Journal:

Article Title: Molecular blockade of VEGFR2 in human epithelial ovarian carcinoma cells

doi: 10.1038/labinvest.2010.52

Figure Lengend Snippet: Increased VEGF-A production in suspension conditions by VEGFR2 OVCAR-3 knockdown cells. (a) ELISA for VEGF-A (Mean ± SEM) detected no change in the amount of VEGF produced by the three OVCAR-3 transfected constructs growing in adhesive monolayer culture. The middle graph shows a significant increase in the production of VEGF-A by shRNAKDR1 clones growing in suspension and the bottom graph shows a significant increase in VEGF-A production by spheroids formed from shRNAKDR1 cells compared shRNAKDR5 and shKDRGAPDH. (b) Short term blockade of VEGFR2 signaling in OVCAR-3 cells with pharmacological inhibitor ZM323881 lead to reduced expression of VEGFR2, NRP-1 and VEGF. (c) Proliferation assay using AlamarBlue (Mean ± SEM) demonstrates significant cell growth in shRNAKDR1 cells compared to other clones. (d) Upper panel shows ascites (OVCAR-3 cells) collected from the intraperitoneal cavity of mice; bottom panel shows western blot of collected cells, demonstrating the stability of VEGFR2 knockdown in the recovered ascites cells. (e) ELISA results (Mean ± SEM) showing higher VEGF-A production in shRNAKDR1 ascites supernatant and lysed cells recovered from intraperitoneal cavities of injected mice, compared to samples from other stable lines.

Article Snippet: For short-term inhibition of VEGFR2 signaling, the small molecule tyrosine kinase inhibitor ZM323881 hydrochloride (Tocris Bioscience, Ellisville, MS, USA) was used as previously reported ( 21 ).

Techniques: Suspension, Knockdown, Enzyme-linked Immunosorbent Assay, Produced, Transfection, Construct, Adhesive, Clone Assay, Expressing, Proliferation Assay, Western Blot, Injection

Journal: Frontiers in Endocrinology

Article Title: Culture in Glucose-Depleted Medium Supplemented with Fatty Acid and 3,3′,5-Triiodo- l -Thyronine Facilitates Purification and Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.3389/fendo.2017.00253

Figure Lengend Snippet: High purity of cardiomyocytes can be obtained under glucose-depleted and fatty acid (with or without T 3 )-supplemented conditions. (A) The schematic for purification and maturation using metabolic selection. Glycogen synthase kinase 3 inhibitor CHIR99021 and Wnt pathway inhibitor IWP2 (gray boxes) were added to the differentiation medium on differentiation days 1 and 4, respectively. On differentiation day 10, cells were dissociated and plated on glass cover slips. After 3 days recovery, cells were cultured with metabolic selection medium (No-glucose DMEM medium supplemented with lactate, fatty acid, and fatty acid + T 3 ). On differentiation day 18, cells were collected for patch clamping experiments. On differentiation day 21, cells were collected for other experiments. (B) Representative fluorescence-activated cell sorting (FACS) analyses of cardiac Troponin T expression in the human pluripotent stem cell (hPSC)-derived cells on day 0 with routine culture medium, and on day 8 with metabolic selection medium. In the control group, the isotype control (mouse IgG) was used instead of the primary antibody. (C) Time courses of selection efficiency using the lactate-supplemented condition (red), the fatty acid-supplemented condition (green), and the fatty acid with T 3 -supplemented condition (purple); cells cultured with routine medium (blue) were set up as the control groups ( n = 3). The asterisk means each test group is significantly different from controls, P < 0.01.

Article Snippet: Briefly, hPSC were treated with small molecule CHIR99021 (Tocris, 4423, final concentration 10 μM) in the RPMI-BSA medium [RPMI 1640 Medium (HyClone, SH30027.01) supplemented with 213 μg/ml AA2P ( l -ascorbic acid 2-phosphate magnesium) (A8960, Sigma) and 0.1% bovine serum albumin (BSA) (A1470, Sigma)] for 24 h, then were incubated with RPMI-BSA medium for 48 h. On differentiation day 4, cells were treated with the small molecule IWP2 (Tocris, 3533, final concentration 5 μM) in RPMI-BSA medium.

