small molecule inhibitor ml141  (BioMimetic Therapeutics)

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: Inhibition of Cdc42 in 3D biomimetic angiogenic model. (A) Schematic of our 3D biomimetic model of angiogenesis. A device is consisted of 2 channels fully embedded inside 2.5mg/ml collagen gel. (B) Cdc42 activity was reduced in half in the presence of 15 μM Cdc42 inhibitor ML141. (C) Representative phase images of sprouts guided by a gradient of angiogenic cocktail including MCP-1, VEGF, PMA, and S1P at Day 4 for control DMSO and Cdc42-inhibited devices. Average invading distance of invading cells into matrix was reduced in the presence of ML141 (N=4 individual experiments); * (p<0.05) indicates statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Inhibition, Activity Assay, Control

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: The effects of Cdc42 on sprout length and density during angiogenesis sprouting. (A) Quantification of sprout density between control DMSO and Cdc42 inhibition conditions. ML141 was initiated at onset of sprouting over a course of 4 days (n=4 individual experiments). The presence of Cdc42 inhibitor slightly decreased the sprout density. (B) Sprout length was quantified at day 4 using images acquired from confocal microcopy. Sprout length was halved when Cdc42 activity was partially inhibited (n=4 individual experiments). (C) Quantification of average sprout angle between DMSO and ML141 devices (n=4 individual experiments) revealed unaltered directional migration of the multicellular sprout structures. (D) Quantification of the number of invading cells demonstrated inhibition of Cdc42 reduced migrating cells into the interstitial matrix (n=4 individual experiments). (E) Representative 3D projections of Z-stack confocal images of sprouts in DMSO and ML141 conditions at day 4. White arrowheads indicate single migrating cells. Scale bar is 100 μm. (F) Quantification of single cell migration among migrating cells in the interstitial matrix revealed a significant increase in the fraction of single migrating cells (n=4 individual experiments). Unit area is 300 μm2. * indicates statistical significance (P<0.05); ns indicates no statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Control, Inhibition, Activity Assay, Migration

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: The effects of antagonizing Cdc42 on branching morphogenesis of angiogenic sprouting. (A) A schematic of two different branching structures (branch and intersegmental branch) observed in angiogenic sprouting in our model guided by a gradient of angiogenic cocktail. (B) Number of branch points is quantified for DMSO vs ML141 conditions. (C) The fraction of sprouts with branches was reduced in the presence of ML141. (D) The fraction of sprouts with intersegmental branches was also reduced when Cdc42 activity was perturbed with ML141. (E) Average length of branch was unaffected by the inhibition of Cdc42. (F) Average length of intersegmental branches was also unaffected by the inhibition of Cdc42. N=4 individual experiments; * (p<0.05) indicates statistical significance; ns indicates no statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Activity Assay, Inhibition

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: Filopodia formation of endothelial cell sprouting upon Cdc42 inhibition. (A) Representative confocal images of phalloidin-stained sprout tip cells showing filopodia-like extensions in DMSO and ML141 conditions. Sprouting was initiated for 3 days. Then 22.5μM ML141 was added for 4hrs before fixation. (B) Average angle of filopodia of sprout tip cells remained unchanged upon inhibition of Cdc42 (n=4 individual experiments). (C) The number of filopodial extensions per sprout tip cells in DMSO and ML141 conditions (n=4 individual experiments) displayed a surge in filopodia-like extension in ML141 treatment. (D) Histogram showing distribution of the filopodia-like extension numbers per sprout tip cells for DMSO and ML141 conditions (n=4 individual experiments). (E) Average length of filopodia-like extensions is quantified for DMSO and ML141 conditions (n=4 individual experiments). (F) Histogram showing distribution of the length of the filopodia-like extensions for DMSO and ML141 conditions. * (p<0.05) and *** (p< 0.001) indicate statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Inhibition, Staining

