Journal: The Journal of Neuroscience

Article Title: Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

doi: 10.1523/JNEUROSCI.3402-12.2013

Figure Lengend Snippet: Regulation of FKBP38 and PHD2 by PSEN1 and PSEN2 in normoxia and hypoxia. A, Total cell extracts from PSEN1/2 wt and ko MEFs were analyzed for PHD2, FKBP38, and β-actin protein levels by immunoblotting (left). Relative band intensities of three independent experiments were quantified by densitometry (right). Data are shown as mean ± SD values. *p < 0.05 (t test). **p < 0.005 (t test). B, Total RNA was extracted from PSEN1/2 wt and ko MEFs. Transcript levels of FKBP38 and PHD2 were quantified by quantitative RT-PCR and normalized to ribosomal protein S12 mRNA levels. *p < 0.05 (t test). n.s., Not significant. C, PSEN1/2 wt and ko MEFs were cultured in normoxia or hypoxia for the time indicated, and PHD2 and FKBP38 protein levels were analyzed by immunoblotting. D, Relative band intensities of three independent experiments were quantified by densitometry. The 0 h time point of each cell line was defined as 1. E, Total RNA was derived from organs of mice that were kept at 20% or 8% oxygen for the time indicated. PSEN1 and PSEN2 transcript levels were quantified by quantitative RT-PCR and normalized to the ribosomal protein S12 mRNA levels. Data are shown as mean ± SEM values of three independent RNA extractions from different mice.

Article Snippet: Primary antibodies used were rabbit anti-APP, C-terminal (Sigma A8717), rabbit anti-human PHD2 (Novus Biologicals), rabbit anti-mouse PHD2 (Novus Biologicals), rabbit anti-FKBP38 ( Edlich et al., 2005 ), rabbit anti-HIF-1α (Novus Biologicals), mouse anti-N-cadherin (BD Biosciences), mouse anti-β-actin (Sigma).

Techniques: Western Blot, Quantitative RT-PCR, Cell Culture, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

doi: 10.1523/JNEUROSCI.3402-12.2013

Figure Lengend Snippet: HIF transcriptional response to hypoxia in PSEN1/2-deficient cells. A, wt, PSEN1/2 ko (ko), PSEN1 ko (1ko), and two reconstituted clones of PSEN1/2 ko (C1 or C2) MEFs were transiently transfected with the HIF-dependent reporter pH3SVL and pRL-SV40 constructs and cultured in 20% or 0.2% O2 for 16 h before relative luciferase activities were determined. The results are shown as mean ± SEM values of three independent experiments performed in triplicates (top). HIF-1α and β-actin protein levels were determined by immunoblotting (bottom). B, PSEN1/2 wt and ko MEFs were cultured in 20% or 0.2% O2 for 4, 8, 16, and 32 h and total RNA was extracted. CAIX, PHD2, NDRG1, and BNIP3 transcript levels were quantified by RT-PCR and normalized to the expression of ribosomal protein S12 mRNA. Data are shown as mean ± SEM values of five independent experiments. Student's t tests were used to statistically evaluate the reduction (if any) of these HIF target genes by PSEN1/2 deficiency at each time point. *p < 0.05. **p < 0.01. C, PSEN1/2 wt and ko MEFs were cultured in 20%, 5%, or 0.2% O2 for 16 h before HIF-1α, PHD2, and β-actin protein levels were analyzed by immunoblotting.

Article Snippet: Primary antibodies used were rabbit anti-APP, C-terminal (Sigma A8717), rabbit anti-human PHD2 (Novus Biologicals), rabbit anti-mouse PHD2 (Novus Biologicals), rabbit anti-FKBP38 ( Edlich et al., 2005 ), rabbit anti-HIF-1α (Novus Biologicals), mouse anti-N-cadherin (BD Biosciences), mouse anti-β-actin (Sigma).

