Figure 1. α-syn-YFP aggregation in iNs... | Rockefeller University Press

Figure 1.

$α-syn-YFP aggregation in iNs exposed to PFFs. (A and B) Immunofluorescence images obtained using spinning disc confocal microscopy depict maximum z-projections made from consecutive optical planes spaced 270 nm apart of chemically fixed parental iPSCs (A) and iNs (B) treated with MAP2-specific antibody for neuronal identification. Cell perimeters in B are outlined with white dotted lines. Images are representative of two biological replicates. Scale bar: 40 µm. (C–E) iNs transiently expressing α-syn-YFP display a diffuse cytoplasmic distribution of α-syn-YFP without PFF treatment (C), compared to distinct α-syn-YFP aggregates mostly in soma and fewer in neurites after 3 days of incubation with 4 µg/ml (∼0.3 µM) PFFs (D). Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of three biological replicates. Scale bar: 10 µm. Insets provide 2× magnification. Bar graph in E quantifies the fraction of cells containing α-syn-YFP aggregates for the specified number of cells; each dot represents data from each replicate. (F–H) iNs stably expressing α-syn-YFP, when incubated without (F) and with (G) 4 µg/ml PFFs for 3 days, show puncta consistent with α-syn-YFP aggregates. Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of two biological replicates. Scale bar: 10 µm. Insets offer 2× magnification. Bar graph in H quantifies the fraction of cells containing α-syn-YFP aggregates; each dot represents data from a biological replicate. (I–K) iPSCs stably expressing α-syn-YFP exhibit a diffuse cytosolic α-syn-YFP signal in the absence (I) or after a 3-day incubation with 4 µg/ml PFFs (J). Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of two biological replicates. Scale bar: 10 µm. Insets show 2× magnification. Bar graph in K quantifies the fraction of cells containing α-syn-YFP aggregates for the specified number of cells; each dot represents data from each replicate. (L–N) SVG-A cells stably expressing α-syn-YFP show diffuse cytoplasmic α-syn-YFP signal in (L) the absence of or (M) after a 3-day incubation with 4 µg/ml PFFs. Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of three biological replicates. Scale bar: 10 µm. Insets show 2× magnification. Bar graph in N quantifies the fraction of cells containing α-syn-YFP aggregates for the specified number of cells; each dot represents data from each replicate. Statistical significance (P < 0.005) is indicated by a star; “ns” denotes no statistical difference.$

α-syn-YFP aggregation in iNs exposed to PFFs . (A and B) Immunofluorescence images obtained using spinning disc confocal microscopy depict maximum z-projections made from consecutive optical planes spaced 270 nm apart of chemically fixed parental iPSCs (A) and iNs (B) treated with MAP2-specific antibody for neuronal identification. Cell perimeters in B are outlined with white dotted lines. Images are representative of two biological replicates. Scale bar: 40 µm. (C–E) iNs transiently expressing α-syn-YFP display a diffuse cytoplasmic distribution of α-syn-YFP without PFF treatment (C), compared to distinct α-syn-YFP aggregates mostly in soma and fewer in neurites after 3 days of incubation with 4 µg/ml (∼0.3 µM) PFFs (D). Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of three biological replicates. Scale bar: 10 µm. Insets provide 2× magnification. Bar graph in E quantifies the fraction of cells containing α-syn-YFP aggregates for the specified number of cells; each dot represents data from each replicate. (F–H) iNs stably expressing α-syn-YFP, when incubated without (F) and with (G) 4 µg/ml PFFs for 3 days, show puncta consistent with α-syn-YFP aggregates. Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of two biological replicates. Scale bar: 10 µm. Insets offer 2× magnification. Bar graph in H quantifies the fraction of cells containing α-syn-YFP aggregates; each dot represents data from a biological replicate. (I–K) iPSCs stably expressing α-syn-YFP exhibit a diffuse cytosolic α-syn-YFP signal in the absence (I) or after a 3-day incubation with 4 µg/ml PFFs (J). Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of two biological replicates. Scale bar: 10 µm. Insets show 2× magnification. Bar graph in K quantifies the fraction of cells containing α-syn-YFP aggregates for the specified number of cells; each dot represents data from each replicate. (L–N) SVG-A cells stably expressing α-syn-YFP show diffuse cytoplasmic α-syn-YFP signal in (L) the absence of or (M) after a 3-day incubation with 4 µg/ml PFFs. Live-cell spinning-disc confocal microscopy images depict maximum z-projections and are representative of three biological replicates. Scale bar: 10 µm. Insets show 2× magnification. Bar graph in N quantifies the fraction of cells containing α-syn-YFP aggregates for the specified number of cells; each dot represents data from each replicate. Statistical significance (P < 0.005) is indicated by a star; “ns” denotes no statistical difference.