Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: RT-PCR of human and murine megakaryocytes and platelets. (A) Murine RT-PCR studies of total RNA from (1) megakaryocyte, (2) platelets, (3) WBCs, (4) NIH-3T3 cells, known to express LRP1,20 and (5) water for LRP1 (first gel), PF4 (second gel), and ITGB2 (third gel). Expected band size is approximately 400 base pair (bp) for LRP1 and ITGB2, and approximately 300 bp for PF4. White space indicates where lanes were removed for ease of presentation. (B) Human RT-PCR studies of total RNA from (6) megakaryocytes, (7) platelets, (8) WBCs, (9) 293T cells, embryonic kidney line known to express LRP1,21 (10) water, and (11) no reverse transcriptase controls for LRP1 (first gel), PF4 (second gel), and ITGB2 (third gel).

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: Flow cytometry and Western blot of human and murine megakaryocytes and platelets. (A) Representative examples of flow cytometry of murine bone marrow–derived megakaryocytes (top) stained with a biotin labeled anti-hLRP1 antibody known to cross-react with mouse LRP131 and then stained with streptavidin, PE–Alexa 647 secondary antibody. The gray line represents unstained cells. The broken black line represents secondary antibody alone. The solid black line is megakaryocytes with both antibodies. The bottom graph shows flow cytometry of platelets similarly performed. (B) As in panel A but for human cultured megakaryocytes and human peripheral blood platelets. LRP1 antibody was directly labeled with Alexa 647 for these experiments. As in panel A, the solid gray line represents unstained cells. The broken black line represents cells with isotype control antibody. The solid black line represents cells stained with the LRP1 antibody. (C) Western blot for LRP1 and actin as a control for protein loading. (1) Megakaryocytes, (2) platelets, and (3) WBCs. LRP1 band is expected at approximately 85 kDa and actin at approximately 25 kDa.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Flow Cytometry, Western Blot, Derivative Assay, Staining, Labeling, Cell Culture

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: In vitro studies of the effect of RAP and anti-LRP1 antibodies on megakaryopoiesis. (A) The effect of RAP on megakaryocyte colony formation. GST indicates empty GST without conjugated RAP. Graphed is the mean percentage of megakaryocytes per well plus 1 SD. Number of experiments, each performed in duplicate, is indicated in each bar. *P = .004 versus WT cultures without PF4; **P < .003 compared with WT culture with PF4. (B) The effect of anti-LRP1 antibody (MA5A6). Ig is isoimmune control for the anti-LRP1 antibody. Mean percentage of megakaryocytes per well plus 1 SD is graphed. Number of experiments done in duplicate is indicated in each bar. *P = .004 compared with WT without PF4; **P < .003 compared with WT with PF4; ***P = .04 compared with WT without PF4.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: In Vitro

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: shRNA suppression of LRP1 and megakaryocyte colony formation. (A) Representative flow cytometry of 3T3 cells (positive control for LRP1) stably transfected with different shRNA viral vectors. The solid gray line represents cells that were unstained. The broken gray line is LRP1 expression on cells expressing the empty lentiviral vector. Solid black line shows the decrease in surface LRP1 expression after stable transfection with the LRP1 shRNA virus. (B) Same as panel A except for murine bone marrow cells after culture in media containing TPO and puromycin for 5 days. The solid gray line represents isotype control. The broken gray line is cells transfected with the negative viral vector. The solid dark line represents cells transfected with LRP1 shRNA. (C) Quantitation of change in mean fluorescence index (MFI) in murine bone marrow cells transfected with virus. Data represent mean +1 SD for 3 independent experiments. Viral titers were between 1 to 2 × 1011 viral particles/mL. (D) Effect of LRP1 shRNA on megakaryopoiesis (meg) using mPF4−/− bone marrow and hPF4High bone marrow expressed relative to megakaryopoiesis with the control empty vector. ■ is relative level of megakaryocyte seen after transfection with the empty lentiviral vector, and □ is relative level after transfection with the LRP1 shRNA vector. Data represent mean +1 SD for 4 experiments, each performed in duplicate. *P < .006 for LRP1 versus negative control for hPF4. shRNA had no effect on colony formation in mPF4−/− bone marrow. (E) Effect of LRP1 shRNA megakaryocyte colony formation in mPF4−/− bone marrow treated with exogenous PF4 (25 μg/mL). Percentage of meg colonies were normalized as in panel D. Ctl indicates control studies with empty vector. Data represent mean +1 SD. Numbers in bars represent times experiments were performed (each in duplicate). *P < .008 for LRP1 versus empty virus in the presence of PF4.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: shRNA, Flow Cytometry, Positive Control, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Fluorescence, Negative Control

