Figure 1.
Influence of anti-prion compounds on the amount of PrP-res.
(A) ScN2a-3-22L cells grown on 12-well plates were cultured in the presence or absence of mAb 44B1, PPS, CPZ, or U18666A at the indicated concentration for 72 h. The samples were subjected to immunoblotting and dot-blotting for PrP-res detection or β-actin detection for endogenous control. Representative blots for each compound are shown on the left. The graph on the right shows PrP-res levels relative to the control samples. The means and standard deviations (SDs) of four independent experiments (PrP-res was detected by dot-blotting) are indicated. Graphs on the upper right show the logistic curve fitted to the data of PrP-res levels by dot-blotting (B) ScN2a-3-22L cells were cultured with anti-prion compounds at the EC65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for the indicated time and subjected to dot-blotting for PrP-res. Representative dot-blotting is shown on the left, and the graph on the right shows the levels of PrP-res relative to the samples from ScN2a-3-22L cells cultured without anti-prion compounds for 72 h. The means and SDs of four independent experiments are depicted. Asterisks indicate a significant difference compared with the control at the same time point (Student’s t-test, p<0.05).
Figure 2.
Influence of anti-prion compounds on the intracellular PrPSc distribution.
ScN2a-3-22L cells grown on a chambered coverglass for 48 h were cultured with or without an anti-prion compound at the EC65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6, 24, or 48 h. The cells were subjected to PrPSc-specific detection by direct immunostaining with rIgG132-EGFP. The cell nuclei were counterstained with DAPI. The panels show the merged images of PrPSc (green) and nuclei (blue). Scale bars: 10 µm.
Figure 3.
Influence of anti-prion compounds on the localization of PrPSc and PrPC.
N2a-3 cells or ScN2a-3-22L cells were cultured with or without an anti-prion compound at the EC65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6 h. The cells were fixed and stained with 31C6-Af555 to detect PrPC, and subsequently subjected to PrPSc-specific detection with rIgG132-EGFP. The cell nuclei were counterstained with DAPI. The merged images of PrPSc (green) and nuclei (blue) are shown on the left, those of PrPC (red) and nuclei are shown in the middle, and those of PrPSc, PrPC, and nuclei are shown on the right. Scale bars: 10 µm.
Figure 4.
Influence of anti-prion compounds on PrPC levels.
N2a-3 cells were cultured with an anti-prion compound at the EC65 (mAb 44B1, 0.4 µg/ml; PPS, 0.1 µg/ml; CPZ, 10 µM; U18666A, 5 µM) for 6–72 h and subjected to dot-blotting for the detection of PrPC or GAPDH for endogenous control. Representative dot-blot images are shown on the left, and the graph on the right shows the PrPC levels relative to that of 72-h mock-treated cells. The means and SDs of three independent experiments are depicted. Asterisks indicate a significant difference between the cells treated with each anti-prion compound and mock-treated control cells at the same time point (Student’s t-test, p<0.05).
Figure 5.
Co-localization of PrPSc with Snx1.
ScN2a-3-22L cells grown on a chambered coverglass for 48 h were incubated with 7.5 µg/ml mAb 44B1, 10 µg/ml PPS, 10 µM CPZ, or 5 µM U18666A or without an anti-prion compound for 2 h. The cells were subjected to PrPSc-specific staining with rIgG132-EGFP and immunostaining for Snx1. Nuclei were counterstained with DAPI. The leftmost column presents a lower-magnification merged image of PrPSc (green), Snx1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Arrows denote representative examples of co-localization of PrPSc with Snx1. Scale bars: 10 µm.
Figure 6.
Co-localization of PrPSc or PrPC with EEA1.
ScN2a-3-22L cells were cultured with 7.5 µg/ml mAb 44B1, 10 µg/ml PPS, 10 µM CPZ or 5 µM U18666A or without an anti-prion compound for 6 h. The cells were subjected to direct immunostaining of PrPC and PrPSc with 31C6-Af555 and rIgG132-EGFP, respectively and subsequently to immunostaining for EEA1 and nuclei. The leftmost column shows a lower-magnification merged image of PrPSc (green), PrPC (cyan), EEA1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Arrows or arrowheads denote representative examples of the co-localization of PrPSc with EEA1 or PrPC with EEA1, respectively. Scale bars: 10 µm.
Figure 7.
Co-localization of PrPSc or PrPC with Lamp1.
