Journal: PLoS ONE

Article Title: Early Treatment with Fumagillin, an Inhibitor of Methionine Aminopeptidase-2, Prevents Pulmonary Hypertension in Monocrotaline-Injured Rats

doi: 10.1371/journal.pone.0035388

Figure Lengend Snippet: (A) Primary RPASMC were serum-starved in basal medium for 24 h to synchronize all cells in G0. Cells were then released from G0 by addition of 10% FBS supplemented with or without PDGF (10 ng/mL) or fumagillin (20 nM). Cells were fixed after 0, 1, 2, and 3 days of culture and then stained with SYBR Green as described in . Fluorescent intensity, which correlates with relative cell number, was measured by a fluorimetric plate reader. Data represent mean + SEM, n = 4. At days 2 and 3, fumagillin significantly reduced cell number in cells incubated in the absence of PDGF (* P <0.01) or in the presence of PDGF (** P <0.001). (B–C) RPASMC were incubated in the absence of serum followed by addition of serum (B) or serum with PDGF (10 ng/mL) (C) with or without fumagillin at the indicated concentrations. Cells were incubated for an additional 48 h and pulsed with BrdU for the final 30 minutes then fixed. BrdU+ cells were quantified as described in . Data represent mean + SEM. Data analyzed by ANOVA followed by Tukey's multiple comparison test (* P <0.05, fumagillin v vehicle AND fumagillin + PDGF v. vehicle + PDGF). (D) Immunoblotting for MetAP2 of RPASMC cultured in the presence of 10% serum and exposed to MetAP2-targeting siRNA (lanes 1 and 2) or non-targeting RNA oligonucleotides (lanes 3 and 4). Cells in lanes 1 and 3 were incubated with the vehicle control, and cells in lanes 2 and 4 were incubated with fumagillin 20 nM. Note the increased band intensity of MetAP2 in the fumagillin conditions. (E) Incorporation of BrdU in RPASMC during silencing of MetAP2 gene expression by siRNA. During suppression of MetAP2 gene expression, BrdU incorporation was decreased 27% compared to the non-targeting control (** P <0.05, ANOVA followed by Neuman-Keuls post-hoc test compared to Non-targeting + VEH). In the presence of fumagillin, silencing of MetAP2 did not lead to additional inhibition of proliferation.

Article Snippet: SYBR Green dye and Rabbit IgG against MetAP2, and Alexa Fluor 488–conjugated goat anti-mouse IgG were from Invitrogen (Carlsbad, CA).

Techniques: Staining, SYBR Green Assay, Incubation, Western Blot, Cell Culture, Expressing, BrdU Incorporation Assay, Inhibition

Journal: PLoS ONE

Article Title: Early Treatment with Fumagillin, an Inhibitor of Methionine Aminopeptidase-2, Prevents Pulmonary Hypertension in Monocrotaline-Injured Rats

doi: 10.1371/journal.pone.0035388

Figure Lengend Snippet: RPASMC were incubated in basal medium with 0.5% FBS. Cells were then stimulated with PDGF (10 ng/mL) for 18 hours. Cells were lysed in detergent and subjected to SDS-PAGE and immunoblotting for MetAP2 and Actin. (A) A representative immunoblot showing an increase in MetAP2 expression by PDGF. (B) Quantitative densitometry was performed as described in and expressed as fold-increase compared to cells incubated in the absence of PDGF (mean + SEM, n = 3,* P <0.03). Data were analyzed by paired t-test.

Article Snippet: SYBR Green dye and Rabbit IgG against MetAP2, and Alexa Fluor 488–conjugated goat anti-mouse IgG were from Invitrogen (Carlsbad, CA).

Techniques: Incubation, SDS Page, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Early Treatment with Fumagillin, an Inhibitor of Methionine Aminopeptidase-2, Prevents Pulmonary Hypertension in Monocrotaline-Injured Rats

doi: 10.1371/journal.pone.0035388

Figure Lengend Snippet: Immunohistochemistry for MetAP2 was performed on formalin-fixed paraffin-embedded samples. Sections were counterstained with hematoxylin. Representative sections are shown, n = 3: (A) normal human lung (B) Pulmonary arterial hypertension ( inset non-immune rabbit IgG) (C) normal rat lung (D) MCT-injured rat lung ( inset non-immune rabbit IgG). Magnification ×400, bar = 20 µm. Note staining for MetAP2 in endothelial cells (black arrows) and cells in the medial layer (yellow arrows).

Article Snippet: SYBR Green dye and Rabbit IgG against MetAP2, and Alexa Fluor 488–conjugated goat anti-mouse IgG were from Invitrogen (Carlsbad, CA).