Techniques: Clone Assay, Transfection, Construct, Cell Culture, Luciferase, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: The Journal of Neuroscience

Article Title: Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

doi: 10.1523/JNEUROSCI.3402-12.2013

Figure Lengend Snippet: HIF-1α regulation in PSEN1/2-deficient cells. A, PSEN1/2 wt and ko MEFs were cultured in 20% or 0.2% O2 for 16 h before treatment with 100 μm cycloheximide. Total cell extracts were prepared after 0.5, 1, 2, and 4 h of treatment. HIF-1α, and β-actin protein levels were analyzed by immunoblotting. B, Relative band intensities of three independent experiments were quantified relative to the β-actin levels and normalized to the 0 h hypoxia time points. Mean values ± SEM of three independent experiments are shown. C, Quantification of HIF-1α mRNA levels in PSEN1/2 wt and ko MEFs by quantitative RT-PCR. Transcript levels were normalized to the mRNA levels of ribosomal protein S12, and the wt level was defined as 1. D, PSEN1/2 wt and ko MEFs were incubated at 0.2% O2 for 16 h before 5 μg/ml actinomycin D was added to the cells. Total RNA was extracted after 0, 4, 8, 16, and 24 h of actinomycin D treatment, and HIF-1α, PHD2, and VEGFA transcript levels were quantified by RT-PCR. mRNA levels were normalized to ribosomal protein S12 mRNA, and the 0 h time point was defined as 1. The results are shown as mean ± SEM values of three independent experiments. E, PSEN1/2 wt, ko, and two PSEN1/2 reconstituted clones were transiently cotransfected with the Hif1a promoter-driven pGL3–885Hif1a or the promoterless pGL3-basic plasmid together with the pSV40-RL control vector. Data were normalized to the Renilla luciferase activities and are shown as mean ± SEM values of three independent experiments performed in triplicates. *p < 0.05,(t test). n.s., Not significant (t test).

Article Snippet: Primary antibodies used were rabbit anti-APP, C-terminal (Sigma A8717), rabbit anti-human PHD2 (Novus Biologicals), rabbit anti-mouse PHD2 (Novus Biologicals), rabbit anti-FKBP38 ( Edlich et al., 2005 ), rabbit anti-HIF-1α (Novus Biologicals), mouse anti-N-cadherin (BD Biosciences), mouse anti-β-actin (Sigma).

Techniques: Cell Culture, Western Blot, Quantitative RT-PCR, Incubation, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Control, Luciferase

Journal: The Journal of Neuroscience

Article Title: Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

doi: 10.1523/JNEUROSCI.3402-12.2013

Figure Lengend Snippet: Requirement of γ-secretase enzymatic activity for presenilin-dependent regulation of HIF-1α, FKBP38, and PHD2. A, PSEN1/2 wt and ko MEFs were pretreated with 0, 2, or 4 μm of the γ-secretase inhibitor DAPT before culturing at 0.2% O2 for 12 h. HIF-1α, FKBP38, PHD2, N-cadherin, and β-actin protein levels were determined by immunoblotting. B, SH-SY5Y cells were cultured at 20% or 0.2% O2 for 16 h in the presence of 0, 2, or 4 μm DAPT, and HIF-1α, PHD2, and β-actin protein levels were analyzed by immunoblotting. C, D, HeLa/trTAA/TRE-N1-ICD cells were cultured for 24 h in the presence or absence of 1 μm doxycycline before exposure to 20% or 0.2% O2 for 16 h. Thereafter, HIF-1α, PHD2, and β-actin protein levels were determined by immunoblotting (C) and PHD2, GLUT1, CAIX, and Snail mRNA levels were quantified by quantitative RT-PCR (D). Transcript levels were normalized to the mRNA levels of ribosomal protein L28. The untreated normoxic control was defined as 1. Data are shown as mean ± SEM values of three independent experiments. E, PSEN1/2 wt, ko, and ko MEFs stably expressing PSEN1 or PSEN2 FAD mutations (ΔE9, A246E, L166P, G384A, and N141I) were transiently cotransfected with the hypoxia response element-driven luciferase reporter plasmid (pH3SVL) together with the pSV40-RL control vector. Cells were cultured for 16 h at 20% or 0.2% O2 before luciferase activity was determined. The results are shown as mean ± SEM values of three independent experiments performed in triplicates.

Article Snippet: Primary antibodies used were rabbit anti-APP, C-terminal (Sigma A8717), rabbit anti-human PHD2 (Novus Biologicals), rabbit anti-mouse PHD2 (Novus Biologicals), rabbit anti-FKBP38 ( Edlich et al., 2005 ), rabbit anti-HIF-1α (Novus Biologicals), mouse anti-N-cadherin (BD Biosciences), mouse anti-β-actin (Sigma).

