Figure 8
The effect of the knockdown of Clint1 on the co-localization of PrPSc with the markers for ERCs and late endosomes/lysosomes. ScN2a-3-22L cells were transfected with siRNA against Clint1 (siClint1) or control siRNA (siNC) similarly as shown in Fig. 7. In order to label ERCs, the cells were incubated with fluorescent-labeled Tfn for 15 min and then fixed with PFA (a). To label the late endosomes and lysosomes, the cultured cells were stained for Lamp1 after fixation with PFA (b). The fixed cells were subjected to IFA for PrPSc and Clint1. The cell nuclei were counterstained with DAPI. Z-series of the images were acquired at 0.8 µm steps from the top to the bottom of the cells in the five view fields. The panel shows the representative images of the signals of Clint1 (cyan), PrPSc (green), marker molecules (red), and nuclei (blue) in cells transfected with the siRNA indicated on the left. The merged images of Clint1 and nuclei, those of PrPSc and nuclei, and those of Lamp1 and nuclei are shown in the leftmost, the second left, and the third left columns, respectively. The merged images of PrPSc, marker molecules, and nuclei are shown on the second right column. The rightmost column displays the higher-magnification images of the boxed regions in the second right column. The ratios of PrPSc signals co-localized with exogenously introduced Tfn or the ratio of PrPSc signals co-localized with Lamp1 relative to the total signals of PrPSc are shown in the graph (c). The mean and SDs of five view fields are depicted. Asterisks indicate a significant increase or decrease compared to the control (Student’s t-test, p < 0.05). Scale bars: 10 μm.