Techniques: Purification, Selection, Cell Culture, Fluorescence, FACS, Expressing, Derivative Assay, Control

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: SMER28 delays progression of HCMV infection. HFFs were infected with TB40E at an MOI of 0.5 at the time of replating (A), and HAECs were infected with TB40E at an MOI of 3 1 day after replating (B). A total of 25 or 50 μM SMER28 or DMSO (0) for HFFs or 50 μM SMER28 or DMSO (0) for HAECs was added to the medium at the time of infection. Cell supernatants (left) and cell pellets (right) were harvested at 24, 72, and 120 hpi. Infectious virions were measured by a plaque assay. Fold changes indicate differences between treatments with 50 μM SMER28 and DMSO at the time points shown. The dashed line indicates the limit of detection of each plaque assay.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Plaque Assay

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: SMER28 reduces levels of early and late viral proteins in HFFs and HAECs. HFFs (A) or HAECs (B) were infected with TB40E at an MOI of 0.5. SMER28 (25 or 50 μM) or DMSO (0) was added to the medium at the time of infection. Cells were harvested at 24, 72, and 120 hpi and processed for Western blotting with antibodies against the IE1 72 and IE2 86 proteins, UL44, UL57, pp28, the late IE2 proteins IE2 60 and IE2 40, and tubulin as a loading control.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Western Blot, Control

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: SMER28 but not trehalose has a differential effect on viral DNA synthesis in HFFs and HAECs. HFFs (A and B) or HAECs (C and D) were infected with TB40E at an MOI of 0.5. A total of 50 or 100 mM trehalose (A and C) or 25 or 50 μM SMER28 or DMSO (B and D) was added to the medium at the time of infection. Cell pellets were harvested at the indicated times p.i. DNA was extracted from cells, and viral genome copy numbers relative to cellular GAPDH were determined by quantitative real-time PCR using primers against unspliced UL77 and are displayed as genome copies normalized to values for untreated samples at 24 hpi. Fold changes indicate differences between untreated samples and samples treated with the highest concentration of the drug.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: DNA Synthesis, Infection, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: SMER28 treatment blocks production of DNA-filled capsids in the nucleus and mature virions in the cytoplasm and does not induce cytoplasmic vacuolation in HAECs at 120 hpi. HAECs were infected with TB40E at an MOI of 3 in the presence of 50 μM SMER28. Cells were fixed with 2% glutaraldehyde and processed for electron microscopy at 120 hpi. Boxes in the left panels indicate regions of zoom (right panels). Black bars (B to D), 1 μm. Black arrows (D) indicate B capsids.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Electron Microscopy

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: SMER28 does not induce vacuolation of the cytoplasm in infected HFFs. HFFs were infected with TB40E at an MOI of 1 in the presence of DMSO or 50 μM SMER28, as indicated. Cells were fixed with 2% glutaraldehyde and processed for electron microscopy at 96 hpi. Boxes in the left images (C and F) indicate regions of enlargement in the right images (D, E, G, and H). A, B, and C capsids in panels E and H are indicated by white arrowheads, black arrows, and white arrows, respectively.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Electron Microscopy

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: SMER28 treatment blocks production of DNA-filled capsids in the nucleus and mature virions in the cytoplasm and does not induce cytoplasmic vacuolation in HAECs. HAECs were infected with TB40E at an MOI of 3 in the presence of 50 μM SMER28. Cells were fixed with 2% glutaraldehyde and processed for electron microscopy at 96 hpi. Boxes in the left images (C and F) indicate regions of enlargement in the right images (D, E, G, and H). A, B, and C capsids in panels E and H are indicated by white arrowheads, black arrows, and white arrows, respectively.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Electron Microscopy

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: SMER28 treatment modestly increases autophagy in uninfected and infected HAECs but not in HFFs. HFFs or HAECs were mock infected (M) or infected with TB40E at an MOI of 3 (V). A total of 50 μM SMER28 or DMSO was added to the medium at the time of infection. Cells were harvested at 24 and 72 hpi and processed for Western blotting with antibodies against LC3B and tubulin as a loading control. In HFF Western blots, a repeated sample was used to calibrate blotting and exposures between the 2 different gels. Both short and long exposures of LC3B are shown.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Western Blot, Control