Techniques: Activity Assay, Western Blot, Cell Culture, Quantitative RT-PCR, Control, Stable Transfection, Expressing, Luciferase, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

doi: 10.1523/JNEUROSCI.3402-12.2013

Figure Lengend Snippet: Regulation of HIF by the APP/AICD cleavage cascade. A, Hek293-citAICD cells were pretreated with 1 μm tebufenozide for 24 h before culturing at 20% or 0.2% O2 for 16 h and determination of HIF-1α, PHD2, APP, and β-actin by immunoblotting (left). In a separate experiment, normoxic HIF-1α was detected using a prolonged exposure time (right). B, Hek293-citAICD cells were cultured for 24 h in the presence or absence of 1 μm tebufenozide and exposed to 20% or 0.2% O2 for the time indicated, before mRNA levels of HIF-1α, PHD2, CAIX, and AICD were quantified by RT-PCR. The transcript levels were normalized to ribosomal protein L28 mRNA levels, and the zero hour time point of the control cells was defined as 1. C, APP wt, APP ko, and APP/APPLP2 ko MEFs were cultured at 20% or 0.2% O2 for 16 h, and HIF-1α, PHD2, and β-actin protein levels were determined by immunoblotting. D, APP wt, APP ko, and APP/APLP2 ko MEFs were exposed to 0, 4, 8, 16, or 24 h of 0.2% O2, and mRNA levels of HIF-1α, PHD2, and CAIX were quantified by RT-PCR. Transcript levels were normalized to ribosomal protein S12 mRNA levels. E, F, Hek293-citAICD cells were grown in the presence or absence of 1 μm tebufenozide for 16 h before 8 h pretreatment with DMSO or DAPT and subsequent incubation at 20% or 0.2% O2 for 16 h. HIF-1α, PHD2, and AICD were determined by immunoblotting (E), and HIF-1α band intensities were quantified and normalized to β-actin (F). Data are shown as mean ± SEM values of three independent experiments. *p < 0.05 (t test).

Article Snippet: Primary antibodies used were rabbit anti-APP, C-terminal (Sigma A8717), rabbit anti-human PHD2 (Novus Biologicals), rabbit anti-mouse PHD2 (Novus Biologicals), rabbit anti-FKBP38 ( Edlich et al., 2005 ), rabbit anti-HIF-1α (Novus Biologicals), mouse anti-N-cadherin (BD Biosciences), mouse anti-β-actin (Sigma).

Techniques: Western Blot, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control, Incubation

Journal: The Journal of Neuroscience

Article Title: Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

doi: 10.1523/JNEUROSCI.3402-12.2013

Figure Lengend Snippet: PSEN1/2 regulate PHD2/HIFα in the brain. A, Cortical mRNA of control C57BL6/129 and PSEN1/2 cdko mice was quantified by RT-PCR and normalized to the transcript levels of the ribosomal protein S12. B, C, PHD2, FKBP38, and β-actin protein levels were determined by immunoblotting (B), and PHD2 and FKBP38 band intensities were quantified and normalized to β-actin (C). Data are shown as mean ± SEM values of n = 3 animals per group. *p < 0.05 (t test). **p < 0.01 (t test). ***p < 0.001 (t test).

Article Snippet: Primary antibodies used were rabbit anti-APP, C-terminal (Sigma A8717), rabbit anti-human PHD2 (Novus Biologicals), rabbit anti-mouse PHD2 (Novus Biologicals), rabbit anti-FKBP38 ( Edlich et al., 2005 ), rabbit anti-HIF-1α (Novus Biologicals), mouse anti-N-cadherin (BD Biosciences), mouse anti-β-actin (Sigma).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of Neuroscience

Article Title: Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

doi: 10.1523/JNEUROSCI.3402-12.2013

Figure Lengend Snippet: Scheme of the mechanisms involved in the regulation of HIF by PSENs. PSEN1/2 γ-secretase-mediated cleavage of the APP generates Aβ involved in AD as well as the AICD that induces Hif1a gene expression and HIF-1α protein stability but does not regulate FKBP38/PHD2. On the other hand, PSEN1/2 increases PHD2 activity by inhibiting FKBP38 in a γ-secretase-independent manner. These two mechanisms overlap with hypoxic induction of HIF-1α protein stability and finally converge in the downregulation of HIF-dependent target gene expression after deletion or functional mutation of PSEN1/2.

Article Snippet: Primary antibodies used were rabbit anti-APP, C-terminal (Sigma A8717), rabbit anti-human PHD2 (Novus Biologicals), rabbit anti-mouse PHD2 (Novus Biologicals), rabbit anti-FKBP38 ( Edlich et al., 2005 ), rabbit anti-HIF-1α (Novus Biologicals), mouse anti-N-cadherin (BD Biosciences), mouse anti-β-actin (Sigma).

