Journal: PLoS ONE

Article Title: Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0086796

Figure Lengend Snippet: Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam 3 CSK 4 (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. ( A ) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, ( B ) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. ( C ) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

Article Snippet: The human pharyngeal squamous cell carcinoma cell line FaDu (HTB-43; ATCC; Manassa, VA) was cultured in minimum essential medium (MEM) with Earl’s salts and 2 mM L-glutamine (GIBCO, Grand Island, NY), and supplemented with 10% FBS (PAN Biotech, Aidenbach, Germany), and 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture, Expressing, Flow Cytometry, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Functional exploration of colorectal cancer genomes using Drosophila

doi: 10.1038/ncomms13615

Figure Lengend Snippet: ( a , b ) Plot of P -values indicating significance of compound rescue ( a ) and summary of compound response ( b ) of ras G12V and ras G12V p53 Ri pten Ri apc Ri animals. P -values ( a ) were obtained by comparing the dissemination phenotype after compound feeding to dimethyl sulfoxide (DMSO) fed flies (dissemination plots can be found in ). Blue dots represent statistically significant results. ( c ) Quantification of dissemination in ras G12V pten Ri and ras G12V p53 Ri apc Ri animals treated with BEZ235. ( d ) Western blot analysis of PI3K pathway output from hindguts with indicated genotypes 7 days after induction of transgenes and quantification. Syn, Syntaxin (loading control). ( e ) Time-course analysis of PI3K pathway activation status in control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( f ) Western blot analysis of the biochemical response by ras G12V and ras G12V p53 Ri pten Ri apc Ri animals to PI3K pathway inhibitors. ( g ) Quantification of dissemination in indicated genotypes treated with BEZ235 or DMSO. ( h ) Schematic illustration of the mechanism of resistance to BEZ235: genetically activating mTORC1 promotes BEZ235 sensitivity. ( d , e ) Each data point represents the average response of two to five biological replicates with ten hindguts per replicate; error bars: s.e.m. ( a – c , g ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P <0.01 and ** P <0.05 (Fisher's exact test). Compound doses reflect concentrations in the food. Uncropped gels with molecular markers for d and f can be found in .

Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

Techniques: Western Blot, Control, Activation Assay

Journal: Nature Communications

Article Title: Functional exploration of colorectal cancer genomes using Drosophila

doi: 10.1038/ncomms13615

Figure Lengend Snippet: ( a ) Western blot analysis of PI3K signalling pathway output in ras G12V and ras G12V p53 Ri pten Ri apc Ri hindguts after 1 day feeding of SC79 at indicated doses. Syn, Syntaxin (loading control);ten hindguts per replicate. ( b ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after sequential treatment with BEZ235 and indicated doses of SC79. ( c ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after two different treatment schedules of SC79/BEZ235 and each drug alone. ( b , c ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P <0.01 and ** P <0.05 (Fisher's exact test). ( b ) *Variable response; not all replicates show significant rescue. Drug doses reflect concentrations in the food. Uncropped gels used to generate panel a can be found in .

Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

Techniques: Western Blot, Control

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 1. Differentiation of 3D-iMACs and their functional analysis. A. Schematic representation of the protocol for generating 3D-iMACs. All cell culture was performed under hypoxic (5% O2) conditions. B. Bright-filed images at day 4, 10, 17, and 24. Scale bar, 200 μm. C. Surface marker expressions of 3D-iMACs at day 24. Most cells express both of CD163 and CD206. D. Comparison of cytokine production profiles between 3D-iMACs and primary M2-like macrophages. They were not treated (NT) or stimulated with 10 ng/ml LPS for 24 h (LPS). n = 3 biological replicates. iMACs and primary MACs showed similar cytokine profiles with or without LPS stimulation. E. Phagocytic activities of 3D-iMACs were confirmed under a fluorescent microscope. E. coli BioParticles conjugated with pHrodo Green appear bright green when they are taken up by macrophages. Scale bar, 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Functional Assay, Cell Culture, Marker, Comparison, Microscopy

