assay

Journal: Human Molecular Genetics

Article Title: Inherited thrombocytopenia associated with mutation of UDP-galactose-4-epimerase ( GALE )

doi: 10.1093/hmg/ddy334

Figure Lengend Snippet: Enzyme activity and differential scanning calorimetry. Enzyme assays in figures (A) and (B) were performed with 10-ng protein. Substrates were (A) UDP-galactose or (B) UDP-N-acetylgalactosamine. Enzyme activities were measured as fraction product (Supplementary Material, Table S2) and were normalized to wildtype protein measurements. The mean values of normalized enzyme activities were plotted + standard deviation for each allele. Experiments were performed in at least triplicate (23,28,29). (C) Representative endotherm plots for each GALE allele. Endotherm data was normalized for protein concentration, with intrascan baseline correction. Black indicates wildtype protein (normal), red indicates GALE p.R51W, purple indicates GALE p.V94M and orange indicates GALE p.D103G. (D) Size distributions of megakaryocyte colonies after treatment of CD34+ hematopoietic progenitor cells with control or anti-GALE shRNA probes. Cells were plated in MegaCult collagen-based media. Slides were fixed, stained and classified by colony size. Data represents two independent experiments, each with plating and counting in duplicate.

Article Snippet: TaqMan probes for GALE (Hs00166181_m1) and control GAPDH were used.

Techniques: Activity Assay, Standard Deviation, Protein Concentration, shRNA, Staining

Journal: Human Molecular Genetics

Article Title: Inherited thrombocytopenia associated with mutation of UDP-galactose-4-epimerase ( GALE )

doi: 10.1093/hmg/ddy334

Figure Lengend Snippet: N-linked glycosylation pathway and thrombocytopenia. Mutations in critical steps of the N-linked glycan biosynthesis pathway are associated with thrombocytopenia. Glycan biosynthesis begins in the cytoplasm, with subsequent modifications in the endoplastic reticulum and the Golgi. Membrane transporters including SLC35A1, SLC35A2 and SLC35A3 allow building-block sugars to enter the Golgi. Enzymes in the pathway, including B4GALT1, control the levels of these sugars, and add or subtract sugars from the glycan chain. GALE is responsible for interconversion of these sugars in the cytoplasm, in preparation for their entry into the Golgi and integration into glycan chains. Mutations in this pathway cause CDGs. Two forms of CDG include thrombocytopenia among presenting symptoms: CDG-IId, caused by mutation of B4GALT1, and CDG-IIf, caused by mutation of SLC35A1. Genes responsible for steps in the pathway are indicated in green boxes, with the CDG disorder under the gene name. Steps in the pathway with mutations associated with thrombocytopenia are indicated by red Xs. Figure is adapted from Freeze 2006 (73).

Article Snippet: TaqMan probes for GALE (Hs00166181_m1) and control GAPDH were used.

Techniques: Blocking Assay, Mutagenesis

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: Correlation of clinicopathological characteristics with UGP2 expression in the ZZU HCC cohort.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: UGP2 expression is frequently dysregulated in cancer. (a) UGP2 mRNA expression level from the TCGA dataset compared with normal tissues. (b) UGP2 protein expression in pancancer tissues and paired nontumour tissues. (c) Representative UGP2 histological scoring in pancancer tissues and paired nontumour tissues. UGP2: uridine diphosphate-glucose pyrophosphorylase 2; TCGA: The Cancer Genome Atlas.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: UGP2 mRNA levels and protein expression are significantly downregulated in HCC. (a) The UGP2 expression level was significantly lower in HCC tissues than in adjacent nontumour tissues in the TCGA cohort and GEO datasets. (b) A representative IHC image of UGP2 expression in HCC and normal tissues. (c) Representative images of UGP2 staining in HCC tissues. (d) The UGP2 expression level in HCC tissues was lower than that in paired nontumour tissues in the ZZU HCC cohort. UGP2: uridine diphosphate-glucose pyrophosphorylase 2; TCGA: The Cancer Genome Atlas; HCC: hepatocellular carcinoma; GEO: Gene Expression Omnibus; IHC: immunohistochemistry.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing, Staining, Gene Expression, Immunohistochemistry