Techniques: Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Staining

Journal:

Article Title: Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival

doi:

Figure Lengend Snippet: Bcl-2 overexpression and zVAD-fmk protect mesothelioma cells from caspase 3 activation and apoptosis induced by MetAP2 inhibition. A: HM cells were exposed to either vehicle or 0.5 μg/ml of fumagillin for 96 hours either in the presence or in the absence of 100 nmol/L of zVAD-fmk. Nucleosome formation was determined as indicated in Materials and Methods. Results are the mean ± SD from four separate experiments. B: HM cells, HM-PKneo, or HM-Bcl-2.2 clones were incubated with 100 nmol/L of MetAP2 anti-sense oligonucleotide either in the presence or the absence of 100 nmol/L of zVAD-fmk. Caspase 3 activity was determined as indicated in Materials and Methods.

Article Snippet: These were incubated for a minimum of 2 hours with either a monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology) or with a rabbit polyclonal anti-MetAP2 antibody (CM33) (Zymed Inc., Histo-Line Laboratories, Milan, Italy) and subsequently exposed to a horseradish peroxidase-conjugated secondary antibody.

Techniques: Over Expression, Activation Assay, Inhibition, Clone Assay, Incubation, Activity Assay

Journal:

Article Title: Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival

doi:

Figure Lengend Snippet: MetAP expression in normal and malignant mesothelial cells. A: Total RNA, extracted from HUVEC (lane 1), three mesothelioma cell lines (MM-B1, MM-F1, and HM) (lanes 2 to 4), and three primary normal mesothelial cells (NM-1, NM-2, and NM-3) (lanes 5 to 7), was subjected to Northern blotting using specific probes for MetAP1 and MetAP2. β-actin was used as an internal control. B: Densitometric analysis of results in A. C: Forty μg of lysates from cells listed in A were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using a specific MetAP2 polyclonal antibody. The MetAP-2 protein was visualized as a band of 67 kd. β-Actin was used as a control for equal protein loading.

Article Snippet: These were incubated for a minimum of 2 hours with either a monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology) or with a rabbit polyclonal anti-MetAP2 antibody (CM33) (Zymed Inc., Histo-Line Laboratories, Milan, Italy) and subsequently exposed to a horseradish peroxidase-conjugated secondary antibody.

Techniques: Expressing, Northern Blot, Polyacrylamide Gel Electrophoresis

Journal:

Article Title: Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival

doi:

Figure Lengend Snippet: Impact of MetAP2 anti-sense oligonucleotide on mesothelioma cell viability and nucleosome formation. A: Mesothelioma cells (HM) were incubated with increasing concentrations of either a specific MetAP2 anti-sense or a scrambled oligonucleotide. After 24 hours total RNA was extracted and blotted with a specific probe for MetAP2. MetAP2 protein levels were determined by Western blotting after 48 hours exposure to the oligonucleotides. β-actin was used as an internal control. B: HM cells were treated for different periods of time with either lipofectin alone (mock) or with 100 nmol/L of the MetAP2 anti-sense or of a scrambled oligonucleotide. At the end of the incubation, cell viability was assessed by trypan blue exclusion. Data points depict mean values ± SD from two experiments with quadruplicate determinations (*, P = 0.02; **, P < 0.001). C: Normal mesothelial (NM-1) and HM cells were treated as indicated in the legend to B. Nucleosome formation in the presence of lipofectin alone, the MetAP2 anti-sense, or the scrambled oligonucleotide was determined after 96 hours using the cell death detection ELISAPLUS kit as indicated in the Materials and Methods section. Results are the average ± SD from three experiments with duplicate determinations. (*, P = 0.018).

Article Snippet: These were incubated for a minimum of 2 hours with either a monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology) or with a rabbit polyclonal anti-MetAP2 antibody (CM33) (Zymed Inc., Histo-Line Laboratories, Milan, Italy) and subsequently exposed to a horseradish peroxidase-conjugated secondary antibody.

Techniques: Incubation, Western Blot

Journal:

Article Title: Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival

doi:

Figure Lengend Snippet: MetAP2 inhibition reduces Bcl-2 expression in mesothelioma cells. A: Total RNA was extracted from malignant (HM) and normal (NM-1) mesothelial cells treated with either 0.5 μg/ml of fumagillin or 100 nmol/L of MetAP2 anti-sense oligonucleotide for varying periods of time and hybridized with bcl-2-, bax-, and β-actin-specific probes. RNA from HeLa cells expressing bcl-2 and bax was used as a positive control (+). Results are from a representative experiment (n = 3). B: Bcl-2 protein levels (open circle) and nucleosome formation (filled circle) were determined in fumagillin-treated HM cells as described in Experimental Procedures. Data points represent the mean ± SD from three separate experiments with duplicate determinations. (*, P < 0.05; **, P < 0.001).