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: Effect of PF4 on G1ME cells and expression of LRP1. (A) The effect of PF4 on percentage of CD42+ cells after re-expression of GATA-1 by the introduction of a GATA-1-IRES-eGFP MIGR1 retrovirus with and without 25 μg/mL PF4 in serum-free media. Results are for eGFP+-transfected cells. On the left are total CD42+ cells; middle are small, CD42+ cells; and right are large, CD42+ cells (based on forward scatter on flow cytometry). Data are shown as mean +1 SD of 4 experiments. *P < .008 comparing with and without PF4 added. (B) LRP1 expression in G1ME cells both before and after transfection with either a MIGR1 empty retrovirus (□) or MIGR1 retrovirus containing GATA-1 (♦). Figure organized as in panel A. Shown is a representative experiment of 3. (C) LRP1 expression on human cultured megakaryocytes derived from adult CD34+ bone marrow cells. Open triangles show total CD41+ cells, whereas closed triangles show LRP1+/CD41+ cells. Mean +1 SD is shown for 4 independent experiments. (D) Ploidy analysis in relation to LRP1 expression. The gray line represents cells that are CD41−. The broken line is cells that are CD41+ but LRP1−. The solid, dark line represents the cells that are positive for both LRP1 and CD41. Data are from a single experiment, but are representative of results from 5 independent experiments.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Derivative Assay

Journal: Journal of Diabetes Investigation

Article Title: Imeglimin improves hyperglycemia and hypoglycemia‐induced cell death and mitochondrial dysfunction in immortalized adult mouse Schwann IMS32 cells

doi: 10.1111/jdi.70092

Figure Lengend Snippet: Cell viability in IMS32 cells treated with or without 100 μM imeglimin under various glucose conditions. Cell viability was determined by (a) cell count, and (b) MTT assay. Cytotoxicity was determined by (c) the released LDH. (d) To evaluate the effect of osmotic changes on cell viability, MTT assay was performed using 2.5 mM glucose (LG) supplemented with 3 mM mannitol to match the osmolarity of NG (MLG), and 5.5 mM glucose (NG) supplemented with 19.5 mM mannitol to match the osmolarity of HG (MHG). Cells were exposed to NG (5.5 mM glucose), LG (2.5 mM glucose), and HG (25 mM glucose) medium conditions for 1 h. Imeglimin was added to the culture medium for 24 h. Values are presented as mean ± SE ( n = 4‐10). * P < 0.05, ** P < 0.01 compared with NG. # P < 0.05, ## P < 0.01 compared with IME (−) control.

Article Snippet: Imeglimin hydrochloride was acquired from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Cell Counting, MTT Assay, Control

Journal: Journal of Diabetes Investigation

Article Title: Imeglimin improves hyperglycemia and hypoglycemia‐induced cell death and mitochondrial dysfunction in immortalized adult mouse Schwann IMS32 cells

doi: 10.1111/jdi.70092

Figure Lengend Snippet: Cytochrome c distribution in IMS32 cells treated with or without 100 μM imeglimin under high‐ and low‐glucose conditions. (a) Immunostaining was performed with specific anti‐cytochrome c antibody. (b) Protein expression of cytochrome c in mitochondrial and cytosol fractions under various glucose conditions. Normalization was carried out using Cox IV and β‐actin as a loading control. The density of cytochrome c bands in mitochondrial fractions and c cytosolic fractions were measured and the ratio was calculated. Cells were exposed to NG (5.5 mM glucose), LG (2.5 mM glucose), and HG (25 mM glucose) medium conditions for 1 h before measurement. Imeglimin was added to the culture medium for 24 h. Value are presented as mean ± SE ( n = 3). * P < 0.05 compared to 5.5 mM glucose. ## P < 0.01 compared with IME (−) control.