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: ROC curve analysis of UGP2 expression in HCC. (a–f) ROC curve analysis of UGP2 expression in HCC from the TCGA, GSE36376, GSE76297, GSE54236, GSE14520, and GSE64041 datasets. UGP2: uridine diphosphate-glucose pyrophosphorylase 2; TCGA: The Cancer Genome Atlas; HCC: hepatocellular carcinoma; ROC: receiver operating characteristic.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: Downregulated UGP2 expression predicts a poor prognosis in HCC patients in the TCGA cohort. (a, b) Kaplan-Meier survival curves showing the correlation between UGP2 expression levels and the OS/PFS of HCC patients in the TCGA cohort. (c, d) Kaplan-Meier analysis showed that OS/PFS was shorter in HCC patients in the TCGA cohort with low UGP2 expression levels regardless of the TNM stage. (e, f) GSEA of the relationship between low UGP2 expression levels and the survival of HCC patients in the TCGA cohort. UGP2: uridine diphosphate-glucose pyrophosphorylase 2; TCGA: The Cancer Genome Atlas; HCC: hepatocellular carcinoma; OS: overall survival; PFS: progression-free survival; HCC: hepatocellular carcinoma.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: Downregulated UGP2 expression predicts a poor prognosis in HCC patients in the ZZU cohort. (a) Kaplan-Meier survival curves showing the correlation between UGP2 expression levels and the OS of HCC patients in the ZZU cohort. (b) Kaplan-Meier analysis revealed that OS was short in HCC patients in the ZZU cohort with low UGP2 expression levels regardless of the TNM stage. UGP2: uridine diphosphate-glucose pyrophosphorylase 2; HCC: hepatocellular carcinoma; OS: overall survival; TNM: tumour-node-metastasis.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of risk factors for overall survival time in the ZZU HCC cohort.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques:

Journal: Disease Markers

Article Title: Low UGP2 Expression Is Associated with Tumour Progression and Predicts Poor Prognosis in Hepatocellular Carcinoma

doi: 10.1155/2020/3231273

Figure Lengend Snippet: UGP2 has potential roles in fatty acid metabolism. (a) KEGG functional enrichment analysis showing the potential mechanism of UGP2 in HCC. (b) GSEA of the relationship between low UGP2 expression and genes associated with fatty acid metabolism. (c–g) The scatter plots show that UGP2 expression is markedly correlated with genes involved in fatty acid metabolism. UGP2: uridine diphosphate-glucose pyrophosphorylase 2; KEGG: Kyoto Encyclopedia of Genes and Genomes; HCC: hepatocellular carcinoma.

Article Snippet: After 30 min of blocking with 10% bovine serum albumin, sections were incubated with a UGP2 primary antibody (1 : 100; ProteinTech Group, Inc.) at 4°C overnight.

Techniques: Functional Assay, Expressing

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Induced maturation of hepatic progenitor cellsin vitro

doi: 10.1590/1414-431x20132455

Figure Lengend Snippet: Figure 3. Expression of liver cell markers during the induced maturation of hepatic progenitor cells (HPCs). Cells were treated with the combination condition of 2% HS+0.1 mM Dex+10 ng/mL HGF+20 ng/mL FGF4. A, The morphology of untreated and treated cells at D3, D6, D9, and D12 days after induction. Panels 1, 2, 3, and 4 are untreated cells; 7, 8, 9, and 10 are induced cells. Scale bar=200 mm. B, RT-PCR analysis of hepatic-related genes DLK, CK19, AFP, ALB, CK18, TAT, and ApoB at D0, D3, D6, D9, and D12 after induction. RT-PCR results were confirmed in at least three independent experiments, and representative results are shown. C, Immunofluorescence staining of DLK, AFP, ALB, and UGT1A markers at D0, D3, D6, D9, and D12 days after induction. Negative controls (NC) are cells stained with nonspecific IgG. Scale bar=200 mm. D, Real-time PCR analysis of hepatic related genes AFP, ALB, CK18, TAT of the induced cells at D12 compared with untreated cells as negative controls and adult liver cells as positive controls. See Figure 1 for explanation of abbreviations. *P,0.05 induced D12 vs control D12 (two-tailed Student t-test); **P,0.05 LC14d vs induced D12 (two-tailed Student t-test).