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: Cdc42 is required for early divisions and PMC organization in the mesenchyme blastula. A–E) S. purpuratus eggs were injected with mRNAs encoding WT or DN-Cdc42, and embryos were scored for developmental progression 24 h post-fertilization when embryos normally reach the mesenchyme blastula stage (A–E; Bar, 50 μm). Embryos were scored as mesenchyme blastula (1A, blue); blastula (1B, green); embryos where PMCs had ingressed but were scattered within the blastocoel (1C, pink); embryos with abnormal ectodermal epithelium (1D, gray); or embryos with cytokinetic defects (1E, yellow). F) Expression of DN-Cdc42 resulted in an increase in cell division defects (1E and F, yellow bars) as well as defects in PMC retention at the vegetal pole, with disorganized cells distributed throughout the blastocoel (1C; F, pink bars). Each experimental condition represents six experimental replicates, with a minimum of 145 embryos scored per condition. * p < 0.05; *** * p < 0.0001.

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Injection, Expressing

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: Archenteron elongation and PMC organization are disrupted upon depletion of Cdc42. A–E) S. purpuratus eggs were injected with untargeting control or Cdc42 morpholinos, and embryos were scored for developmental progression 48 h post-fertilization, when embryos normally reach the gastrula stage (Bar, 50 μm). Embryos were scored as gastrulae (with archenterons extended at least 75% of total length and spicules present, 2A, blue); mid-gastrulae, with elongating archenteron and spicules (2B, green); embryos with a primary invagination and a lack of organized PMCs (2C, orange); embryos containing disorganized PMCs and lacking an archenteron (2D, pink); and embryos displaying both disrupted mesenchymal cells and epithelia (2E, gray). F) Quantification of phenotypes in embryos injected with control or Cdc42 antisense morpholinos at 48 h post-fertilization. While control embryos were in the gastrula or mid-gastrula stages, in embryos injected with 500 μM MASO showed defects in PMC organization and archenteron elongation (2H, pink bars). These defects were rescued with co-injection of human Cdc42. Each experimental condition represents at least three experimental replicates, with a minimum of 100 embryos per condition. *** p < 0.001; ns: p = 0.9765.

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Injection, Control

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: Cdc42 activity is required for correct PMC migration, filopodia formation and initiation of skeletogenesis. A) Cdc42 activity assay. Lysates from gastrulae treated with carrier control (0.1%) DMSO or the Cdc42 inhibitor ML141 were incubated with PAK beads, and Cdc42 bound to the beads or present in unfractionated lysates was detected by Western blotting. B–U) L. variegatus embryos were treated with 5 μM ML141 at the blastula stage, and then fixed and processed for immunolabeling with PMC-specific (6a9, green) and anti-actin (magenta) antibodies or analyzed live by polarization microscopy. Maximum intensity projections of Lateral (L) or Vegetal (V) views of control or ML141-treated embryos revealed differences in PMC morphology and migration. Control embryos exhibited PMCs organized in ventrolateral clusters (VLCs) (3B, arrows) and in a ring around the vegetal pole (3G, arrow), with extended filopodia contacting the ectoderm (3E and J, arrows). Inhibition of Cdc42 resulted in PMCs organized around VLCs and the vegetal ring, but lacked filopodia (3O and T, arrows), and the tight organization of controls (3L and Q). Imaging by polarization microscopy revealed the presence of birefringent, tri-radiate spicules in controls (3K) but not detectable in ML141-treated embryos (3U). Bar, 50 μm.

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Activity Assay, Migration, Control, Incubation, Western Blot, Immunolabeling, Microscopy, Inhibition, Imaging

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: PMC syncytium formation and skeletogenesis requires Cdc42 activity. A–D) L. variegatus embryos were treated with ML141 at the gastula stage (A), and embryos were scored for developmental progression when controls reached the prism/early larval stage. D) Quantification of phenotypes represented in B and C for three experimental replicates, with an average of 250 embryos scored per condition per experiment. ***, p < 0.0003; *** *, p < 0.0001. E) L. variegatus embryos with treated with 5 μM ML141 at the gastrula stage, and then fixed and processed for immunolabeling with PMC-specific (6a9, green) and anti-actin (magenta) antibodies or analyzed live by polarization microscopy. Control embryos presented a well-formed common synctytium (panels a–d) and larval skeleton (panel f), whereas PMCs in embryos incubated with ML141 failed to form a common syncytium (panels g–j). Deposition of skeletal material was limited to what was generated at the time of treatment (panel l). Bar, 50 μm.

