Journal: Redox Biology

Article Title: Hypoxic acclimation improves cardiac redox homeostasis and protects heart against ischemia-reperfusion injury through upregulation of O-GlcNAcylation

doi: 10.1016/j.redox.2021.101994

Figure Lengend Snippet: HA activated pentose phosphate pathway (PPP) via O-GlcNAcylation in hearts. A , Experimental illustration of the O-glycoproteomics analysis of the Control and HA hearts. B , Bubble chart showing Gene Ontology (GO) annotation results. C , The pentose phosphate pathway. D , The levels of reduced nicotinamide adenine dinucleotides phosphate (NADPH) in cardiac tissue lysates (n = 6 per group). E , The NADPH/NADP + ratio in cardiac tissue lysates (n = 6 per group). F , The Glucose-6-phosphate dehydrogenase (G6PDH) activity in cardiac tissue lysates (Control: n = 6, HA: n = 9). G , The levels of glutathione (GSH) in cardiac tissue lysates (n = 5 per group). H , The GSH/GSSG ratio in cardiac tissue lysates (n = 5 per group). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 for indicated comparisons.

Article Snippet: Membrane was blocked in 5% non-fat milk and incubated with primary antibodies at 4 °C overnight, including anti- O -GlcNAC (#ab2739, Abcam), anti-OGT (#11576-2-AP, Proteintech, China), anti-OGA (#14711-1-AP, Proteintech), anti-GFAT1 (#14132-1-AP, Proteintech), anti-GALE (#14414-1-AP, Proteintech), anti-HK2 (#22029-1-AP, Proteintech), anti-IL-1β (#AF4006, Affbiotech, China), anti-IL-6 (#DF6087, Affbiotech), anti-G6PDH (#DF6444, Affbiotech), anti-GAPDH (#10494-1-AP, Proteintech), anti-Tubulin (#11224-1-AP, Proteintech) and anti-β-actin (#20536-1-AP, Proteintech) antibodies.

Techniques: Activity Assay

Journal: Redox Biology

Article Title: Hypoxic acclimation improves cardiac redox homeostasis and protects heart against ischemia-reperfusion injury through upregulation of O-GlcNAcylation

doi: 10.1016/j.redox.2021.101994

Figure Lengend Snippet: PPP was enhanced via O-GlcNAcylation-mediated activation of G6PDH. A , Representative blots of G6PDH in cardiac tissues and H9c2 cells (n = 4 per group). B–C , Representative blots of O-GlcNAc modified G6PDH ( B ) and G6PDH activity ( C ) in H9c2 cells treated with IL-1β for 4 or 24 h (n = 6 per group). D-E , Representative blots of O-GlcNAc modification ( D ) and the G6PDH activity ( E ) in H9c2 cells treated with IL-6 for 4 or 24 h (n = 6 per group). F , The compounds used in this study for targeting HBP pathway enzymes. G-I , Representative blots of O-GlcNAc-modified G6PDH ( G ), G6PDH activity ( H ), and NADPH/NADP + ratio ( I ) in H9c2 cells with indicated treatments (n = 4–5 per group). J , Representative blots of O-GlcNAc modified G6PDH in the mouse hearts with indicated treatments (n = 3 per group). K-M , The G6PDH activity ( K ), NADPH/NADP + ratio ( L ), and GSH/GSSG ratio ( M ) in the mouse hearts with indicated treatments (n = 4–6 per group). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 for indicated comparisons. NS: no significant difference.

Article Snippet: Membrane was blocked in 5% non-fat milk and incubated with primary antibodies at 4 °C overnight, including anti- O -GlcNAC (#ab2739, Abcam), anti-OGT (#11576-2-AP, Proteintech, China), anti-OGA (#14711-1-AP, Proteintech), anti-GFAT1 (#14132-1-AP, Proteintech), anti-GALE (#14414-1-AP, Proteintech), anti-HK2 (#22029-1-AP, Proteintech), anti-IL-1β (#AF4006, Affbiotech, China), anti-IL-6 (#DF6087, Affbiotech), anti-G6PDH (#DF6444, Affbiotech), anti-GAPDH (#10494-1-AP, Proteintech), anti-Tubulin (#11224-1-AP, Proteintech) and anti-β-actin (#20536-1-AP, Proteintech) antibodies.