Article Snippet: As previously described (13), methanol was used to fix the treated cells at -206C for 15 min, 5% goat serum was used to block cells at room temperature (RT) for 1 h. Then, cells were incubated with primary antibodies against delta-like protein (DLK), ALB, a-fetoprotein (AFP), or glucuronosyltransferase 1 A (UGT1A) (Santa Cruz Biotechnology, USA) at RT for 1 h, followed by incubation with DyLight 488-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, USA) at RT for 30 min.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Molecular Reproduction and Development

Article Title: Endometrial glycogen metabolism during early pregnancy in mice

doi: 10.1002/mrd.23634

Figure Lengend Snippet: Levels of glycogen metabolizing enzymes in uterine homogenates. (a–d) Western blots for hexokinase 1 (HK1, a), phospho‐glycogen synthase (pGYS, b), glycogen synthase (GYS, c), and glycogen phosphorylase (PYG, d) in uteri collected from mice at proestrus (PROE) and days postcoitum (DPC) 1.5, 3.5, and 5.5. n = 4.

Article Snippet: Glycogen synthase , 3886 Cell signaling , WB , 1:500 , BSA.

Techniques: Western Blot

Journal: Molecular Reproduction and Development

Article Title: Endometrial glycogen metabolism during early pregnancy in mice

doi: 10.1002/mrd.23634

Figure Lengend Snippet: Localization of glycogen synthesizing enzymes in the endometrium from proestrus (PROE) until days postcoitum (DPC) 5.5. Immunohistochemistry for hexokinase 1 (HK1) and glycogen synthase (GYS) in uteri collected at PROE, DPC 1.5, DPC 3.5, and DPC 5.5 IIS. n = 4. Scale bar = 50 µm. IIS, interimplantation site; Neg, negative control.

Article Snippet: Glycogen synthase , 3886 Cell signaling , WB , 1:500 , BSA.

Techniques: Immunohistochemistry, Negative Control

Journal: Molecular Reproduction and Development

Article Title: Endometrial glycogen metabolism during early pregnancy in mice

doi: 10.1002/mrd.23634

Figure Lengend Snippet: Immunostaining for glycogen metabolizing enzymes at the implantation site (IS) and the interimplantation site (IIS) on DPC 5.5. Immunohistochemistry for hexokinase 1 (HK1), glycogen synthase (GYS), glycogen phosphorylase (PYG), and glucose‐6‐phosphatase (G6PC) in uteri at DPC 5.5. n = 4. Scale bar = 50 µm. DPC, days postcoitum; Neg, negative control.

Article Snippet: Glycogen synthase , 3886 Cell signaling , WB , 1:500 , BSA.

Techniques: Immunostaining, Immunohistochemistry, Negative Control

Journal: Molecular Reproduction and Development

Article Title: Endometrial glycogen metabolism during early pregnancy in mice

doi: 10.1002/mrd.23634

Figure Lengend Snippet: Western blots comparing levels of glycogen metabolizing enzymes in the uterus at the interimplantation site (IIS) and implantation site (IS) on DPC 5.5. Levels of hexokinase 1 (HK1, a), phospho‐glycogen synthase (pGYS, b), glycogen synthase (GYS, c), and glycogen phosphorylase (PYG, d). * p < 0.05 relative to DPC 5.5‐IIS. n = 4–5. DPC, days postcoitum.

Article Snippet: Glycogen synthase , 3886 Cell signaling , WB , 1:500 , BSA.

Techniques: Western Blot

Journal: Molecular Reproduction and Development

Article Title: Endometrial glycogen metabolism during early pregnancy in mice

doi: 10.1002/mrd.23634

Figure Lengend Snippet: Localization of glycogen metabolizing enzymes in the decidualized and undecidualized uterine horn in an artificial decidualization model. Immunohistochemistry for hexokinase 1 (HK1), glycogen synthase (GYS), glycogen phosphorylase (PYG), and glucose‐6‐phosphatase (G6PC) in hormonally primed mice. One horn was simulated to decidualize. The unstimulated horn served as a nondecidualized control. n = 4. Scale bar = 50 µm.

