Journal: Oncotarget

Article Title: EglN2 contributes to triple negative breast tumorigenesis by functioning as a substrate for the FBW7 tumor suppressor

doi: 10.18632/oncotarget.14290

Figure Lengend Snippet: A, B, C . qRT-PCR (A), representative images (B) and quantification (C) of soft agar assays from C3Tag cells infected with the lentivirus encoding EglN2 shRNAs (744, 746) or Control (Ctrl) shRNA. The statistical significance was calculated using student's t test. *** denotes p value of < 0.005. Error bars represent one SEM. D, E . Representative tumor gross appearance and tumor weight plots for C3Tag cells infected with the lentivirus encoding EglN2 shRNA (744) or Control (Ctrl) shRNA implanted into the mammary fat pads in FVB/NJ mice. * denotes p value of <0.05 for comparison between two groups by using unpaired two sample t-test. Error bars represent one SEM. F . Kaplan-Meier survival curves for C3Tag: EglN2 +/+ and C3Tag: EglN2 −/− mice. The p value was determined by the Log-rank (Mantel-Cox) test.

Article Snippet: Rabbit EglN2 antibody (NB100-310) was from Novus Biological.

Techniques: Quantitative RT-PCR, Infection, Control, shRNA, Comparison

Journal: Oncotarget

Article Title: EglN2 contributes to triple negative breast tumorigenesis by functioning as a substrate for the FBW7 tumor suppressor

doi: 10.18632/oncotarget.14290

Figure Lengend Snippet: A . Immunoblots from a panel of indicated breast cancer cell lines. B . Immunoblots of cell lysates from 293T cells transfected with FLAG tagged EglN2, HA tagged FBW7 or both followed by either DMSO (-) or MG132 (10 μM) treatment for overnight. GFP plasmids were transfected as the normalization control for equal transfection efficiency. C . 293T cells were transfected with HA-tagged EglN2 in the presence of dominant negative FLAG tagged Cul1, 2, 3, 4a, 4b, 5 or control (-). Cell lysates were harvested followed by immunoblots as indicated. D . HCT-116 WT or FBW7 −/− cells were treated with either control (-) or MG132 (10 μM) followed by immunoblots as indicated. Dotted vertical lane for EglN2 indicates that there is a non-essential lane that have been removed from a single original gel.

Article Snippet: Rabbit EglN2 antibody (NB100-310) was from Novus Biological.

Techniques: Western Blot, Transfection, Control, Dominant Negative Mutation

Journal: Oncotarget

Article Title: EglN2 contributes to triple negative breast tumorigenesis by functioning as a substrate for the FBW7 tumor suppressor

doi: 10.18632/oncotarget.14290

Figure Lengend Snippet: A . Immunoblot analysis of MDA-MB-453 cells infected with the lentivirus encoding FBW7 shRNA (#1, #6) or control (Ctrl) shRNA. B-C . qRT-PCR (B) or immunoblot (C) or analysis of MCF-7 cells infected with the lentivirus encoding either FBW7 shRNA (#1, #4, #5) or control (Ctrl) shRNA. D-E . qRT-PCR (D) or immunoblot (E) analysis of MCF-10A cells infected with the lentivirus encoding FBW7 shRNA (#1, #4, #6) or control (Ctrl) shRNA. F-G . Immunoblot analysis from cycloheximide (CHX) pulse chase of MCF-10A cells infected with the lentivirus encoding FBW7 shRNA (#1) or control (Ctrl) shRNA (F) or HCT-116 WT or FBW7 −/− cells expressing exogenous Flag tagged EglN2 (G).

Article Snippet: Rabbit EglN2 antibody (NB100-310) was from Novus Biological.

Techniques: Western Blot, Infection, shRNA, Control, Quantitative RT-PCR, Pulse Chase, Expressing