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: Trehalose, but not SMER28, induces acidification of cytoplasmic compartments. HFFs (A) or HAECs (B) were mock infected or infected with TB40E at an MOI of 3 in the presence of 50 μM SMER28 or 100 mM trehalose or were left untreated and plated onto coverslips at the time of infection. At 96 hpi, cells were stained with LysoTracker and fixed. Nuclei were counterstained with Hoechst 33342 dye. Bars, 50 μm.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Staining

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: Trehalose, but not SMER28, induces a diffuse localization of Rab7 in infected HFFs. HFFs were mock infected (Mock) or infected with TB40E at an MOI of 0.5 (Virus) in the presence of 50 μM SMER28 or 100 mM trehalose or were left untreated and plated onto coverslips at the time of infection. At 96 hpi, coverslips were fixed and processed for immunofluorescence analysis using antibodies against IE1 72 and Rab7. Nuclei were counterstained with Hoechst 33342 dye. Images of representative cells are shown. Bars, 50 μm.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Infection, Virus, Immunofluorescence

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

doi: 10.1128/JVI.02015-17

Figure Lengend Snippet: Model for inhibition of HCMV infection by trehalose and SMER28. (A) Diagram of the viral life cycle showing gene expression stages and viral envelopment and egress involving Rab11-dependent trafficking. (B) Trehalose treatment in HFFs and HAECs interferes with the HCMV life cycle by reducing late protein levels and shifting intracellular traffic from the plasma membrane to lysosomes associated with decreased Rab11 and increased Rab7 levels. (C) SMER28 treatment of HFFs blocks expression of early and late proteins, and few enveloped virions are produced. (D) SMER28 treatment of HAECs interferes with early protein expression and viral genome replication, highlighting a cell type specificity in the stage at which HCMV replication is inhibited. DNA rep, DNA replication.

Article Snippet: Culture medium for each cell type was supplemented with SMER28 (Tocris) prepared in DMSO and added to the medium at the concentrations indicated in the figure legends.

Techniques: Inhibition, Infection, Gene Expression, Clinical Proteomics, Membrane, Expressing, Produced

Journal: The Journal of Biological Chemistry

Article Title: Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

doi: 10.1016/j.jbc.2021.101097

Figure Lengend Snippet: The roles of phosphatases PTPN2 and PTPN22 in restraining GITR signaling. A , splenic T cells isolated from littermate control (LMC) or T-Traf3 −/− mice were pretreated with DMSO ( upper-left ), inhibitors (5 μM) of PTPN2 (SF1670, upper-right ), PTPN22 (LTV-1, bottom-left ) or ERK (U0126) ( bottom-right ) before being stimulated via GITR for the indicated times. Whole cell lysates were subjected to SDS-PAGE and Western blot analysis. Representative blots of four similar experiments are shown. B–D , quantification of four independent experiments including representative blots shown in A . Curves represent mean ± SEM of relative band intensities, where statistical significance was determined by two-way ANOVA. ∗ p < 0.05; ∗∗ p < 0.01; n.s indicates no significant difference. E , subclones of HuT28.11 or Traf3 −/− HuT28.11 T cells expressing hCD40-GITR (FLAG-tagged) were stimulated via anti-hCD40 Ab-conjugated protein G beads (see ) for times indicated and the hCD40-GITR signaling complex was immunoprecipitated. IPs ( left ) were subjected to SDS-PAGE and Western blotting for TRAF3, PTPN2 and PTPN22. Blots of whole cell lysates are shown on the right . Ctrl = IP with protein G beads conjugated with isotype control mAb. Positions of TRAF3 and PTPN2 bands are indicated by arrows . ∗ indicates the heavy chain of the IP Ab remaining in the IP sample. Blots in E are representative of ≥ 3 similar experiments. Quantification of band intensity is summarized in Fig. S4 .

