Journal: The FASEB Journal

Article Title: PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis

doi: 10.1096/fj.201601291R

Figure Lengend Snippet: PHD3−/− mice show increased incidence of disc degeneration and decreased expression of select HIF-1 target genes in NP tissue. A) Schematic drawing of spinal motion segment and coronal cross section showing vertebral bodies and the intervertebral disc with its central NP, circumferential AF, and superior and inferior cartilaginous endplates (CEP). B, C) Representative Safranin-O/Fast Green/hematoxylin–stained sections of 12.5-mo-old WT (B) and PHD3−/− (C) mice. The PHD3−/− mouse showed a decreased number of NP cells and some changes in cell morphology. D) PHD3−/− mice showed increased incidence of discs with a higher grade of degeneration, as assessed by the Thompson grading scale. Four discs/animal were scored from 3 pairs of littermate animals. E–L') Representative immunofluorescence images from 12.5-mo-old WT (PHD3+/+) and knockout (PHD3−/−) mice showing NP tissue areas with a comparable number of cells. There was decreased staining of HIF-1 targets VEGF-A (E–F'), LDHA (G–H'), and GLUT1 (I, J) in knockout mice compared to WT mice. In contrast, another HIF-1 target, ENO1, shows comparable expression in PHD3+/+ (K, K') and PHD3−/− (L, L') mice. Krt19 was used as a marker to label the NP tissue compartment with VEGF-A, LDHA, and ENO1 (independent channel not shown). Scale bars, 100 μm (B, C) and 50 μm (E–L'). Images are representative from 3 independent littermate groups.

Article Snippet: Sections were then sequentially incubated with antibodies [VEGF, ab46154; LDHA, ab52488; and GLUT1, ab40084 (all from Abcam), or ENO1, NB100-65252 (Novus Biologics, Littleton CO, USA), all at a concentration of 1:100) with an NP marker anti-keratin (Krt)19 antibody (1:3, TROMA-III; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) in 5% normal donkey serum in 1× PBS, 0.4% Triton-X at 4°C overnight ( 22 ).

Techniques: Expressing, Staining, Immunofluorescence, Knock-Out, Marker

Journal: The FASEB Journal

Article Title: PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis

doi: 10.1096/fj.201601291R

Figure Lengend Snippet: PHD3 controls expression of a select set of HIF-1 targets in NP cells through a p300-dependent mechanism. A, B) Bright-field (A) and fluorescent (B) images of rat NP cells after transduction with lentivirus coexpressing shPHD3 and enhanced green fluorescent protein. Scale bars, 10 μm. C) Measurement of PHD3 mRNA expression after transduction of NP cells with shRNA targeting PHD3 (n = 7). D) Measurement of mRNA expression of HIF-1 target genes in PHD3-silenced NP cells cultured in NX or HX for 72 h (n = 7). E) Hypoxic expression of Pgk1, Gapdh, and Eno1 in PHD3-silenced cells did not change (n = 3). F) Measurement of activity of Eno1 promoter containing 2 well-characterized HIF-1 binding sites after PHD3 knockdown (n = 7). G, H) Western blot (G) and corresponding densitometric analysis (H) of select HIF-1 targets in NP cells after stable knockdown of PHD3 (n = 4). I) Location of HRE sites within promoters of Vegfa, Slc2a1, and Ldha and location of ChIP primers used in J. J) HIF-1α enrichment at HRE sites within Vegfa, Slc2a1, and Ldha promoters decreases after knockdown of PHD3 during 72 h HX (n = 3). Luciferase assays were performed with 3 technical replicates per experiment; quantitative RT-PCR assays were performed with 2 technical replicates per experiment. Data are means ± sem. *P < 0.05.

Article Snippet: Sections were then sequentially incubated with antibodies [VEGF, ab46154; LDHA, ab52488; and GLUT1, ab40084 (all from Abcam), or ENO1, NB100-65252 (Novus Biologics, Littleton CO, USA), all at a concentration of 1:100) with an NP marker anti-keratin (Krt)19 antibody (1:3, TROMA-III; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) in 5% normal donkey serum in 1× PBS, 0.4% Triton-X at 4°C overnight ( 22 ).

Techniques: Expressing, Transduction, shRNA, Cell Culture, Activity Assay, Binding Assay, Knockdown, Western Blot, Luciferase, Quantitative RT-PCR

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 antibodies inhibit the expression of β-hCG and progesterone in JEG-3 cells. (a) An obvious band signal marked by an arrow was detected by 1 dimension western blot. JEG-3 proteins were used as antigens, 10 μg/ml ATAB-positive IgGs as the primary antibody and goat-anti-human antibodies as the secondary antibody. ENO1 was identified to be one of the most prominent targets with western blot and mass spectrometry. 1D western blots were repeated at least 5 times. (b.c) The production of β-hCG in JEG-3 cells was inhibited by 1, 100 and 1000 ng/ml of anti-ENO1 antibodies while the secretion of progesterone was suppressed by 1 μg/ml of anti-ENO1 antibodies compared to the rabbit polyclonal IgGs by ELISA (P < 0·05, Wilcoxon test, n = 6). Data were shown as mean ± SEM and P-values are shown individual treatment groups compared to isotype control.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Expressing, Western Blot, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Control