Article Snippet: Imeglimin hydrochloride was acquired from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Immunostaining, Expressing, Control

Journal: Journal of Diabetes Investigation

Article Title: Imeglimin improves hyperglycemia and hypoglycemia‐induced cell death and mitochondrial dysfunction in immortalized adult mouse Schwann IMS32 cells

doi: 10.1111/jdi.70092

Figure Lengend Snippet: Mitochondrial oxidative stress in IMS32 cells treated with or without 100 μM imeglimin under high‐and low‐glucose conditions. (a) Representative images of MitoSOX Red‐stained cells were obtained using a confocal microscope. (b) Quantitative analysis of mitochondrial ROS levels under various glucose conditions. Cells were exposed to NG (5.5 mM glucose), LG (2.5 mM glucose), and HG (25 mM glucose) medium conditions for 1 h before measurement. Imeglimin was added to the culture medium for 24 h. Values are presented as mean ± SE ( n = 6). * P < 0.05 compared with NG, ## P < 0.01 compared to IME (−) control.**P < 0.01 compared with NG, ##P < 0.01 compared to IME (−) control.

Article Snippet: Imeglimin hydrochloride was acquired from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Staining, Microscopy, Control

Journal: Journal of Diabetes Investigation

Article Title: Imeglimin improves hyperglycemia and hypoglycemia‐induced cell death and mitochondrial dysfunction in immortalized adult mouse Schwann IMS32 cells

doi: 10.1111/jdi.70092

Figure Lengend Snippet: Mitochondrial function in IMS32 cells treated with or without 100 μM imeglimin under high‐ and low‐glucose conditions. (a) Mitochondrial membrane potential, (b) Oxygen consumption rate (OCR), (c) Complex I activity, (d) ATP levels. Cells were exposed to NG (5.5 mM glucose), LG (2.5 mM glucose), and HG (25 mM glucose) medium conditions for 1 h before measurement. Imeglimin was added to the culture medium for 24 h. Values are presented as mean ± SE ( n = 3–6). * P < 0.05, ** P < 0.01 compared with NG. # P < 0.05, ## P < 0.01 compared with IME (−) control.

Article Snippet: Imeglimin hydrochloride was acquired from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Membrane, Activity Assay, Control

Journal: Journal of Diabetes Investigation

Article Title: Imeglimin improves hyperglycemia and hypoglycemia‐induced cell death and mitochondrial dysfunction in immortalized adult mouse Schwann IMS32 cells

doi: 10.1111/jdi.70092

Figure Lengend Snippet: Schematic representation of mitochondrial dysfunction under hyperglycemic and hypoglycemic conditions and the protective effects of imeglimin. Hyperglycemia and hypoglycemia impair mitochondrial function by increasing ROS production, disrupting the mitochondrial membrane potential, reducing ATP synthesis, and inducing apoptosis. Imeglimin alleviates these dysfunctions by suppressing excessive Complex I activity, reducing ROS production, stabilizing mitochondrial membrane potential, and mitigating ATP depletion and apoptosis.

Article Snippet: Imeglimin hydrochloride was acquired from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Membrane, Activity Assay

Journal: Neuron

Article Title: Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex

doi: 10.1016/j.neuron.2019.01.027

Figure Lengend Snippet:

Article Snippet: Agonists table ft1 table-wrap mode="anchored" t5 Agonist Product number Concentration in pipette for Ca imaging Final concentration in Polaris Concentration in pipette for slice recordings NMDA / Gly Sigma M3262 / G6761 100 mM / 1 mM 1 mM / 10 µM 100 mM / 1 mM AMPA Tocris 0169 10 mM 100 µM 10 mM Acetylcholine Sigma A6625 10 mM 100 µM 10 mM GABA Sigma A2129 100 mM 1 mM 100 mM TCB-2 Tocris 2592 10 mM 1 mM 10 mM MeSADP Tocris 1624 10 mM 100 µM 10 mM Ionomycin Sigma I3909 10 µM - - Open in a separate window Antagonists table ft1 table-wrap mode="anchored" t5 Antagonist Product number Bath Concentration DNQX Sigma D0540 20 µM EMD281014 Tocris 4470 200 µM Suramin Tocris 1472 100 µM D-APV Tocris 0106 100 µM Carbenoxolone Tocris 3096 100 µM BMI Sigma B7561 20 µM Open in a separate window QUANTIFICATION AND STATISTICAL ANALYSIS Specific statistical tests are described in the relevant sections.