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 2. Differentiation of 2D-iMACs and their functional analysis. A. Schematic representation of the protocol for generating 2D-iMACs. B. Bright-field images at days 12 and 19. Scale bar, 200 μm. C. Surface marker expression of 2D- iMACs at days 12 and 19 (WT1323). They are positive for CD45, CD14, and CD11b. D. Polarization protocol and their subsequent surface marker expression. While M2-like iMACs showed high CD163 and low CD80 positivity, the expression patterns were opposite in M1-like iMACs. E. mRNA expression levels of macrophage- related genes in M1-like and M2-like 2D-iMACs. M1-related and M2-related genes are shown in the upper and lower portions respectively. They showed the expected expression patterns except for TGF-β. Expression levels are normalized to the housekeeping gene β-actin. Student's t-test was used for comparison of two groups. **p < 0.01. Data represent mean ± SEM of four independent experiments with technical triplicates. F. Phagocytic activities of M1-like and M2-like 2D-iMACs were confirmed (WTC11). E. coli BioParticles taken up by macrophages appear bright green. Scale bar, 200 μm. G. Comparison of cytokine production profiles between 2D- iMACs and primary macrophages. M1-like and M2-like 2D-iMACs showed similarities to primary M1- and M2-like macrophages respectively. n = 4–6 biological replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Functional Assay, Marker, Expressing, Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 4. Response to PAMP (LPS) and DAMPs (HMGB1, S100A8/A9) stimulation in 2D-iMACs. A. Protocol for testing the response to PAMPs and DMAPs. B. Comparison of cytokine production profiles in response to a PAMP (LPS) and DAMPs (HMGB1, S100A8/ A9) between primary M1-like macrophages and M1-like 2D iMACs. Primary cells and iMACs showed similarities regarding their responses to LPS. NT, HMGB, and S100 labels refer to untreated, stimulated with HMGB1, and stimulated with S100A8/A9, respectively. Primary cells and iMACs showed strong similarities regarding their responses to LPS. C. Comparison of cytokine production profiles in response to PAMP (LPS) and DAMPs (HMGB1, S100A8/A9) between primary M2-like macrophages and M2-like 2D iMACs. n = 2–3 biological replicates for each condition.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 5. Comparison between WT-iMACs and FOP-iMACs. A. Comparison of cytokine production profiles between WT and FOP 2D-iMACs. Cells were not treated (NT) or stimulated with 10 ng/ml LPS for 24 h (LPS) based on the protocol shown in Fig. 4A. n = 6 biological replicates. Differences between WT- and FOP-iMACs were more apparent in M1-like iMACs. B. Cytokines that showed higher production in FOP-M1-like iMACs compared with WT-M1-like iMACs at their baseline state (nontreated, NT). Student's t-test was used for comparison of two groups. *p < 0.05, **p < 0.01. Data represent mean ± SEM of six independent experiments with technical triplicates. C. Cytokine concentrations showing significant differences between WT- and FOP-M1-like iMACs stimulated with 10 ng/ml LPS. Key pro-inflammatory cytokine concentrations were higher in WT-M1-like iMACs when stimulated with 10 ng/ml LPS. Student's t-test was used for comparison of two groups. **p < 0.01Data represent mean ± SEM of six independent experiments with technical triplicates.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Comparison

Journal: Bone

Article Title: ACVR1 R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages.

doi: 10.1016/j.bone.2021.116129

Figure Lengend Snippet: Fig. 6. Activin A concentrations in iMACs and cytokine concentrations in FOP-M1-like iMACs treated with inhibitors of Activin A pathways. A. Activin A concentrations in NT and LPS-stimulated group were analyzed using Human/Mouse/Rat Activin A Quantikine ELISA Kit. Analyzed samples were the same as those used in Fig. 5A. The concentrations of FOP-M1-like iMACs were significantly higher than WT-M1-like iMACs in NT group, while their response to LPS was downregulated. Student's t-test was used for comparison of two groups. *p < 0.05, **p < 0.01. Data represent mean ± SEM of six independent experiments with technical triplicates. B. FOP-M1-like iMACs were treated with 100 ng/ml anti-human/mouse/rat Activin A antibody (Anti) or 10 mM SB431542 (SB) after M1 polarization. Each inhibitor was added every 24 h. After 3 days culture, supernatants were collected and analyzed. There were no significant differences in key pro- inflammatory cytokines that were found elevated in FOP-M1-like iMACs as shown in Fig. 5B. Dunnett's test was used to compare each group to control (CTRL). Data represent mean ± SEM of five independent experiments with technical triplicates.

