Journal: Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics

Article Title: Fasudil inhibits α-synuclein aggregation through ROCK-inhibition-mediated mechanisms.

doi: 10.1016/j.neurot.2025.e00544

Figure Lengend Snippet: Fig. 1. Experimental design and schematic represen- tation of the in vitro experiments. (a): Schematic rep- resentation of the α-synuclein-T/Synphilin-1 method. Co-expression of SynT and V5-Sph-1 leads to the for- mation of inclusions that can be observed after immunofluorescence. (b): Schematic representation of the VN-α-synuclein/α-synuclein-VC BiFC technique. α-synuclein tagged to the VN fragment of venus in- teracts with α-synuclein tagged to the VC fragment of venus leading to fluorescence. (c): Schematic repre- sentation of the RT-QuiC amplification. Small α-syn- uclein aggregates will break after quaking, creating nucleation centers (seeds) that will incorporate monomeric α-synuclein, leading to the formation of bigger aggregates. ThT binds to aggregates of a certain size, changing its conformation and leading to fluo- rescence emission. (d): Schematic representation of the incorporation of EGFP-tagged α-synuclein into microglia. AngII or AngII plus fasudil-treated microglial cells are co-cultured with neuron cells that express EGFP-tagged α-synuclein. EGFP-tagged α-synuclein is then incorporated into microglia, which is then identified by immunofluorescence and flow cytometry.

Article Snippet: Immunofluorescence was visualized with a fluorescence detection system (Molecular Imager ChemiDoc MP imaging System, BioRad).

Techniques: In Vitro, Expressing, Cell Culture, Cytometry

Journal: Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics

Article Title: Fasudil inhibits α-synuclein aggregation through ROCK-inhibition-mediated mechanisms.

doi: 10.1016/j.neurot.2025.e00544

Figure Lengend Snippet: Fig. 4. RT-QuiC analyses. (a–b): Coomassie staining of acrylamide gels shows the presence of PFFs 8 days after starting the preparation process. (c): Quantifi- cation of Coomassie-stained gels showed a higher significant quantity of protein in supernatants before starting the PFFs preparation process and after 4 days. *P < 0.05 relative to the previous column. n ¼ 3. Mann-Whitney U test and t-test. Error bars represent SD. (d): ThT assay of PFFs shows a significant increase in ThT fluorescence in samples collected at 8 days after starting the preparation process. R: Recombinant monomeric protein; sup: supernatant; prec: precipi- tate; PFFs: Pre-formed fibrils (4): Sample after four days of shaking; PFFs (8): Sample after eight days of shaking. *P < 0.05 relative to R. n ¼ 3 independent measurements. One-way ANOVA and Tukey's multiple comparison test. Error bars represent SD. (e): RT-QuiC graph of the monomeric α-synuclein amplification in the presence and absence of 20 μM fasudil. (f–i): Treatment with fasudil does not lead to significant changes in AUC, lag time, aggregation constant, nor in normalized RFU values at the end of the assay for unseeded RT-QuiC reactions. Treatment with fasudil significantly increases the lag time of the aggregation process in seeded RT-QuiC reactions. (j): Treatment with fasudil does not affect already aggregated α-synuclein. (k): RT-QuiC graph of monomeric α-synuclein amplification in the presence and absence of 40 μM fasudil, added at the onset of reaction and after 20 h (dotted line). (l–o): A second fasudil treatment 20 h after the onset of the process does not lead to significant changes in AUC, lag times, aggre- gation constant, nor in normalized RFU values at the end of the assay for seeded RT-QuiC reactions. n ¼ 3–6 independent amplifications. *P < 0.05 relative to Monomer þ PFFs. Error bars represent SEM. Mann- Whitney U test (f, i) or two-tailed unpaired Student's t-test (g-h, l-o).

Article Snippet: Immunofluorescence was visualized with a fluorescence detection system (Molecular Imager ChemiDoc MP imaging System, BioRad).

Techniques: Staining, MANN-WHITNEY, ThT Assay, Recombinant, Comparison, Two Tailed Test