Fig 2: Myo1d mRNA levels in PAN-treated E11 cells. E11 cells were treated with 0–300 µg/mL PAN. Values were normalized relative to 18S rRNA expression. The relative expression level of Myo1d mRNA at 0 µg/mL PAN was given arbitrary value of 1. ** p < 0.01 versus 0 µg/mL PAN. Data represent the means ± SD (n = 3).

Fig 3: Inhibition of Myo1d can reduce the a-syn PFF transmission from BMVECs to pericytes(A and B) Western blot analysis of protein levels of Myo1d in primary BMVECs of control siRNA and Myo1d siRNA with or without a-syn PFF treatment. ß-actin served as a control. n = 6.(C) Representative images of a-syn PFF treatment with control siRNA and Myo1d siRNA in the coculture system of BMVECs to pericytes. Alexa 647 Protein Labeling Kits stained a-syn PFF (white), PDGFR-ß(green) stained acceptor cells (pericytes), and the yellow and red arrows individually indicated spreading or no spreading to acceptor cells. Nuclei were stained with DAPI (blue). Scale bar = 115.8µm.(D) Percent of a-syn positive acceptor cells was presented as the transmitted efficiency of a-syn PFF. n = 4 independent tests with 170–240 individual cells. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA with Bonferroni (B), Student’s t test (D).

Fig 4: a-Syn PFF accumulation in BMVECs activate molecular motors Myo1d(A) Total RNA extracted from BMVECs of PBS and a-syn PFF was analyzed using RNA-seq. Volcano plots of all the identified genes in the RNA-seq with upregulated DEGs in red, downregulated DEGs in green, and non-DEGs in gray.(B) The top 40 of GO functional annotation based on the total DEGs.(C) Heatmap showed the DEGs expression of molecular motors.(D) Quantitative PCR analysis of mRNA levels of Myo1d. n = 4.(E and F) Western blot analysis of protein levels of Myo1d in BMVECs with or without a-syn PFF treatment. ß-actin served as a control. n = 4. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Student’s t test.

Fig 5: Myo1d localized in F-actin-rich protrusions(A) Representative images of BMVECs of control siRNA and Myo1d siRNA with or without a-syn PFF treatment. F-actin labeled using phalloidin (green). Nuclei were stained with DAPI (blue). Scale bar = 115.8µm.(B) Colocalization coefficient of Myo1d and F-actin in BMVECs of control and Myo1d siRNA with or without a-syn PFF treatment. n = 5. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA with Bonferroni.