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Activity Assay, Immunolabeling, Microscopy, Control, Incubation, Generated

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: PMCs resume skeletogenesis upon reversal of Cdc42 inhibition. Viability of PMCs following ML141 treatment was confirmed by treating embryos at the blastula stage with 2.5 μM ML141 and then releasing embryos from Cdc42 inhibition. As viewed by polarization microscopy, ML141-treated embryos that failed to initiate spiculogenesis by 24 h were able to reinitiate skeletogenesis upon removal of ML141 (5 M, arrows). Arrowhead denotes the presence of an ectopic spicule. Bar, 50 μm.

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Inhibition, Microscopy

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: Live-cell imaging of filopodia in the presence or absence of Cdc42 activity. L. pictus gastrulae expressing GFP-Lifeact were imaged by brightfield and confocal microscopy. A) Control embryos form dynamic filopodia along long the length of the spicule (pink and yellow arrowheads), at the distal tips (red arrowhead), and in contacts with non-skeletogenic mesenchymal cells (green arrowhead). Bar, 50 μm. B and C) Observation of ventro-lateral clusters (VLCs) at early maturation stages showed PMCs associated with small tri-radiate spicules (6B, panels d and e, black arrows) and later to the growing spicules (6B, panels c and f). In all stages abundant filopodia extended from the PMCs and spicule rods (6B, panels a–c, white arrows). Embryos incubated with 10 μ ML141 for 20 min exhibited PMCs associated with skeletal rods but with decreased filopodia (6 C, panels a–d) compared to controls (6B, panels a–c). Longer treatments resulted in stronger effects on PMCs organization and filopodia processes, and loss of syncytial cytoplasm associated with the spicule rods (6C, panels e and f). D) Control S. purpuratus gastrulae expressing GFP-Lifeact exhibited dynamic filopodia processes elaborated from PMCs migrating along the ectoderm (6D, panel a, arrows) as well as from the basal membrane of ectodermal cells (6D, panel a, red arrowheads). Treatment with ML141 for 2.5 h inhibited filopodia formation in PMCs (6D, panel b, arrows), whereas ectodermal filopodia were unaffected by Cdc42 inhibition (6D, panel b, red arrowheads). Bar, 25 μm.

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Live Cell Imaging, Activity Assay, Expressing, Confocal Microscopy, Control, Incubation, Membrane, Inhibition

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: Contribution of Cdc42, formins and Arp2/3 in sea urchin PMC filopodia formation. A) L. pictus embryos were treated at the blastula stage with either 0.1% DMSO, 100 μM Arp2/3 inhibitor (CK666, ) or 10 μM formin inhibitor (SMIFH2) for 24 h before being fixed and processed for immunolabeling with PMC-specific (6a9, green) and anti-actin (magenta) antibodies or analyzed live by polarization microscopy. Bar, 50 μm. B) L. pictus gastrulae expressing GFP-Lifeact were treated with 0.1% DMSO, 100 μM CK666 or 50 μM SMIFH2 and imaged by confocal microscopy. Live-cell imaging of PMCs in ventrolateral clusters from L. pictus gastrulae revealed that embryos treated with Arp2/3 and formin inhibitors did not exhibit the dynamic filopodia observed in controls. Bar, 25 μm. C–E) Isolated PMCs from L. variegatus embryos were treated with Cdc42 (10 μM ML141), Arp2/3 (100 μM CK666) and formin (5 μM SMIFH2) inhibitors, and cultures were fixed, probed with 6a9 and the number and length of filopodia were quantified for 30 cells/experiment, 3 experimental replicates. *** p = 0.0001; *** * p < 0.0001. Bar, 10 μm.

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Immunolabeling, Microscopy, Expressing, Confocal Microscopy, Live Cell Imaging, Isolation

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: L. variegatus qPCR Primers

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Sequencing, Ubiquitin Proteomics

Journal: Developmental biology

Article Title: Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo

doi: 10.1016/j.ydbio.2018.03.015

Figure Lengend Snippet: Morpholino sequences

Article Snippet: To block Cdc42 activity, embryos were cultured in the presence of the Cdc42 small molecule inhibitor ML141 (Tocris).

Techniques: Sequencing, Control