Article Snippet: Glycogen synthase , 3886 Cell signaling , WB , 1:500 , BSA.

Techniques: Immunohistochemistry, Control

Journal: Molecular Reproduction and Development

Article Title: Endometrial glycogen metabolism during early pregnancy in mice

doi: 10.1002/mrd.23634

Figure Lengend Snippet: Levels of glycogen metabolizing enzymes in uterine horns stimulated to decidualize or left unstimulated. (a–d) Western blots for hexokinase 1 (HK1, a), phospho‐glycogen synthase (pGYS, b), glycogen synthase (GYS, c), and glycogen phosphorylase (PYG, d) in uterine horns stimulated to decidualization with corn oil or unstimulated after hormonal priming. ** p < 0.01 relative to unstimulated horn. n = 5.

Article Snippet: Glycogen synthase , 3886 Cell signaling , WB , 1:500 , BSA.

Techniques: Western Blot

Journal: Molecular Reproduction and Development

Article Title: Endometrial glycogen metabolism during early pregnancy in mice

doi: 10.1002/mrd.23634

Figure Lengend Snippet: Primary antibodies and summary of conditions used for western blot (WB) and immunohistochemistry (IHC)

Article Snippet: Glycogen synthase , 3886 Cell signaling , WB , 1:500 , BSA.

Techniques: Western Blot, Immunohistochemistry, Blocking Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Carbon Monoxide Protects against Hepatic Ischemia/Reperfusion Injury via ROS-Dependent Akt Signaling and Inhibition of Glycogen Synthase Kinase 3 β

doi: 10.1155/2013/306421

Figure Lengend Snippet: Pretreatment of mice with CO gas inhalation ameliorates liver I/R injury via AKT-GSK3 β activation. Mice were subjected to 90 minutes liver warm ischemia, followed by 6 h reperfusion. (a) Hepatocellular function was evaluated by sALT (IU/L). (b) Representative liver histology of ischemic liver lobes. (c) Liver neutrophil accumulation, assessed by MPO activity. Data represent the mean ± standard deviation (SD) ( N = 4–6 samples/group). ** P < 0.01. (d) Hepatic HMGB1 expression in liver tissue was assessed by Western blot analysis at 1 h and 6 h of reperfusion. Total cell lysates were analyzed for HMGB1 and β -actin protein levels by Western blot analysis. (e) Western-blot analysis of phospho (p)-GSK3 β (Ser 9), p-GS (Ser641), and p-Akt in HepG2 cells after treatment with CORM2 (50 μ M) at the indicated times. (f) RAW264.7 cells were stimulated with 10 ng/mL of LPS for 30 minutes in the absence or presence of CORM2 and the ROS scavenger, N-acetyl-cysteine (NAC). Total cell lysates were analyzed for phosphorylated GS, GSK3 β , and Akt as well as total GS, GSK3 β , Akt, and β -actin protein levels by Western immunoblot analysis. (g) Mice were subjected to 90 minutes of liver warm ischemia, followed by 1 h or 6 h reperfusion. Liver tissue was analyzed by Western blotting of p-Akt and total Akt. β -Actin served as the standard.

Article Snippet: After blocking with 5% skim milk in PBS, membranes were incubated with appropriate dilutions of antibodies at 4°C overnight as follows: polyclonal rabbit anti-phospho glycogen synthase kinase (Ser9), rabbit anti-phospho glycogen synthase (Ser641), mouse anti-glycogen synthase kinase, rabbit anti-glycogen synthase, rabbit anti-phospho CREB (Ser133), rabbit anti-phospho Akt (Ser473), rabbit anti-HMGB1 (Cell Signaling Technology, Danvers, MA), rabbit anti-CBP, rabbit anti-phospho-NF- κ B-p65 (Ser276), and β -actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

Techniques: Activation Assay, Activity Assay, Standard Deviation, Expressing, Western Blot