Journal: Oncotarget

Article Title: EglN2 contributes to triple negative breast tumorigenesis by functioning as a substrate for the FBW7 tumor suppressor

doi: 10.18632/oncotarget.14290

Figure Lengend Snippet: A-B . Immunoblot analysis of Hs-578T (A) and MDA-MB-231 (B) cells co-transfected with HA tagged FBW7 or GFP with either HA-tagged GSK3β or Control. Cell lysates were harvested 48 hours post-transfection. C . 293T cells were transfected with either HA tagged EglN2 or control followed by immunoprecipitation with HA agarose beads (3F10, Roche). Coomassie staining was performed to visualize the purified HA EglN2, which was cut followed by mass spectrometry analysis as described previously. EglN2 Ser401 and Thr405 sites were shown to be phosphorylated. D . Immunoblots for 293T cells co-transfected with various FLAG tagged EglN2 constructs (WT, S401A, T405A, ST-AA, ∧TPT or ∧SQPPTPT) and HA tagged GSK3β or Control (-). Equal amount of GFP was transfected to make sure of comparable transfection efficiency. Cell lysates were harvested 48 hours post-transfection. E . Immunoblot (IB) assays of whole cell extract (WCE) and immunoprecipitation (IP) of 293T cells (expressing Ctrl or HA-FBW7) co-transfected with Flag-EglN2 (F-EglN2), Flag-EglN2 ST-AA, Flag-EglN2 ∧SQPPTPT, Flag c-Jun or Flag c-Jun FS-AF mutants. Cells were treated with MG132 (10 μM) for overnight before harvesting at 48 hours post-transfection. F . In vivo ubiquitination assays of immunoprecipitation (IP with Ni-NTA) of 293T cells (expressing Ctrl or HA-FBW7) co-transfected with Flag-EglN2 (WT), Flag-EglN2 S401A, T405A or ST-AA. Cells were treated with MG132 (10 μM) for overnight before harvesting at 48 hours post-transfection.

Article Snippet: Rabbit EglN2 antibody (NB100-310) was from Novus Biological.

Techniques: Western Blot, Transfection, Control, Immunoprecipitation, Staining, Purification, Mass Spectrometry, Construct, Expressing, In Vivo, Ubiquitin Proteomics

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Decreased expression of prolyl hydroxylase 1 is associated with poor prognosis in colorectal cancers

doi: 10.1007/s00432-023-04717-y

Figure Lengend Snippet: PHD1 expression in CRCs. Representative PHD1 immunostaining of A low and B high immunoreactivity in CRCs

Article Snippet: Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121 °C in pH 7.8 Tris–EDTA-Citrate buffer prior to incubation with antibody PHD1 (polyclonal; rabbit; Novus Biologicals; 1/450 dilution).

Techniques: Expressing, Immunostaining

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Decreased expression of prolyl hydroxylase 1 is associated with poor prognosis in colorectal cancers

doi: 10.1007/s00432-023-04717-y

Figure Lengend Snippet: PHD1 immunostaining in association with clinical parameters in CRCs

Article Snippet: Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121 °C in pH 7.8 Tris–EDTA-Citrate buffer prior to incubation with antibody PHD1 (polyclonal; rabbit; Novus Biologicals; 1/450 dilution).

Techniques: Immunostaining, Expressing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Decreased expression of prolyl hydroxylase 1 is associated with poor prognosis in colorectal cancers

doi: 10.1007/s00432-023-04717-y

Figure Lengend Snippet: Kaplan–Meier analysis of PHD1 protein expression in primary CRCs. A Association between overall survival of patients and tumor stage ( p < 0.0001). B Association between clinical outcome in CRCs and PHD1 expression ( p < 0.0001)

Article Snippet: Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121 °C in pH 7.8 Tris–EDTA-Citrate buffer prior to incubation with antibody PHD1 (polyclonal; rabbit; Novus Biologicals; 1/450 dilution).

Techniques: Expressing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Decreased expression of prolyl hydroxylase 1 is associated with poor prognosis in colorectal cancers

doi: 10.1007/s00432-023-04717-y

Figure Lengend Snippet: Multivariate analysis including PHD1 immunostaining results

Article Snippet: Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121 °C in pH 7.8 Tris–EDTA-Citrate buffer prior to incubation with antibody PHD1 (polyclonal; rabbit; Novus Biologicals; 1/450 dilution).

Techniques: Immunostaining, Staining

Journal: PLoS ONE

Article Title: Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect

doi: 10.1371/journal.pone.0042072

Figure Lengend Snippet: A – Western blotting of HIF1A and its hydroxylated form in LCLs (left panel). The membrane was probed with mouse antibody against HIF1A and the rabbit serum against the hydroxylated form of HIF1A (HIF1A-OH). Notice the absence of HIF1A-OH in LCLs (first lane) and the lack of effect of proteasome inhibition. In contrast, high levels of hydroxylated HIF1A were detected in control MCF7 cells upon proteasome inhibition (right panel). B – Western blotting of whole cell lysates of CD40+IL4-activated B-cells, freshly EBV-infected B-cells and LCLs. Cells were treated with 1 mM NiCl 2 , mimicking hypoxia-like conditions. Notice that HIF1A, PHD1 and PHD2 levels in LCLs did not change upon treatment with NiCl 2 , in contrast to freshly infected cells. C – HIF1A expression in CD40+IL4-activated (ABC), freshly EBV-infected (EBC) B-cells and LCLs (LCL) measured by Q-PCR.