Article Snippet: On day 4, medium was removed in the same way as described above and cultured on a new low-attachment plate in medium consisting of PromoCell basal media (PromoCell) supplemented with Cytokine Mix E (PromoCell), 10 ng/ml VEGF (Calbiochem), 10 ng/ml bFGF (Peprotech), 25 ng/ml IL-6 (Peprotech), and 10 ng/ml IL-11 (Peprotech).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Molecular Cancer

Article Title: Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients

doi: 10.1186/s12943-015-0381-6

Figure Lengend Snippet: Silencing of KLK6 expression in FaDu cells promotes tumor cell proliferation. ( A ) KLK6 expression in human HNSCC (FaDu, Cal27, SCC25) and HeLa cervix carcinoma cells was monitored on protein level by Western blot analysis with cell culture supernatants (upper panel), and on transcript level by semi-quantitative (middle panels) as well as quantitative RT-PCR (lower graph). Detection of LMNB1 amplicons served as control for cDNA quality and quantity for semi-quantitative RT-PCR (lower panel), while transcript levels of three independent reference genes ( ACTB, LMNB1, TBP ) were used for quantitative RT-PCR data. ( B ) KLK6 expression in stable FaDu-Mock and FaDu-shKLK6 clones is given by semi-quantitative (upper panel) and quantitative RT-PCR (lower graph) and was determined as described in ( A ). Differences in tumor cell proliferation between stable FaDu-Mock and FaDu-shKLK6 clones was monitored by quantification of cell counts over a time period of six days ( C ) and a BrdU incorporation assay ( D ). The graph represents mean values and standard deviations (SD) of the percentage total of BrdU-positive cells from three independent FaDu-shKLK6 clones and mock controls, respectively

Article Snippet: Human HNSCC cell lines (FaDu, Cal27 and SCC25 purchased from ATCC) and the cervix carcinoma cell line HeLa were maintained in Dulbecco’s Modified Eagle’s Medium (Sigma, Germany) supplemented with 10 % fetal bovine serum (Sigma, Germany), 2 mM L-Glutamine (Sigma, Germany) and 50 μg/ml Penicillin-Streptomycin (Sigma, Germany) in a humidified atmosphere of 6-8 % CO 2 at 37 °C.

Techniques: Expressing, Western Blot, Cell Culture, Quantitative RT-PCR, Control, Clone Assay, BrdU Incorporation Assay

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Impaired induction of HIF-1α in Psen1-/- immortalized mouse embryonic fibroblasts . In panel A, the time course of HIF-1α induction in Psen1+/+ (wt) and Psen1-/- (-/-) immortalized mouse embryonic fibroblasts is shown. Cultures were treated with 100 μm cobalt chloride for the indicated times (min). Lysates were prepared and Western blotting was performed using an anti-HIF-1α antibody. The lower panel shows the blot reprobed for β-tubulin. A representative blot is shown from experiments that were performed multiple times. In panels B and C, Psen1+/+ (wt) and Psen1-/- (-/-) cultures were treated with 100 μm cobalt chloride for 4 hours and analyzed for HIF-1α as in (A). The lowest panel in B shows representative samples of wt and -/- fibroblasts probed with the Psen1 antibody 33B10 to confirm the lack of detectible Psen1 expression in Psen1-/- fibroblasts. Panel C shows quantitation of the levels of HIF-1α in data derived from four independent experiments. In panels D and E cultures were serum starved overnight and then treated with insulin for 4 hours after which lysates were prepared and Western blotting performed as in (A). Panel E shows quantitation of the experiment shown in (D). In panel F, Psen1+/+ and Psen1-/- fibroblasts were treated with BAPTA-AM for 1.5 hours. Results from an experiment performed in triplicate is shown. The lower panel shows the blot reprobed for β-tubulin. All panels show representative blots from experiments that were performed multiple times. Western blots for HIF-1α were performed using a rabbit polyclonal antibody. The quantitative results are expressed as the ratio of HIF-1α to β-tubulin and are presented as + SEM. Asterisks in panels C and E indicate values that are different from the corresponding untreated cells (p < 0.05, unpaired t-tests). Other statistical comparisons are discussed in the text.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Quantitation Assay, Derivative Assay

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Reduced activation of HIF-1α downstream target genes in Psen1-/- fibroblasts treated with cobalt chloride . Psen1+/+ (wt) or Psen1-/- fibroblasts were treated with cobalt chloride for the indicated times (hours). Levels of Vegf (A) and Glut-1 (B) RNA were determined by qPCR (n = 9 cultures per group untreated; n = 3 per group treated). (*) indicates p < 0.05 and (**) p < 0.01 vs. untreated control by Dunnett's post-test. Statistical comparisons between groups are discussed further in the text.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Activation Assay