Techniques: Activation Assay, Modification, Activity Assay

Journal: Redox Biology

Article Title: Hypoxic acclimation improves cardiac redox homeostasis and protects heart against ischemia-reperfusion injury through upregulation of O-GlcNAcylation

doi: 10.1016/j.redox.2021.101994

Figure Lengend Snippet: A schematic illustration of the working model of this study . The present study demonstrated that hypoxia acclimation-associated inflammatory cytokines upregulate the hexosamine biosynthetic pathway (HBP) and activate G6PDH by increasing the O-GlcNAcylation, which improves pentose phosphate pathway (PPP)-mediated redox homeostasis, and alleviates acute cardiac I/R injury.

Article Snippet: Membrane was blocked in 5% non-fat milk and incubated with primary antibodies at 4 °C overnight, including anti- O -GlcNAC (#ab2739, Abcam), anti-OGT (#11576-2-AP, Proteintech, China), anti-OGA (#14711-1-AP, Proteintech), anti-GFAT1 (#14132-1-AP, Proteintech), anti-GALE (#14414-1-AP, Proteintech), anti-HK2 (#22029-1-AP, Proteintech), anti-IL-1β (#AF4006, Affbiotech, China), anti-IL-6 (#DF6087, Affbiotech), anti-G6PDH (#DF6444, Affbiotech), anti-GAPDH (#10494-1-AP, Proteintech), anti-Tubulin (#11224-1-AP, Proteintech) and anti-β-actin (#20536-1-AP, Proteintech) antibodies.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor

doi: 10.1073/pnas.1707965114

Figure Lengend Snippet: 11βHSD2 and 11βHSD1 interconvert OCDO and CT. (A, Upper) 11HSD2 catalyzes the dehydrogenation of cortisol into cortisone. 11HSD1 realizes the reverse reaction. H6PD is the enzyme that produces the cofactor NADPH necessary for the reductase activity of 11HSD1. (A, Lower) 11HSD2 produces OCDO from CT and 11HSD1 produces CT from OCDO. (B–M and O) HEK293T (HEK) or MCF7 cells were transfected with plasmids encoding the indicated enzyme or the empty vector (mock) or shRNA. (B–F) The production of the indicated metabolites in transfected HEK (B–E) or MCF7 cells (F) was analyzed as in Fig. 1, n = 5. (G) The proliferation of transfected MCF7 cells was analyzed as in Fig. 3 C–F, n = 8. (H–M) shCA- and shHSD2A-MCF7 cells were assayed for: (H) cortisone or (I) OCDO production, as measured in Fig. 1, n = 5; (J–L) cell proliferation by (J) counting cell numbers, n = 5, or (K–L) as in G, n = 8; or (M) cell clonogenicity (n = 3), with or without 5 µM OCDO or cortisone. (N) shCA or shHSD2A-MCF7 tumors engrafted into mice (10 per group) were treated with solvent vehicle (control) or OCDO (16 µg/kg, 5 d/wk). Data are representative of three independent experiments. (O) Control (mock) or HSD2 overexpressing MCF7 cell proliferation was analyzed as in G. (P) The growth of control (mock) or 11HSD2 overexpressing MCF7 tumors engrafted into mice (10 mice per group) were compared over time. Data are representative of three independent experiments. (B–M and O) Data are the mean (±SEM) of five separate experiments and were analyzed (B, C, F, and H–J) by a Student’s t test, two-tailed, or (D, E, G, and K–O) by one-way ANOVA, Tukey’s posttest. *P < 0.05, **P < 0.01, ***P < 0.001. (N and P) Mean tumor volumes (±SEM) are shown, and data were analyzed by two-way ANOVA, Bonferroni posttest. *P < 0.05, **P < 0.01, ***P < 0.001. In N, letters indicate the comparison between: a: shCA vs. shHSD2A; b: shCA vs. shCA + OCDO; c: shHSD2A vs. shHSD2A + OCDO.

Article Snippet: The NEON Transfection system was purchased from Invitrogen, the BrdU cell proliferation Elisa kit was from Roche Diagnostics or Sigma, and the following plasmids were from OriGene: 11HSD1: sc109325; 11HSD2: sc122552 or RG207796; H6PD: sc117481; DHCR7: sc110871; and D8D7I (EBP): sc116006. pCMV-RORγ plasmid (NBP2-25278) was from Novus biologicals; pSG5-hGR was a gift from H. Richard-Foy, CNRS, Toulouse, France; pSG5-hRORα was a gift from V. Laudet, CNRS, Banyuls-sur-Mer, France; RORE-Luc was from B. Staels, the Pasteur Institute, Lille, France; pSG5-hRXRγ was a gift from M. Oulad-Abdelghani, INSERM, Strasbourg, France; and pSG5-hFXR and tk-EcRE-Luc were gifts from R. M. Evans, The Salk Institute, La Jolla, CA.

