cdc42 inhibitor—ml141  (Tocris)

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: Cdc42 is responsible for the activation of PAK2-MLC signaling and viral entry. (A) Cdc42 was activated by NDV during the viral entry process. HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cell lysates were harvested and the GTPase activity of Cdc42 was analyzed using a Cdc42 activation assay kit followed by Western blotting using anti-Cdc42 antibody. GAPDH was used as a control. (B and C) Treatment with Cdc42 inhibitor ML141 inhibited NDV-induced activation of Cdc42, PAK2, and MLC. HD11 cells were pretreated with either DMSO or ML141. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of GTP-Cdc42 (B), p-PAK2 (T402), PAK2, p-MLC (S19) and MLC (C) were determined by a Cdc42 activation assay kit and Western blotting. GAPDH was used as a control. (D and E) Treatment with the Cdc42 inhibitor, ML141, reduced the level of NDV internalization. HD11 cells were pretreated with ML141 or mock-treated with DMSO, after which NDV adsorption (D) and internalization (E) assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. (F) Overexpression of a DN mutant Cdc42 reduced the internalization of NDV. HD11 cells transfected with the BFP-tagged WT Cdc42 or DN mutant Cdc42 T17N were inoculated with DiOC-labelled NDV. The internalization assay was performed as described in Fig. 4A. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Article Snippet: ML141 , Cdc42 activation inhibitor , MCE , HY-12755.

Techniques: Activation Assay, Activity Assay, Western Blot, Control, Adsorption, Flow Cytometry, Over Expression, Mutagenesis, Transfection

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: NDV activates Cdc42-PAK2-MLC signaling axis via Src, rather than Cdc42-N-WASP-Arp2/3, to promote viral entry. (A and B) Treatment with Src inhibitor, Saracatinib, inhibited NDV-induced activation of Cdc42, PAK2, and MLC. HD11 cells were pretreated with either DMSO or Saracatinib. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of GTP-Cdc42 (A), p-PAK2 (T402), PAK2, p-MLC (S19), and MLC (B) were determined with a Cdc42 activation assay kit and Western blotting. GAPDH was used as a control. (C and D) Treatment with the N-WASP inhibitor, Wiskostatin, and (E and F) Arp2/3 complex inhibitor, CK636, had no effect on the adsorption and internalization of NDV. HD11 cells were pretreated with Wiskostatin, CK-636 or mock-treated with DMSO, and NDV adsorption and internalization assays were performed as described above. (G) Schematic of NDV-activated Cdc42-PAK2-MLC signaling axis during entry into HD11 cells. The bars represent the means ± SD from three independent experiments (*P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Article Snippet: ML141 , Cdc42 activation inhibitor , MCE , HY-12755.

Techniques: Activation Assay, Western Blot, Control, Adsorption

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: Schematic of NDV entry pathways into HD11 cells. (A) NDV entry through CavME. By binding to gangliosides located in caveolae, Src tyrosine kinase was activated by NDV, leading to activation of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase, RhoA and Cdc42, and downstream effectors, CFN and MLC, orchestrating the endocytic entry of NDV. The endocytosed virus-containing caveolar vesicles were subsequently trafficked to the early endosome, where viral-cell membrane fusion occurs [12]. (B) By binding to glycoproteins, viral-cell membrane fusion occurs at the PM. Src-mediated actin cytoskeletal rearrangement also contributes to the direct fusion of the NDV with the cell PM.

Article Snippet: ML141 , Cdc42 activation inhibitor , MCE , HY-12755.

Techniques: Binding Assay, Activation Assay, Virus, Membrane

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: Inhibitors and activators used in this study

Article Snippet: ML141 , Cdc42 activation inhibitor , MCE , HY-12755.

Techniques: Activation Assay

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: Primers used for plasmid construction b

Article Snippet: ML141 , Cdc42 activation inhibitor , MCE , HY-12755.

Techniques: Plasmid Preparation, Sequencing, Amplification

Journal: Journal of Virology

Article Title: Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

doi: 10.1128/jvi.01915-23

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: ML141 , Cdc42 activation inhibitor , MCE , HY-12755.

Techniques:

Journal: bioRxiv

Article Title: Microglial low-affinity FcγR mediates the phagocytic elimination of dopaminergic neurons in Parkinson’s disease degeneration