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Reintroduction of Psen1 into Psen1-/- fibroblasts . Psen1-/- fibroblasts were infected with lentiviruses carrying the human Psen1 cDNA (hPS1-LV) for 24 hours and grown in complete medium for 48 hours. The infected cells were then treated for 4 hours with 100 μM cobalt chloride along with non-infected Psen1-/- cells grown in parallel. The cells were lysed and analyzed for HIF-1α induction by Western blot using anti HIF-1α polyclonal antibodies. Blots were reprobed for anti β-tubulin as a loading control. Psen1 expression was analyzed using the NT.1 mouse monoclonal antibody. FL: full length, NTF: N-terminal fragment. A representative blot is shown in panel A. Panel B shows quantitation of the HIF-1α response (n = 3 samples per group non-infected; n = 4 infected). P values (unpaired t-tests) are indicated for selected comparisons which are discussed further in the text.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Infection, Western Blot, Expressing, Quantitation Assay

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Impaired induction of HIF-1α in immortalized mouse embryonic fibroblasts lacking both Psen1 and Psen2 . Wild type , Psen1-/- (PS1-/-) and Psen1/Psen2-/- (PS-/-) immortalized fibroblasts were treated with 100 μm cobalt chloride for 4 hours (A) or 2.4 μg/ml of insulin for 6 hours (B). Lysates were prepared and Western blotting was performed using an anti HIF-1α antibody. Lower panels show blots reprobed for β-tubulin. Asterisks (*) indicate lanes that were under loaded based on β-tubulin levels. Note the lowered induction of HIF-1α in the cells lacking both presenilins. Representative blots are shown from experiments that were performed multiple times.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: γ-secretase activity is not needed for insulin or cobalt induction of HIF-1α . Psen1+/+ immortalized fibroblasts (A) were treated overnight with the γ-secretase inhibitor XXI and then stimulated with insulin for 6 hours. Western blotting for HIF-1α was performed (upper panel) followed by reprobing of the blot for β-tubulin (middle panel). The same samples were then reblotted and probed for N-cadherin (lower panel). A band for the full length N-cadherin (FL) is indicated. A band that corresponds to the N-cadherin CTF is visible in the γ-secretase inhibitor treated lanes. In panel B, Psen1+/+ fibroblasts were treated overnight with the γ-secretase inhibitor L-685,458 and then stimulated with cobalt chloride for 6 hours. The samples were blotted and probed for HIF-1α (upper panel) and β-actin (middle panel) and then reblotted and probed for N-cadherin (lower panel). In panel C, the experiment in (A) was quantitated with the levels of HIF-1α normalized to β-tubulin. Analysis of the data in panel C (n = 3 per group) revealed significant increases in HIF-1α following treatment with insulin vs. untreated control (p = 0.004, unpaired t-test) or XXI treated vs. insulin + XXI (p = 0.01). There was no difference between insulin treated vs. insulin plus XXI treated cultures (p = 0.29) although XXI treatment alone modestly decreased HIF-1α levels vs. untreated control (p = 0.006). Representative experiments are shown. Asterisks (*) indicate values that are significantly different from corresponding samples that were not treated with insulin.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Activity Assay, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Normal activation by IGF-1 and insulin of PI3K/Akt in Psen1-/- fibroblasts . Immortalized fibroblast cell lines were treated with IGF-1 (A) or insulin (B) for the indicated times. Western blotting was performed for p-Akt (Ser473) followed by reprobing for total Akt. Panels show representative blots from experiments that were performed multiple times.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Activation Assay, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Basal levels of HIF-1α are decreased in Psen1-/- fibroblasts . Shown are prolonged exposures of Western blots from non-stimulated Psen1+/+ and Psen1-/- fibroblasts. A representative experiment that was performed multiple times is shown in panel A. In panel B, the experiment in (A) is quantified (asterisk indicates p = 0.0005, unpaired t-test). In panel C, levels of HIF-1α RNA were determined in Psen1 +/+ and Psen1-/- fibroblasts by qPCR (n = 3 cultures per group). Samples were run in triplicate and normalized to the geometric mean of Ppia and Gusb. Asterisk (*) indicates p = 0.0008.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Synthesis rates of HIF-1α are unchanged in Psen1-/- fibroblasts . Psen1+/+ (A) and Psen1-/- (B) fibroblasts were treated with the proteasome inhibitor MG132 for the indicated times (min). Lysates were prepared and Western blotting was performed using an anti-HIF-1α antibody. The lower panels show the blots reprobed for β-tubulin. In panels C and D, the rate of accumulation of HIF-1α normalized to β-tubulin levels from the experiments in panels A and B is shown either as the raw data (C) or normalized to basal levels of HIF-1α in the respective cell types (D). Note that although basal levels of HIF-1α are lower in Psen1-/- fibroblasts, HIF-1α accumulated at similar rates in Psen1+/+ and Psen1-/- fibroblasts. A representative example is shown from experiments that were performed multiple times.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Accelerated degradation of HIF-1α in Psen1-/- fibroblasts . Psen1+/+ and Psen1-/- fibroblasts were treated with cycloheximide. In panel A, a representative experiment is shown with cells treated for the indicated times. Due to the difference in basal HIF-1α levels between Psen1 +/+ and Psen1-/- cells, total protein loaded (15 μg for Psen1+/+ and 30 μg for Psen1 -/- ) and blot exposure times were adjusted so that the signal at time 0' would be of similar intensity in Psen1+/+ and Psen1-/- cells to facilitate comparison. In panel B, the % HIF-1α remaining at each time point normalized to β-tubulin is shown. Data is derived from the average of three independently performed experiments.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Derivative Assay