Techniques: Activity Assay, Transfection, Plasmid Preparation, shRNA, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor

doi: 10.1073/pnas.1707965114

Figure Lengend Snippet: Measuring the expression or extinction of the proteins of interest and their activity. (A) The expression of endogenous HSD2 and HSD1 was analyzed by immunoblotting in the indicated mammary tumor cells (related to Table S1). (B) HEK293T cells were transfected with a plasmid encoding HSD2 or the empty vector (mock) and the expression of the enzyme was confirmed by immunoblotting. (C) HEK293 cells were transfected with a plasmid encoding HSD1, H6PD, or the empty vector and the expression of the enzyme was confirmed by immunoblotting. (D) MCF7 cells were transfected with a plasmid encoding 11HSD1 and the expression of the enzyme was confirmed as in C. (E) MCF7 cells were transfected with two different shRNA plasmids targeting HSD2 (shHSD2), or with a control shRNA (shC) (SureSilencing ShRNA plasmid; Qiagen). The decreased expression of the enzyme was confirmed by immunoblotting and by qRT-PCR. Two clones (shHSD2A and shHSD2B) were selected. (F–J) shCB-MCF7 and shHSD2B-MCF7 cells were assayed for (F and G) cortisone or OCDO production, respectively, as described in Fig. 4 B and C; cell proliferation (H and I) by counting cell numbers (n = 5) or using a colorimetric BrDu assay, respectively (n = 8), or (J) cell clonogenicity (n = 3); with or without 5 µM OCDO, as indicated. Data were analyzed as described in Fig. 4 H–K and M and are the mean (±SEM) of five separate experiments. (K) The growth of shCB or shHSD2B-MCF7 tumors engrafted into mice (10 per group) were compared. OCDO treatment (16 µg/kg, 5 d/wk) increased shCB-MCF7 and reversed the growth inhibition of shHSD2-MCF7 tumors. Data are representative of three independent experiments. (L and M) MCF7 cells were transfected with a plasmid encoding 11HSD2 or the empty vector (mock). (L) Two stable clones were selected for the overexpression of 11HSD2 (HSD2A and HSD2B) and were compared with control cells (mockA and mockB) by immunoblotting. Mock-MCF7 or HSD2-MCF7 cells were incubated with [14C]CT and the production of OCDO was analyzed as in Fig. 1. Data are the mean (±SEM) of three experiments. (M) The growth of Mock-MCF7 or HSD2-MCF7 tumors engrafted into mice (10 per group) were compared. Mean tumor volumes (±SEM) are shown. Data are representative of three independent experiments. (E and H) Data were analyzed by a Student’s t test, two-tailed, or (I and J) by one-way ANOVA, Tukey’s posttest. *P < 0.05, **P < 0.01, ***P < 0.001. (K and M) Mean tumor volumes (±SEM) are shown, and data were analyzed by two-way ANOVA, Bonferroni posttest. *P < 0.05, **P < 0.01, ***P < 0.001. In K, letters indicate the comparison between: a: shCB vs. shHSD2B; b: shCB vs. shCB + OCDO; c: shHSD2B vs. shHSD2B + OCDO. ns, not significant.

Article Snippet: The NEON Transfection system was purchased from Invitrogen, the BrdU cell proliferation Elisa kit was from Roche Diagnostics or Sigma, and the following plasmids were from OriGene: 11HSD1: sc109325; 11HSD2: sc122552 or RG207796; H6PD: sc117481; DHCR7: sc110871; and D8D7I (EBP): sc116006. pCMV-RORγ plasmid (NBP2-25278) was from Novus biologicals; pSG5-hGR was a gift from H. Richard-Foy, CNRS, Toulouse, France; pSG5-hRORα was a gift from V. Laudet, CNRS, Banyuls-sur-Mer, France; RORE-Luc was from B. Staels, the Pasteur Institute, Lille, France; pSG5-hRXRγ was a gift from M. Oulad-Abdelghani, INSERM, Strasbourg, France; and pSG5-hFXR and tk-EcRE-Luc were gifts from R. M. Evans, The Salk Institute, La Jolla, CA.