Article Snippet: These were incubated for a minimum of 2 hours with either a monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology) or with a rabbit polyclonal anti-MetAP2 antibody (CM33) (Zymed Inc., Histo-Line Laboratories, Milan, Italy) and subsequently exposed to a horseradish peroxidase-conjugated secondary antibody.

Techniques: Inhibition, Expressing, Positive Control

Journal:

Article Title: Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival

doi:

Figure Lengend Snippet: MetAP2 inhibition decreases telomerase activity in mesothelioma cells. A: Mesothelioma (HM) cells were exposed to either 0.5 μg/ml of fumagillin or 100 nmol/L of MetAP2 anti-sense for the indicated periods of time. Telomerase activity in cell extracts (0.1 μg) was determined as described in Experimental Procedures. Values are expressed as percentage of the telomerase activity present in untreated or scrambled oligo-treated HM cells (white bar) and represent the mean ± SD from three experiments with duplicate determinations (*, P = 0.015; **, P < 0.001). B: TRAP assay of mesothelioma (HM) and normal mesothelial (NM-2) cell extracts from cultures treated for 96 hours with either vehicle or 0.5 μg/ml of fumagillin. Negative control refers to HM cell extracts that were incubated with 200 μg/ml of RNase before the TRAP assay. Positive control was 0.02 μg of HeLa cell extract. Results are from one experiment representative of three.

Article Snippet: These were incubated for a minimum of 2 hours with either a monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology) or with a rabbit polyclonal anti-MetAP2 antibody (CM33) (Zymed Inc., Histo-Line Laboratories, Milan, Italy) and subsequently exposed to a horseradish peroxidase-conjugated secondary antibody.

Techniques: Inhibition, Activity Assay, TRAP Assay, Negative Control, Incubation, Positive Control

Journal:

Article Title: Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival

doi:

Figure Lengend Snippet: Schematic representation of intracellular pathways controlled by MetAP2. MetAP2 positively regulates Bcl-2 expression. This, on one side contributes to the up-regulation of telomerase activity in the nucleus, and on the other inhibits caspase activation. On the other hand, telomerase activity down-regulates caspase activity. Whether telomerase activity may have also an impact on Bcl-2 expression in this model remains to be established.

Article Snippet: These were incubated for a minimum of 2 hours with either a monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology) or with a rabbit polyclonal anti-MetAP2 antibody (CM33) (Zymed Inc., Histo-Line Laboratories, Milan, Italy) and subsequently exposed to a horseradish peroxidase-conjugated secondary antibody.

Techniques: Expressing, Activity Assay, Activation Assay

Journal: medRxiv

Article Title: The Proteome Landscape of Human Placentas for Monochorionic Twins with Selective Intrauterine Growth Restriction

doi: 10.1101/2022.08.29.22278892

Figure Lengend Snippet: A . IPA identified the “angiogenesis” annotation to be affected in IUGR twin placentas. B . Immunostaining of CD34 in human placentas from IUGR twins and normal cotwins. CD34 is limited to endothelial cells. Scale bar, 50 μm. C . Placental MVD in IUGR twins and normal cotwins. D . Placental vascular area density in IUGR twins and normal cotwins. E . Immunohistochemical images of EFNB2, VIM, and METAP2 in placental shares from IUGR twins and normal cotwins. Scale bar, 100 μm. F – H . EFNB2, VIM, and METAP2 immunostaining intensity per tissue area is represented by the fold-decrease relative to the normal control mean. I – K . qRT-PCR analysis showed that the EFNB2, VIM , and METAP2 transcriptional levels were significantly lower in placentas of IUGR twins compared with normal cotwins. All data are shown as mean ± SD with 6 samples per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. MVD, microvessel density; NC, negative control; SD, standard deviation.

Article Snippet: After deparaffinization, antigen retrieval and endogenous peroxide blocking, the sections (5 μm in thickness) were incubated with primary anti-CD34 antibody (Catalog No. ZM-0046, ZSGB-BIO, Beijing, China), anti-EFNB2 antibody (1:50; Catalog No. HPA008999, Atlas Antibodies, Bromma, Sweden), anti-METAP2 antibody (1:3000; Catalog No. HPA019095, Atlas Antibodies), anti-VIM antibody (Catalog No. ZM-0260, ZSGB-BIO) and anti-MTDH antibody (1:150; Catalog No. ab124789, Abcam, Cambridge, UK).

Techniques: Immunostaining, Immunohistochemical staining, Control, Quantitative RT-PCR, Negative Control, Standard Deviation