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Physical interaction between HIF-1α and Psen1 . Expression vectors carrying no insert, Psen1 or HIF-1α were transfected into HEK 293 cells in the indicated combinations. In panel A, immunoprecipitations (IP) were performed with a polyclonal anti-Psen1 antibody which recognizes the Psen1 NTF or with rabbit IgG as control and Western blotting was performed for HIF-1α. Total extracts without IP (-) (5% of input) were loaded in the two far right lanes (input). Note the co-immunoprecipitation of HIF-1α in the cultures co-transfected with HIF-1α and Psen1. A similar experiment was performed in (B), except that immunoprecipitations were performed with the monoclonal antibody NT.1 which recognizes the Psen1 NTF or with an antibody against the Psen1 loop region. In panel C, immunoprecipitation was done with a monoclonal HIF-1α antibody followed by Western blotting with an antibody against the Psen1 NTF (NT.1) or CTF (33B10). Panels labeled "none" show blots of total extracts prior to immunoprecipitation (input). Bands marked with an asterisk (*) are likely IgGs based on the rate of migration. Panel D shows the co-immunoprecipitation of endogenous HIF-1α by endogenous Psen1. Wild type fibroblasts were treated for 4 hours with 100 μM CoCl 2 and 10 μM MG132 to induce HIF-1α accumulation and co-immunoprecipitations were performed as in (A).

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Labeling, Migration

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Altered activation of HIF-1α in fibroblasts harboring the M146V PSEN1 FAD mutation . Fibroblast cell lines harboring human wild type Psen1 (HuPS1Wt) or the M146V FAD mutant both on the mouse Psen1-/- background were treated with cobalt chloride (A) or insulin (C) as in Figure 1 for the indicated times (hours). Representative blots are shown from experiments that were performed multiple times. The experiment in panel A is quantitated in panel B. In panel D, the insulin induction time course in panel C was quantitated. Asterisks (*) indicate p = 0.04 at 3 hours and p = 0.02 at 6 hours (unpaired t-tests).

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Activation Assay, Mutagenesis

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Basal levels of HIF-1α are unchanged in M146V FAD mutant fibroblasts . Shown are prolonged exposures of Western blots from the fibroblast lines studied in Figure 11. In panel A, representative blots are shown of samples from individual cultures harboring the human wild type Psen1 (HuPS1Wt) or the M146V FAD mutant. The lower panel shows the blot reprobed for β-tubulin. In panel B, levels of HIF-1α were determined by quantitative Western blotting (n = 5 cultures per genotype) with the levels of HIF-1α normalized to the levels of β-tubulin. Studies were performed both in the presence of serum (FBS) and after overnight serum starvation (-FBS). There were no statistically significant differences between the HuPS1Wt and M146V FAD mutant cell lines.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Mutagenesis, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Decreased insulin-induced activation of the insulin receptor, Akt and GSK-3β in fibroblasts harboring the M146V Psen1 FAD mutation . In panel A, human wild type Psen1 (HuPS1Wt) or M146V FAD mutant cell lines were serum starved overnight and then stimulated with insulin for the indicated times. Western blotting for the phosphorylated IR receptor β (Tyr 1150/1151) or total IR is shown in experiments run in duplicate. The pro-insulin receptor and processed IR are indicated. In panel B, duplicate experiments are shown following Western blotting for p-Akt (Ser473) and total Akt. p-Akt panels are shown at two different exposures. Lower panel shows the blot reprobed for β-tubulin as a loading control. In panel C, the extracts were blotted and probed for p-GSK-3β (Ser9) and total GSK-3β. In panels D, E and F the relative increase in p-IR/total IR (D), p-Akt/total Akt (E) and p-GSK-3β/total GSK-3β (F) are shown for the experiments in A-C.