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Plasmid Preparation, shRNA, Quantitative RT-PCR, Clone Assay, BrdU Staining, Inhibition, Over Expression, Incubation, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor

doi: 10.1073/pnas.1707965114

Figure Lengend Snippet: Antibodies and methods used for IHC analyses

Article Snippet: The NEON Transfection system was purchased from Invitrogen, the BrdU cell proliferation Elisa kit was from Roche Diagnostics or Sigma, and the following plasmids were from OriGene: 11HSD1: sc109325; 11HSD2: sc122552 or RG207796; H6PD: sc117481; DHCR7: sc110871; and D8D7I (EBP): sc116006. pCMV-RORγ plasmid (NBP2-25278) was from Novus biologicals; pSG5-hGR was a gift from H. Richard-Foy, CNRS, Toulouse, France; pSG5-hRORα was a gift from V. Laudet, CNRS, Banyuls-sur-Mer, France; RORE-Luc was from B. Staels, the Pasteur Institute, Lille, France; pSG5-hRXRγ was a gift from M. Oulad-Abdelghani, INSERM, Strasbourg, France; and pSG5-hFXR and tk-EcRE-Luc were gifts from R. M. Evans, The Salk Institute, La Jolla, CA.

Techniques: Staining

Journal: PLoS ONE

Article Title: Evaluation of the Diagnostic Accuracy of CareStart G6PD Deficiency Rapid Diagnostic Test (RDT) in a Malaria Endemic Area in Ghana, Africa

doi: 10.1371/journal.pone.0125796

Figure Lengend Snippet: An outline comparing some common factors of importance between the methods used for G6PD screening in the study.

Article Snippet: The CareStart G6PD deficiency RDT and Trinity Quantitative methods were controlled with two levels of commercially prepared G6PD quality control (QC) (normal level, Cat. No. PD 2618 and deficient level Cat. No. PD 2617 controls) samples from Randox.

Techniques:

Journal: PLoS ONE

Article Title: Evaluation of the Diagnostic Accuracy of CareStart G6PD Deficiency Rapid Diagnostic Test (RDT) in a Malaria Endemic Area in Ghana, Africa

doi: 10.1371/journal.pone.0125796

Figure Lengend Snippet: Performance of the CareStart G6PD deficiency RDT kit to other commercially available methods for screening G6PD deficiency .

Article Snippet: The CareStart G6PD deficiency RDT and Trinity Quantitative methods were controlled with two levels of commercially prepared G6PD quality control (QC) (normal level, Cat. No. PD 2618 and deficient level Cat. No. PD 2617 controls) samples from Randox.

Techniques:

Journal: PLoS ONE

Article Title: Evaluation of the Diagnostic Accuracy of CareStart G6PD Deficiency Rapid Diagnostic Test (RDT) in a Malaria Endemic Area in Ghana, Africa

doi: 10.1371/journal.pone.0125796

Figure Lengend Snippet: Diagnostic performance of CareStart G6PD RDT by gender compared to Trinity Biotech Quantitative and Qualitative methods for G6PD deficiency screening.

Article Snippet: The CareStart G6PD deficiency RDT and Trinity Quantitative methods were controlled with two levels of commercially prepared G6PD quality control (QC) (normal level, Cat. No. PD 2618 and deficient level Cat. No. PD 2617 controls) samples from Randox.

Techniques: Diagnostic Assay

Journal: PLoS ONE

Article Title: Evaluation of the Diagnostic Accuracy of CareStart G6PD Deficiency Rapid Diagnostic Test (RDT) in a Malaria Endemic Area in Ghana, Africa

doi: 10.1371/journal.pone.0125796

Figure Lengend Snippet: Area under the Receiver Operator Characteristics (ROC) curve for G6PD RDT performance considering malaria status of participants.

Article Snippet: The CareStart G6PD deficiency RDT and Trinity Quantitative methods were controlled with two levels of commercially prepared G6PD quality control (QC) (normal level, Cat. No. PD 2618 and deficient level Cat. No. PD 2617 controls) samples from Randox.

Techniques:

Journal: PLoS ONE

Article Title: Evaluation of the Diagnostic Accuracy of CareStart G6PD Deficiency Rapid Diagnostic Test (RDT) in a Malaria Endemic Area in Ghana, Africa

doi: 10.1371/journal.pone.0125796

Figure Lengend Snippet: Estimates of G6PD enzyme activity levels by malaria parasite status.

Article Snippet: The CareStart G6PD deficiency RDT and Trinity Quantitative methods were controlled with two levels of commercially prepared G6PD quality control (QC) (normal level, Cat. No. PD 2618 and deficient level Cat. No. PD 2617 controls) samples from Randox.

Techniques: Activity Assay