Techniques: Gene Expression, Activity Assay, Targeted Gene Expression, Functional Assay, Mutagenesis

Journal: Medicine

Article Title: Tumor PHD2 Expression Is Correlated With Clinical Features and Prognosis of Patients With HCC Receiving Liver Resection

doi: 10.1097/MD.0000000000000179

Figure Lengend Snippet: Kaplan–Meier curves in patients with HCC according to their PHD2 expression status. Compared with patients with HCC having high PHD2 expression, those with low PHD2 expression had a longer (A) DFS period (25.5 ± 3.6 vs 16.7 ± 3.4 months, log-rank, P < 0.001). (B) Patients with low PHD2 expression had longer OS times, whereas those with high PHD2 expression had shorter survival times (37.5 ± 3.6 vs 29.5 ± 4.7 months, log-rank, P < 0.001). DFS = disease-free survival, HCC = hepatocellular carcinoma, OS = overall survival, PHD2 = prolyl hydroxylase domain protein.

Article Snippet: After immunoblot analysis, membranes were immunoblotted with anti-PHD2 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Medicine

Article Title: Tumor PHD2 Expression Is Correlated With Clinical Features and Prognosis of Patients With HCC Receiving Liver Resection

doi: 10.1097/MD.0000000000000179

Figure Lengend Snippet: PHD2 protein expressions in HCC cell lines (QGY7703, Bel7404, and Hep3B) by Western blot assays. (A) Our data show that the HCC cell lines had significantly higher PHD2 expressions than normal hepatic cell line (Lo2). (B) After si-RNA transfection, the PHD2 protein in HCC cell lines was significantly inhibited. HCC = hepatocellular carcinoma, PHD2 = prolyl hydroxylase domain protein.

Article Snippet: After immunoblot analysis, membranes were immunoblotted with anti-PHD2 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection

Journal: Medicine

Article Title: Tumor PHD2 Expression Is Correlated With Clinical Features and Prognosis of Patients With HCC Receiving Liver Resection

doi: 10.1097/MD.0000000000000179

Figure Lengend Snippet: Biological behaviors of HCC cells after PHD2 inhibition. (A) Cell growth rate significantly inhibited in HCC cell lines after PHD2 si-RNA transfection by MTT assay. (B) Cell migration abilities significantly decreased after PHD2 silencing. (C) PHD2 gene silencing dramatically inhibited the invasion abilities of these HCC cells. (D) PHD2 inhibition markedly increased cell apoptosis in cultured HCC cell lines compared to control si-RNA transfection. HCC = hepatocellular carcinoma, PHD2 = prolyl hydroxylase domain protein.

Article Snippet: After immunoblot analysis, membranes were immunoblotted with anti-PHD2 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology).

Techniques: Inhibition, Transfection, MTT Assay, Migration, Cell Culture, Control

Journal: Medicine

Article Title: Tumor PHD2 Expression Is Correlated With Clinical Features and Prognosis of Patients With HCC Receiving Liver Resection

doi: 10.1097/MD.0000000000000179

Figure Lengend Snippet: Representative images of migration assay using transwell (magnification: ×100). Cells were added to the top transwell chamber and allowed to migrate for 24 hours. Our data show that the PHD2 inhibition in (A) QGY7703, (B) Bel7404, and (C) Hep3B considerably reduced their migration abilities. Migrated cells were labeled with Calcein-AM. The nucleus was counterstained with DAPI. Migrated cells were quantified in 3–5 fields/well with 2–3 wells/condition. DAPI = 4’,6-diamidino-2-phenylindole, PHD2 = prolyl hydroxylase domain protein.

Article Snippet: After immunoblot analysis, membranes were immunoblotted with anti-PHD2 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology).

Techniques: Migration, Inhibition, Labeling

Journal: Medicine

Article Title: Tumor PHD2 Expression Is Correlated With Clinical Features and Prognosis of Patients With HCC Receiving Liver Resection

doi: 10.1097/MD.0000000000000179

Figure Lengend Snippet: Representative images of stained cells on the underside of the insert in invasion assay (magnification: ×100). Our data show that the PHD2 inhibition in (A) QGY7703, (B) Bel7404, and (c) Hep3B considerably reduced their invasive abilities. Invading cells were labeled with Calcein-AM and quantified in 3 to 5 fields/well with 2 to 3 wells/condition. PHD2 = prolyl hydroxylase domain protein.

Article Snippet: After immunoblot analysis, membranes were immunoblotted with anti-PHD2 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology).

Techniques: Staining, Invasion Assay, Inhibition, Labeling