streptomycin  (Thermo Fisher)

Journal: PLoS ONE

Article Title: Sirtuin 1 Facilitates Generation of Induced Pluripotent Stem Cells from Mouse Embryonic Fibroblasts through the miR-34a and p53 Pathways

doi: 10.1371/journal.pone.0045633

Figure Lengend Snippet: SIRT1 expression during iPSC formation and differentiation of ESCs and iPSCs in mouse model. (A) Temporal expression of Sirt1 mRNA on day 0 to day 20 after DOX treatment. MEF without DOX on day 20 (20-DOX) and mESC were included. (B) Western blotting analysis of SIRT1, OCT4 and PCNA in 2°F/1B MEF without (-DOX) and with (+DOX) DOX treatment for 15 days, serially passaged iPSC from passages 4 (P4), 5–7 (P5, P6, P7) and differentiated colonies at passage 4 (Diff-P4). (C) The relative expression levels of SIRT1 protein in MEF, miPSC and mESC. (D) Relative SIRT1 protein expressions in embryoid bodies collected from mESC and miPSC on days 2, 5, 8, 11, 14 and 17 after differentiation. D0 are the undifferentiated cell control. *p

Article Snippet: L4 and miPSC were cultured in mESC medium [DMEM with high glucose, 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco, Life Technologies), 0.1 mM MEM non-essential amino-acids (Gibco), sodium pyruvate (110 mg/L, Gibco), 50 µM beta-mercaptoethanol, 15% FBS (Gibco) and 1000 units/ml LIF (Millipore).

Techniques: Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: Analysis of cell spreading following PTP-PEST targeting in fibroblasts. Both PTP-PEST (+/−) and (−/−) were allowed to spread on fibronectin-coated tissue culture dishes, and the number of spread cells was assayed after 10, 15, and 30 min. Phase-contrast of random fields after 10 (a and b) and 30 (c and d) min are shown for PTP-PEST (+/−; a and c) and PTP-PEST (−/−; b and d) cells. PTP-PEST (−/−) cells showed significantly higher cell spreading. A quantitative evaluation was expressed as the number of cells spread as a percentage of the total number of cells in the field (e). Average number of cells per field: 300; n = 8. Error bars represent the standard deviation of the mean. Bar, 200 μm.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: Cytokinesis defect in PTP-PEST (−/−) cells. Unsynchronized PTP-PEST (−/−) cells were plated on uncoated tissue culture glass slides and stained using rhodamine-conjugated phalloidin to highlight the actin-rich cleavage furrows (a). An antiphosphotyrosine immunostaining of cells in the same population is shown in b. Bar, 30 μm.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Staining, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: Constitutive hyperphosphorylation of PSTPIP in unsynchronized PTP-PEST (−/−) cells. PSTPIP was immunoprecipitated from PTP-PEST (+/−) and PTP-PEST (−/−) cell lysates and probed with a HRP-conjugated antiphosphotyrosine antibody (top). PSTPIP was hyperphosphorylated in the (−/−) cells. (Bottom) Anti-PSTPIP blot of the same membrane to ensure equal loading of the protein.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: Gene targeting of the PTP-PEST suppresses fibroblast motility on the extracellular matrix fibronectin. Monolayers of each cell line were wounded (a and b) and maintained at 37°C for 24 h before fixing (c and d). The ability to migrate into the wound was monitored by phase-contrast microscopy of unstained cells which were photographed (×100). The aspect of each wound represents the typical result obtained after five independent experiments. In a chamber-type assay (e), the bottom side of the polycarbonate membrane was coated with fibronectin, and 10 5 cells were added to the top chamber. After 5 h, the cells that translocated to the bottom side of the membrane were counted and the result is shown as an average of eight fields from four independent experiments (error bars: standard deviation). PTP-PEST (−/−) cells stably overexpressing wild-type PTP-PEST were also tested by the same method. (Inset) Western blotting against PTP-PEST in the two cell lines. The band that appears represents overexpression since the antibody is known not to detect endogenous levels of PTP-PEST. Bar, 200 μm.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Microscopy, Standard Deviation, Stable Transfection, Western Blot, Over Expression

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: Actin and focal adhesions staining in PTP-PEST (+/−) (a, b, e, and f) and (−/−) (c, d, g, and h) cells plated on fibronectin. After 20 min (a–d), there are no qualitative differences on the actin filaments, stained using a rhodamine-phalloidin conjugate (a and c) or in focal adhesions, stained with an antivinculin antibody and highlighted using a FITC-conjugated second antibody (b and d). However, when the cells were left for 3 h before fixing, the (+/−) cells became rounded (e) and only formed punctual focal adhesions at their periphery (f), whereas the (−/−) cells continued to spread, forming numerous stress fibers (g), and numerous focal adhesion plaques scattered throughout their ventral surface (h). The number of focal adhesions after 3 h in each cell was counted from photographs and the results are shown in i. Bar, 20 μm.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: (a) Constitutive hyperphosphorylation of FAK and paxillin in PTP-PEST (−/−) cells. Left column shows extent of tyrosine phosphorylation of immunoprecipitates as detected by the 4G10 antiphosphotyrosine mAb; right column shows evaluation of the amount of protein loaded using the same antibody as the immunoprecipitation. (b) Rescue of p130 CAS hyperphosphorylation in PTP-PEST (−/−) overexpressing wild-type PTP-PEST. p130 CAS was already shown to be a substrate for PTP-PEST ( Garton et al., 1996 ) and to be hyperphosphorylated in the PEST (−/−) cells ( Côté et al., 1998 ). Left panel shows phosphorylation levels of p130 CAS in each cell line; right panel shows loading control.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: Constitutive interactions of FAK, paxillin, and p130 CAS with the SH2-domains of Crk, Grb2, and Src in binding assays. TNE cell lysates were prepared from exponentially growing PTP-PEST (+/−) and PTP-PEST (−/−) cells. Lysates were incubated with the indicated GST fusion proteins coupled to glutathione-agarose as described in Materials and Methods. The precipitates were analyzed by sequential stripping and immunoblotting of the same membrane with the indicated antibodies.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Binding Assay, Incubation, Stripping Membranes