Article Snippet: After rehydration in PBS, cells were stained with rabbit anti-PHD1, anti-PHD2 and mouse anti-FLAG (Rockland, Gilbertsville, PA) antibodies.

Techniques: Western Blot, Inhibition, Infection, Expressing

Journal: PLoS ONE

Article Title: Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect

doi: 10.1371/journal.pone.0042072

Figure Lengend Snippet: A – GST pulldown assay and Western blotting from lymphoblastoid cell lysates. EBNA-3 was detected in a complex with HIF1A protein. As a positive control, 5% of the crude lysate was loaded. B – Immunoprecipitation and Western blotting from lymphoblastoid cell lysates. HIF1A was detected in a complex with EBNA-3, but not with EBNA-5, on the surface of CNBr beads with immobilized anti-EBNA-3 and anti-EBNA-5 antibodies, respectively. As a positive control, 7% of the crude lysate was loaded. C – Immunostaining of MCF7 cells transfected with GFP-HIF1A (green) and RedDs-EBNA-5 (red). Rabbit anti-PHD1 antibody, followed by biotinylated goat anti-rabbit antibody and AMKA streptavidin (white). EBNA-5 and HIF1A partially colocalize in the nucleus. In doubly transfected cells, the normally cytoplasmic PHD1 is located in the nucleus, colocalized with GFP-HIF1A and RedDS-EBNA-5. D – MCF7 cells transfected with GFP-HIF1A (green) and FLAG-EBNA-3 (white) were stained with rabbit anti-PHD1 antibody, followed by biotinylated goat anti-rabbit antibody and AMKA streptavidin (blue). The mainly cytoplasmic PHD2 redistributed to the nucleus in doubly transfected cells, colocalizing with GFP-HIF1A and FLAG-EBNA-3.

Article Snippet: After rehydration in PBS, cells were stained with rabbit anti-PHD1, anti-PHD2 and mouse anti-FLAG (Rockland, Gilbertsville, PA) antibodies.

Techniques: GST Pulldown Assay, Western Blot, Positive Control, Immunoprecipitation, Immunostaining, Transfection, Staining

Journal: PLoS ONE

Article Title: Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect

doi: 10.1371/journal.pone.0042072

Figure Lengend Snippet: A – Western blotting following the GST pulldown assay from lymphoblastoid cell lysates. PHD2 and a small portion of PHD1 were precipitated on the beads with GST-EBNA-3 from cell lysate. Positive control shows 15% the crude lysate. B – Western blotting following the GST pulldown assay from LCL lysates. PHD1 was precipitated on the support with immobilized GST-EBNA-5 protein from the lymphoblastoid cell lysate. Positive control shows 15% of the crude lysate.

Article Snippet: After rehydration in PBS, cells were stained with rabbit anti-PHD1, anti-PHD2 and mouse anti-FLAG (Rockland, Gilbertsville, PA) antibodies.

Techniques: Western Blot, GST Pulldown Assay, Positive Control

Journal: PLoS ONE

Article Title: Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect

doi: 10.1371/journal.pone.0042072

Figure Lengend Snippet: A – HIF1A is hydroxylated by the PHDs under normoxic conditions. The hydroxylated HIF1A is recognized by the VHL, E3 ubiquitin ligase, and HIF1A is degraded on proteasomes in activated B-cells. B – Upon EBV infection, EBNA-3 and EBNA-5 bind to PHD2 and PHD1, respectively, and inhibit HIF1A hydroxylation and degradation. The stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT and transactivates genes such as GLUT1 , PDK1 and LDHA . This results in conversion of pyruvate to lactate, i.e., aerobic glycolysis.

Article Snippet: After rehydration in PBS, cells were stained with rabbit anti-PHD1, anti-PHD2 and mouse anti-FLAG (Rockland, Gilbertsville, PA) antibodies.

Techniques: Infection