Journal: Cell Death and Differentiation

Article Title: Endothelial cells-derived SEMA3G suppresses glioblastoma stem cells by inducing c-Myc degradation

doi: 10.1038/s41418-025-01534-3

Figure Lengend Snippet: A The ubiquitination level of c-Myc in GSC07 cells treated with or without ML141 (10 μmol/l) for 24 h. B , C c-Myc level in T98G cells transfected with GFP-Tagged Cdc42 WT , Cdc42 T17N or Cdc42 Q61L . D Schematic diagram depicting the plasmid construction for bimolecular fluorescence complementation assays. E Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between Cdc42 and WWP2 . F HEK-293 cells were transfected with CrN173-tagged Cdc42 T17N or Cdc42 Q61L , and VC155-tagged WWP2 for 48 h. CFP signal that represents Cdc42 binding to WWP2 were determined using confocal. Scale bar, 5 μm. G HEK-293 cells were transfected with Flag-tagged c-Myc, HA-tagged ubiquitin, MYC-tagged WWP2 and GFP-tagged Cdc42 T17N or Cdc42 Q61L for 48 h. The ubiquitin level of c-Myc and the binding of Cdc42 to WWP2 were determined by co-immunoprecipitation. H Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between WWP2 and Cdc42 or c-Myc. I HEK-293 cells were incubated with or without SEMA3G (200 ng/ml) for 48 h after co-transfected with CrN173-tagged Cdc42 WT or VN173-tagged c-Myc and VC155-tagged WWP2 for 12 h. CFP signal that represents Cdc42 binding to WWP2 and Venus signal that represents WWP2 binding to c-Myc were captured using confocal. Scale bar, 5 μm. J HEK-293 cells that were co-transfected with GFP-tagged Cdc42 WT , MYC-tagged WWP2, Flag-tagged c-Myc, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The binding of Cdc42 WT to WWP2 and the ubiquitination level of c-Myc were determined by co-immunoprecipitation. K HEK-293 cells were co-transfected with GFP-tagged Cdc42 Q61L and MYC-tagged WWP2, Flag-tagged c-MYC, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The protein level of c-Myc and ubiquitination level of c-Myc were determined by co-immunoprecipitation. L Schematic diagram illustrating the potential mechanism by which Cdc42 regulates WWP2 binding to c-Myc and promotes c-Myc degradation. All the western blot bands represent one of the three independent experiments.

Article Snippet: USA), or ML141 (MedChemExpress, NJ, USA) according to the experimental design.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Binding Assay, Immunoprecipitation, Incubation, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Apolipoprotein E promotes white matter remodeling via the Dab1‐dependent pathway after traumatic brain injury

doi: 10.1111/cns.13298

Figure Lengend Snippet: ApoE induces the activation of Dab1 and Cdc42 after CCI. (A) Representative bands and quantitative analysis of the ratio of phosphorylated Dab1(pDab1) at 3 days after brain injury. Dab1 phosphorylation was significantly suppressed in the APOE −/− group compared to that in the APOE +/+ group, whereas RAP, a competitive receptor blocker known to strongly inhibit ligand binding to ApoE receptors, abolished this difference. (B) The level of Dab1 in APOE +/+ mice was suppressed by Dab1 shRNA lentivirus in the APOE +/+ +Dab1 shRNA group. Following injury, a higher level of GAP43 was observed in the APOE +/+ group than in the APOE −/− group. However, GAP43 levels were lower in the APOE +/+ +Dab1 shRNA group than in the APOE +/+ group. (C) The ratio of GTP‐Cdc42/Cdc42 at 3 days following brain injury was measured. The level of activated Cdc42 was significantly higher in the APOE +/+ group than in the APOE −/− group. However, compared with the level in the APOE +/+ group, activated Cdc42 was lower in the APOE +/+ +Dab1 shRNA group, in which Dab1 was downregulated. (D) ML141 (a Cdc42 activation inhibitor) treatment showed a similar effect as that of Dab1 shRNA. GAP43 levels were lower in the ML141 treatment group than in the APOE +/+ group. The expression levels of each protein were normalized to those in the APOE −/− group. N = 6/group. *, P < .05

Article Snippet: ML141 (Selleckchem) at 10 mg/kg body weight was dissolved in dimethylsulfoxide (DMSO) and was intraperitoneally injected at 1‐7 days postinjury.

Techniques: Activation Assay, Phospho-proteomics, Ligand Binding Assay, shRNA, Expressing

Journal: Nature communications

Article Title: Polarized NHE1 and SWELL1 regulate migration direction, efficiency and metastasis.

doi: 10.1038/s41467-022-33683-1

Figure Lengend Snippet: Fig. 5 | Cdc42 facilitates the reversal of migration direction by controlling NHE1 repolarization. a Migration velocity and b fraction of vehicle control and ML141- treated MDA-MB-231 cells displaying negative velocity before (gray) and after (red) hypotonic shock (165 mOsm/l) at the leading edge in confinement. Data represent the a mean ± SD for the indicated number of cells or b mean ± SEM from 3 (a) or 4 (b) independent experiments. c Representative images and d quantification of NHE1 polarization in vehicle control and ML141-treated MDA-MB-231 cells inside confining channels before (340 mOsm/l) and after (165 mOsm/l) application of hypotonic shock at the leading edge. NHE1 is polarized at the cell leading edge in control and ML141-treated cells before osmotic shock (yellow arrowheads) and repolarizes to the “new” leading edge in response to osmotic shock only in control (red arrowhead), but not ML141-treated (white arrowhead),cells. Data represent the mean ± SD from three independent experiments. e Time-lapse montage of an MDA-

Article Snippet: In select experiments, cells were treated with the following pharmacological agents or corresponding vehicle controls: 5-(N-Ethyl-N-isopropyl) amiloride (EIPA, 40 μM, Sigma–Aldrich), DCPIB (40 μM, Tocris Biosciences), bumetanide (30μM, Santa Cruz Biotechnology), Lat-A (2 μM, Sigma–Aldrich), ML141 (10μM, Santa Cruz Biotechnology).

Techniques: Migration, 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: 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