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Activation Assay, Mutagenesis, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Altered induction of HIF-1α in Psen1-/- primary neuronal cultures . Primary neuronal cultures were prepared from E15.5-16 embryonic brains and maintained in Neurobasal medium/B27 supplement. Experiments were performed after 5-6 days in vitro . In panel A, cultures were treated with 100 μm cobalt chloride for the indicated times (hrs). In panels B and C, cells were cultured without B27 overnight and then treated with insulin (B) or IGF-1 (C) for six hours. In panel D, cells were cultured without B27 overnight and then treated with IGF-1 for the indicated times. Western blotting for HIF-1α was performed as in Figure 1 and the lower panels show the same blots reprobed for β-tubulin or actin. In panels E and H, the time course of the studies in (A) and (D) are quantitated. The insulin and IGF-1 studies in panels B and C are quantitated in (F) and (G). All panels show representative blots from experiments that were performed multiple times. Asterisks indicate p < 0.05 vs. untreated control (unpaired t-tests).

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: In Vitro, Cell Culture, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Activation of HIF-1α downstream target genes in Psen1-/- neurons treated with cobalt chloride or insulin . Levels of Vegf RNA were determined by qPCR following three hours of treatment of wild type or Psen1-/- primary neuronal cultures with cobalt chloride (A, n = 3 cultures per group) or seven hours with insulin (B, n = 2 cultures per group). Samples were run in triplicate and normalized to Ppia. Asterisks indicate p < 0.01 vs. untreated control (unpaired t-tests).

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Activation Assay

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Basal levels of HIF-1α RNA and protein are not significantly changed in Psen1-/- neurons . Basal HIF-1α RNA and protein were measured in neurons in the presence and absence of B27 supplement. In panel A, HIF-1α RNA was measured in neuronal cultures (n = 3/genotype) by qPCR. Samples were run in triplicate and normalized to the geometric means of Ppia and Gusb. In panel B, basal levels of HIF-1α protein were measured in neurons by Western blotting (n = 6 for cultures with B27 and n = 10 for B27 starved cultures). No statistically significant differences were found among the samples (unpaired t-tests). In panel C, representative Western blots are shown of cells cultured with B27 or without (-B27).

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Western Blot, Cell Culture

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: Normal activation by insulin of PI3K/Akt in Psen1-/- neurons . Primary neuronal cultures were treated with insulin for the indicated times. In panel A, Western blotting was performed for p-Akt (Ser473) followed by reprobing for total Akt. Shown are representative blots from experiments that were performed multiple times. In panel B, the time course of induction of p-Akt in primary neuronal cultures (n = 2 per genotype per time point) is shown as a ratio of p-Akt/total Akt. There were no significant differences at any time point (unpaired t-tests).

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Activation Assay, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Presenilin-1 regulates induction of hypoxia inducible factor-1α: altered activation by a mutation associated with familial Alzheimer's disease

doi: 10.1186/1750-1326-5-38

Figure Lengend Snippet: No change in HIF-1α levels in Psen1-/- embryonic brain . HIF-1α levels were determined by Western blotting of extracts from E14.5 (A) or E18.5 (B) brain from wild type (wt) or homozygous (-/-) Psen1 mutant mice. Representative experiments are shown. Lower panels show the same blots reprobed for β-tubulin. HIF-1α levels were quantitated as a ratio to β-tubulin at E14.5 (C) and E18.5 (D) (n = 4 wild type and 2 Psen1 -/- at each age). There were no differences between wild type (wt) and Psen1-/- at either age (unpaired t-tests).

Article Snippet: An expression ready plasmid for mouse HIF-1α (clone ID 4019056) was obtained from Open Biosystems (Huntsville, AL, USA) and for human Psen1 (clone sc125532) from Origene (Rockville, MD, USA).

Techniques: Western Blot, Mutagenesis