doi: 10.1101/2024.07.04.602092

Figure Lengend Snippet: A. CD16/32 accumulates at the leading front of motile microglia. Top panel shows maximum intensity projection of a BV2 cell marked with CD16/32 and DAPI. Leading lamellas (left pole) and the uropod (opposite pole) indicate its direction. Bottom panel shows a 0.5 mm optical plane represented in a scale of 16 colors. Scale on the bottom (white represents the highest value and black the lowest). Leading front lamellas (1) and uropod (2) are indicated. B. Illustration of characteristic motile microglial cell showing essential cytoskeletal elements (Modified from Roig-Martinez et al., 2019) . C. Plot of relative fluorescence of motile microglia measured (white broken line) from image on the left. Note the higher fluorescence at the leading front (1) and a smaller peak at the uropod (2). All images scale bars equal 10 μm. D. Representative image of a BV2 microglial cell expressing high density of Cdc42 (red) in a F-actin-rich protrusion. The nucleus was counterstained with DAPI (blue). Scale bar: 3 μm. E. Plot profile of relative fluorescence displays high expression of Cdc42 corresponding with high fluorescence of F-actin at the protruding edge (2) compared with the nucleus (1). F. Confocal image of microglial cell establishing a phagocytic cup around a dopaminergic cell (1). The PC12 DA cell is marked with dopaminergic neuronal marker, TH (green), and the microglial cell is stained by F-actin (magenta). (2) Diagram of the engulfing process depicted in 1. (3) Image of F-actin fluorescence intensity of the engulfing microglia seen in 1. Note the higher intensity is displayed at the borders of the phagocytic cup. (4) higher magnification of the phagocytic cup to visualize the characteristic accumulations of F-actin. Scale bar: 10 mm. (5) 3D reconstruction of the phagocytic event displaying the microglia arranging the F-actin rich phagocytic cup around a dopaminergic cell (yellow arrowhead). G. Increasing concentrations of ML141 were safe for BV2 cell viability. MTT cell viability assay for BV2 microglial cells submitted to increasing concentrations of ML141 was performed to test their safety: 500 μg of H 2 O 2 resulted in approximately 50% decrease of the cell viability. But no significant decrease was appreciated after the administration of ML141 or vehicle (*** p˂0.001 H 2 O 2 significant against every other treatment). H. Quantification of the number of PC12 DA cells at 60 min. of interaction with BV2 microglia. BV2 were treated either before or after activation with Cdc42 inhibitor ML141. The proinflammatory-mediated elimination of DA cells was prevented when BV2 cells were incubated with ML141. (Pre-activation inhibition ** p˂0.01 IFN-γ + LPS vs. ML141/IFN-γ +LPS and ML141. Post-activation inhibition $$$ p˂0.001 IFN-γ + LPS vs. every condition and **** p˂0.0001 control vs. IFN-γ + LPS).

Article Snippet: To analyze how the inhibition of the protein Cdc42 affects the preservation of dopaminergic cells, the inhibitor ML141 (Cdc42/Rac1 GTPase Inhibitor, Calbiochem, San Diego, CA, USA) was used in BV2/PC12 co-cultures.

Techniques: Modification, Fluorescence, Expressing, Marker, Staining, Viability Assay, Activation Assay, Incubation, Inhibition, Control

Journal: bioRxiv

Article Title: Microglial low-affinity FcγR mediates the phagocytic elimination of dopaminergic neurons in Parkinson’s disease degeneration

doi: 10.1101/2024.07.04.602092

Figure Lengend Snippet: A. Diagram of the procedure used for the CD16/32 passive immunotherapy. Neuropathological histology was performed in the SNpc (red square). B. Representative confocal images showing the SNpc labeled with TH and counterstained with DAPI. Depletion of DA neurons can be appreciated in MPTP-treated animals. However, a protective effect can be seen in mice treated with CD16/32 neutralizing monoclonal antibodies (aCD16/32) (Scale bar: 30 mm). C. Quantification of the DA neurons in the SNpc demonstrating a significant decrease in the MPTP group which is prevented by the administration of CD16/32 (*** p˂0.001 MPTP vs. every treatment except MPTP + Isotype, ### p˂0.001 MPTP + Isotype vs. every treatment except MPTP). D. 3D reconstruction of representative microglial cells expressing Iba-1 in different treatments of the experiment (Scale bar: 10 µm). C. Quantification of the area of Iba-1 in the SNpc, indicating variation of microglial size when the animals were intoxicated with MPTP. F. Diagram of the procedure used for Cdc42 inhibition. G. Representative confocal images of histological analysis. Upper panel: Representative images of TH positive neurons of the SNpc in every treatment (scale bar: 30 µm). Lower panel: 3D reconstructions illustrating morphology and changes of size in microglial cells expressing Iba-1 (scale bar: 10 µm). H. Quantification of TH positive neurons indicating a significant decrease in the MPTP group and prevention by the previous administration of Cdc42 inhibitor ML141 (** p <0.01 with respect to saline). I. Quantification of the Iba-1 expressing area, which represents changes in microglial size. Microglial activation is present in the MPTP group evidenced by the increased area of Iba-1, which is decreased in the animals intoxicated with MPTP but previously treated with ML141 (*** p<0.001 with respect to controls, $$ p<0.01 with respect to ML141).

Article Snippet: To analyze how the inhibition of the protein Cdc42 affects the preservation of dopaminergic cells, the inhibitor ML141 (Cdc42/Rac1 GTPase Inhibitor, Calbiochem, San Diego, CA, USA) was used in BV2/PC12 co-cultures.

Techniques: Labeling, Expressing, Inhibition, Saline, Activation Assay