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 antibodies are a member of ATAB. (a) The signals of ENO1 in JEG-3 cells were reduced by incubation with a mixture of antibodies (10 μg/ml rabbit anti-ENO1 antibodies and 10 μg/ml human ATAB-positive IgGs) compared with only 10 μg/ml incubating human purified IgGs or 10 μg/ml rabbit anti-ENO1 antibodies. Different secondary antibodies were used in western blots: goat-anti-human antibody, goat-anti-human antibody and goat-anti-rabbit antibody (from left to right rows). (b.c) The diagram shows that in the flow cytometry mixed antibodies (0·42 mg/ml anti-ENO1 antibodies with 2 mg/ml isolated IgGs) bind less JEG-3 cells as ATAB-positive IgGs (P < 0·05, Wilcoxon test, n = 3). (d.e) Pre-incubating 20 μg recombinant human ENO1 protein with 2 mg/ml ATAB-positive IgGs decreases the binding of ATAB-positive IgGs to JEG-3 cells (P < 0·05, Wilcoxon test, n = 3). The MCS values are showed with mean ± SD and P-values are shown individual treatment groups compared to incubation with ATAB-positive IgGs.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Incubation, Purification, Western Blot, Flow Cytometry, Isolation, Recombinant, Binding Assay

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 antibodies inhibit the expression of β-hCG and progesterone in human villous trophoblast (HVT) cells. (a) The production of β-hCG in HVT cells was inhibited by 1, 10 and 100 ng/ml of anti-ENO1 antibodies in a dose dependent manner. (each P value <0·05, Wilcoxon test, n = 6). (b) The secretion of progesterone in HVT cells was suppressed by 1, 10 and 100 ng/ml of anti-ENO1 antibodies in a dose dependant manner. (each P value <0·05, Wilcoxon test, n = 6). Values are expressed as shown as mean ± SEM and P-values are shown individual treatment groups compared to isotype control.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Expressing, Control

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: Anti-ENO1 titers are higher in the sera of ATAB-positive patients. (a) Anti-ENO1 antibodies were increased in the 16 uRM patients with positive ATAB compared with 17 cases with negative ATAB (P = 0·007, student's t -test). (b.c) The levels of anti-γ-Enolase antibodies and anti-citrullinated fibrinogen (CFG) antibodies were not changed in 36 ATAB-positive patients and 30 ATAB-negative cases (both P > 0·05, student's t -test). (d) Anti-MCV antibodies were higher in the sera of 34 ATAB-positive patients than 28 ATAB-negative cases (P = 0·018, student's t -test). Data were shown as mean ± SEM and statistically significant differences between two uRM groups were marked with P values.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques:

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: ENO1 expression is increased in the decidua of uRM patients in the first trimester. Immunohistological analyses of ENO1 expression in the first trimester placenta were measured among 20 uRM patients and 22 healthy controls by IRS scores. (a–c) In the syncytium, the staining of ENO1 did not show significant difference in uRM patients (a) and controls (b), which is represented as bar charts (P > 0·05, Mann-Whitney test). The strong staining of ENO1 in the syncytium is marked with red arrows. (d–f) In the decidua, the expression of ENO1 was higher in uRM patients (d) as in controls (e, P = 0·038, Mann-Whitney test). Magnification × 10 lens, scale bar = 200 μm; magnifications of inserted pictures are ×25 lens, scale bar = 100 μm. uRM = unexplained recurrent miscarriages. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: ENO1 coexpresses with β-arrestin in the extravillous trophoblasts (EVT) of uRM patients and healthy controls. Confocal microscopy images showed a co-localization of ENO1 (red) and β-arrestin (green) in EVT of the first trimester placenta in uRM women (a–d) and healthy controls (e–h), which are shown with white arrows. HLA-G is used as the marker for EVT (magenta), magnification x20, scale bar =20 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Confocal Microscopy, Marker

Journal: EBioMedicine

Article Title: Anti α-enolase antibody is a novel autoimmune biomarker for unexplained recurrent miscarriages

doi: 10.1016/j.ebiom.2019.02.027

Figure Lengend Snippet: The possible role of ENO1 in unexplained recurrent miscarriages (uRM). Anti-ENO1 antibodies are among the antitrophoblast antibodies (ATAB) and are higher expressed in the sera of positive-ATAB uRM patients. The translocation (1), overexpression (2) or posttranslational modifications (3) of ENO1 in the extravillous trophoblasts may be the reason for induction of anti-ENO1 antibody expression in the sera of uRM patients. Anti-ENO1 antibodies can inhibit the production of β-hCG and progesterone in trophoblast cells, and may stimulate the expression of PAI-1. All these changes may eventually contribute to recurrent miscarriages.

Article Snippet: On the basis of 1D western blotting, a competition assay was performed with similar procedures except for the primary antibodies, where a mixture of ATAB-positive IgGs and polyclonal rabbit anti-ENO1 antibodies (Novus Biologicals, Littleton, Colorado, USA) both at a concentration of 10 μg/ml was used.

Techniques: Translocation Assay, Over Expression, Expressing