Journal: The Journal of Cell Biology

Article Title: Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts

doi:

Figure Lengend Snippet: PTP-PEST translocates to the membrane periphery following fibronectin stimulation of COS-1 cells. a, c, e, and g are phase-contrast images while b, d, f, and h are HA epitope indirect immunofluorescence. (a and b) Untransfected cells. (c and d) Transfected, unstimulated cells. (e and f) Transfected cells starved for 16 h in 0.1% serum, then stimulated for 10 min with 100 ng/ml of EGF. (g and h) Transfected cells plated on fibronectin slides for 45 min before fixing and staining (see Materials and Methods). The arrow in h shows an example of membrane periphery translocation of the immunofluorescence staining. Bar, 20 μm.

Article Snippet: Stable Overexpression of PTP-PEST in PTP-PEST (−/−) Cells Full-length, wild-type PTP-PEST was inserted in a vector containing a selectable Zeocin resistance marker, pcDNA3.1/Zeo (+) (Invitrogen Corp.).

Techniques: Immunofluorescence, Transfection, Staining, Translocation Assay

Journal: Respiratory Research

Article Title: Characterization of proximal pulmonary arterial cells from chronic thromboembolic pulmonary hypertension patients

doi: 10.1186/1465-9921-13-27

Figure Lengend Snippet: Proximal pulmonary vascular cell proliferating and migrating capacity . (A) Mitogenic activity of proximal PASMC isolated from lung donors (n = 4), non-thromboembolic PH (n = 4) and CTEPH patients (n = 4). Subconfluent cells were starved for 24 h in 0.2% FBS medium. Mitogenic activity was measured in the presence of 0.2% (control) or 10% FBS plus 0.5 μCi.mL -1 of [ 3 H]-thymidine for 36 h. ANOVA, p = 0.02; *p

Article Snippet: Cell culture media EC were cultured in M199 medium (Life Technologies, Gent, Belgium), supplemented with 20% fetal bovine serum (FBS), 100 U.mL-1 penicillin, 100 μg.mL-1 streptomycin, 1.25 μg.mL-1 fungizone (Life Technologies), 10 U.mL-1 heparin (Aventis, Brussels, Belgium) and 5 ng.mL-1 α-FGF (R & D Systems, Abington, UK).

Techniques: Activity Assay, Isolation

Journal: bioRxiv

Article Title: Homing Choices of Breast Cancer Cells Revealed by Tissue Specific Invasion and Extravasation Lab-on-a-chip Platforms

doi: 10.1101/2020.09.25.312793

Figure Lengend Snippet: Extravasation of metastatic breast cancer cells into lung, liver and breast microenvironments. a) 3D schematic of the EX-chip. Zoom-in image of 3D EX-chip showing the side 2 view (Cancer cells: Red, Endothelial cells: Green, Direction of extravasation: Blue arrow). b) Representative Z-stack images showing side 2 views of endothelial monolayer formation by HUVEC-C cells (green) and extravasated (arrow head) or associated (arrow) MDA-MB-231 cells (red) into lung, liver or breast microenvironments generated by WI-38, BRL-3A and MCF-10A cell lines, respectively (Scale bar: 200 μm). c) The number of extravasated and associated MDA-MB-231 cells. Each black dot represents the cell number for one post gap within the EX-chip, while the red dot is the average number of cells for each condition (n=9).

Article Snippet: MDA-MB-231, its derivatives and MCF-7 were cultured in DMEM high glucose (11965092, Gibco) with Fetal Bovine Serum (FBS, 10%) (A3840001, Gibco) and Penicillin/Streptomycin (15070063, Gibco, 1%); MCF-10A was cultured in DMEM-F12 high glucose (11330057, Gibco) with Horse Serum (04-004-1A, Biological Industries, 5%), Insulin (I9278, Sigma, 10 μg mL-1 ), Cholera Toxin (C8052, Sigma, 100 ng mL-1 ), EGF (E9644, Sigma, 20 ng mL-1 ), Hydrocortisone (H0888, Sigma, 0.5 μg mL-1 ) and Penicillin/Streptomycin (1%).

Techniques: Chromatin Immunoprecipitation, Multiple Displacement Amplification, Generated