Techniques: Concentration Assay

Journal: Neuron

Article Title: Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex

doi: 10.1016/j.neuron.2019.01.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Agonists table ft1 table-wrap mode="anchored" t5 Agonist Product number Concentration in pipette for Ca imaging Final concentration in Polaris Concentration in pipette for slice recordings NMDA / Gly Sigma M3262 / G6761 100 mM / 1 mM 1 mM / 10 µM 100 mM / 1 mM AMPA Tocris 0169 10 mM 100 µM 10 mM Acetylcholine Sigma A6625 10 mM 100 µM 10 mM GABA Sigma A2129 100 mM 1 mM 100 mM TCB-2 Tocris 2592 10 mM 1 mM 10 mM MeSADP Tocris 1624 10 mM 100 µM 10 mM Ionomycin Sigma I3909 10 µM - - Open in a separate window Antagonists table ft1 table-wrap mode="anchored" t5 Antagonist Product number Bath Concentration DNQX Sigma D0540 20 µM EMD281014 Tocris 4470 200 µM Suramin Tocris 1472 100 µM D-APV Tocris 0106 100 µM Carbenoxolone Tocris 3096 100 µM BMI Sigma B7561 20 µM Open in a separate window QUANTIFICATION AND STATISTICAL ANALYSIS Specific statistical tests are described in the relevant sections.

Techniques: Plasmid Preparation, RNAscope, HD Assay, RNA Sequencing Assay, Software

Journal: Oncogene

Article Title: FOXC2 promotes colorectal cancer metastasis by directly targeting MET.

doi: 10.1038/onc.2014.368

Figure Lengend Snippet: Figure 4. FOXC2 activates MET signaling pathway in CRC cells. (a) RNA from three separate experiments was extracted and analyzed using a whole-genome expression microarray. Gene expression was analyzed using Ingenuity Pathway Analysis. The signaling pathways activated by FOXC2-overexpressing in SW480 cells were shown. (b) Heatmap analysis showing part of the genes regulated by FOXC2 as identified by microarray profiling. (c) Protein–protein interaction network showing MET is in the central role of regulating the proteins upregulated by FOXC2. (d) Western blot analysis of the expression levels of indicated genes in the indicated CRC cell lines. (e) Real-time RT-PCR analyses of MET expression in the indicated cells. (f) SW480 cells were transiently transfected with the indicated amounts of pBabe/FOXC2. After 48 h, the expression of FOXC2, MET and p-MET was determined by western blotting (left), and the expression of MET mRNA was analyzed by real-time RT-PCR (right).

Article Snippet: MET inhibitor treatment We treated the SW480 cells with MET inhibitor (EMD 1214063, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a concentration of 0.5 μmol/l.

Techniques: Expressing, Microarray, Gene Expression, Protein-Protein interactions, Western Blot, Quantitative RT-PCR, Transfection

Journal: Oncogene

Article Title: FOXC2 promotes colorectal cancer metastasis by directly targeting MET.

doi: 10.1038/onc.2014.368

Figure Lengend Snippet: Figure 6. Inhibition of MET reduces the invasive and metastatic ability of FOXC2-overexpressing cells. (a, b) Western blotting analyzed the expression of the indicated proteins in SW480 (a) or HCT15 (b) cells with indicated treatments. (c, d) Mice were orthotopically transplanted with indicated cells (n = 6 in each group). Representative gross images of the intestines, livers and lungs were shown. Sections of the liver and lung were stained with hematoxylin and eosin (H&E). Arrows show the primary tumors in the intestines, and macrometastases or micrometastases in the liver and lungs. The numbers of liver and lung macrometastases or micrometastases were determined using a dissection microscope. Error bars represent mean ± s.d. **Po0.001.

Article Snippet: MET inhibitor treatment We treated the SW480 cells with MET inhibitor (EMD 1214063, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a concentration of 0.5 μmol/l.

Techniques: Inhibition, Western Blot, Expressing, Staining, Dissection, Microscopy