Article Snippet: The small-molecule inhibitors for PTPN2 (SF1670) and PTPN22 (LTV-1) were purchased from Tocris Bioscience.

Techniques: Isolation, Control, SDS Page, Western Blot, Expressing, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

doi: 10.1016/j.jbc.2021.101097

Figure Lengend Snippet: Model: TRAF3 suppresses GITR expression and signaling in T cells. GITR transcription is stringently regulated by activity of NFκB2. In normal T cells ( left panel ), the major upstream kinase for activation of NFκB2, NIK is constitutively polyubiquitinated (represented as chain of black dots attached to NIK) and targeted for degradation by an E3 ubiquitination complex containing TRAF2, TRAF3, and cIAP. Engagement of GITR with GITRL leads to recruitment of TRAF3 and disassembly of the TRAF2/TRAF3/cIAP E3 complex, which releases NIK and activates downstream NFκB2. In addition, phosphatases PTPN2 and PTPN22 attenuate the signaling strength of the GITR complex independent of TRAF3. Negative regulation of GITR signaling by TRAF3 may also involve competition with TRAF2 and TRAF5 ( yellow and blue ovals ), both of which can promote GITR signaling . In the absence of TRAF3 ( right panel ), NFκB2 is constitutively active and upregulates GITR transcription. Increased levels of the GITR receptor and lack of TRAF3 regulation of the GITR signaling cascade lead to elevated GITR signaling in TRAF3-deficient T cells.

Article Snippet: The small-molecule inhibitors for PTPN2 (SF1670) and PTPN22 (LTV-1) were purchased from Tocris Bioscience.

Techniques: Expressing, Activity Assay, Activation Assay, Ubiquitin Proteomics

Journal: Cell reports

Article Title: Live Imaging Reveals that the First Division of Differentiating Human Embryonic Stem Cells Often Yields Asymmetric Fates.

doi: 10.1016/j.celrep.2017.09.044

Figure Lengend Snippet: Figure 2. Innate Resistance to WNT Signaling in One Daughter Cell Leads to Asymmetric Primitive Streak Specification in Daughter Cell Pairs (A) Overview of the WNT pathway. (B) Stills taken from live imaging of hESCs differentiating toward primitive streak (top); staining of fixed cells reveals asymmetric LEF1 protein expression (bottom). Scale bar represents 10 mm. (C) Ratio of LEF1 protein expression in pairs of daughter cells derived from hESCs exposed to primitive streak differentiation media (ACTIVIN, BMP4, FGF2, and WNT3A); the ratio of LEF1 expression is plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric daughter cell pairs. (D) Live imaging of hESCs carrying a Tcf/Lef-TurboGFP:NLS:d2PEST reporter allele revealed that upon treatment with primitive streak differentiation media (ACTIVIN, BMP4, FGF2, and WNT3A), WNT/b-catenin signaling is often only activated in one cell in a daughter cell pair. Scale bar represents 10 mm. (E) Ratio of Tcf/Lef-TurboGFP fluorescence in pairs of daughter cells, plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric daughter cell pairs. (F) MIXL1-GFP hESCs differentiated in primitive streak differentiation media (ACTIVIN, BMP4, FGF2) containing the GSK3 inhibitor CHIR99021 (3 mM) still differentiated through asymmetric divisions, as shown by still images from live imaging. Immunostaining of fixed cells revealed that the MIXL1-GFP+ daughter also expressed BRACHYURY protein. Scale bar represents 5 mm. (G and H) Ratio of MIXL1-GFP (G) and BRACHYURY (H) protein expression in pairs of CHIR99021-treated daughter cells, plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric divisions. Data are representative of three to five independent experiments.

Article Snippet: Atop this minimal primitive streak induction condition, WNT3A protein (made in house from WNT3A-producing L cells), RSPO2 protein (10–20 ng/mL; R&D Systems), and/or the small-molecule WNT agonist CHIR99021 (3 mM; Tocris) were added as indicated to potentiateWNT signaling and to drive primitive streak specification.

Techniques: Imaging, Staining, Expressing, Derivative Assay, Immunostaining