ScN2a-3-22L cells were cultured under the same condition as described in Figure 6. The cells were subjected to direct immunostaining of PrPC and PrPSc with 31C6-Af555 and rIgG132-EGFP, respectively and subsequently to immunostaining for Lamp1 and nuclei. The leftmost column shows a lower-magnification merged image of PrPSc (green), PrPC (cyan), Lamp1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Arrows or arrowheads denote representative examples of the co-localization of PrPSc with Lamp1 or PrPC with Lamp1, respectively. Scale bars: 10 µm.
Figure 8.
Co-localization statistics of PrPSc or PrPC with organelle markers.
Co-localization analyses of the images shown in Figures 7 and 8 and Figure S3 were conducted. (A) Ratio of double-positive areas for PrPSc and markers to the sum of PrPSc-positive areas. (B) Ratio of double-positive areas for PrPC and markers to the sum of PrPC positive areas. The means and SDs of the value acquired in five or six view fields are shown. Single (p<0.05) and double (p<0.01) asterisks indicate a significant difference compared with the control.
Figure 9.
Induction of autophagy by CPZ or U18666A treatment.
ScN2a-3-22L cells were cultured under the same conditions as described in Figure 6. The cells were subjected to immunostaining for LC3 and Lamp1 and counterstained with DAPI. The leftmost column shows the lower-magnification merged image of LC3 (green), Lamp1 (red), and nuclei (blue). Individual and merged high-magnification images of the boxed regions are shown on the right. Scale bars: 10 µm.
Figure 10.
Fate of PrPSc after treatment with CPZ or U18666A.
ScN2a-3-22L cells were cultured with 10 µg/ml PPS, 10 µM CPZ or 5 µM U18666A or without an anti-prion compound for 24 or 48 h. The cells were subjected to the PrPSc-specific indirect immunostaining of PrPSc with mAb 132 and immunostaining for Lamp1 and nuclei. (A) Localization of PrPSc. The left columns show a lower-magnification merged image of PrPSc (green), Lamp1 (red), and nuclei (blue). The high-magnification images of the boxed regions are shown on the right. Arrows indicate representative co-localization of PrPSc with Lamp1. Arrows with asterisks indicate swollen Lamp1-positive vesicles positive for PrPSc. Scale bars: 10 µm. (B) Intensity of PrPSc in Lamp1-positive vesicles. The graph represents the values of the fluorescent intensities of PrPSc in Lamp1-positive vesicles per cell relative to those of PrPSc in Lamp1-positive vesicles per cell in mock-treated cells after 24 h of treatment. The means and SDs of the value acquired in six view fields are shown. Double asterisks indicate a significant difference between cells treated for 24 and 48 h (Student’s t-test, p<0.01).
Figure 11.
Effect of lysosomal hydrolysis inhibition on the decrease of PrPSc levels induced by CPZ or U18666A treatment.
(A) Immunoblot analysis of PrP-res and cathepsin D. ScN2a-3-22L cells were cultured with 10 µM CPZ or 5 µM U18666A or without compounds for 24 h. Subsequently, monensin (Mon) or bafilomycin A1 (BafA1) was added to the culture at a final concentration of 100 or 5 nM, respectively. Following an additional incubation for 36 h with or without Mon or BafA1, the cells were subjected to immunoblotting for PrP-res, cathepsin D, or β-actin. Representative immunoblot images are shown on the left. The bracket in the immunoblot of cathepsin D denotes the pro- and/or intermediate forms of cathepsin D (Pro/Int). The arrowhead denotes the mature form of cathepsin D (M). The upper right graph shows the levels of PrP-res relative to the control. The lower right graph shows the ratio of mature to pro−/intermediate forms of cathepsin D. The means and SDs of three independent experiments are depicted. Asterisks indicate a significant difference between Mon- or BafA1-treated samples and untreated samples (non-treated) (Student’s t-test, p<0.05). (B) Localization of PrPSc. ScN2a-3-22L cells grown on a chambered coverglass were cultured with 10 µM CPZ for 24 h. Subsequently, Mon or BafA1 was added at a final concentration of 100 or 5 nM, respectively, and the cells were cultured for additional 24 h in the presence of CPZ and Mon or BafA1. The cells were subjected to double-staining of PrPSc and Lamp1 before (left) and after the treatment with Mon (middle) or BafA1 (right). The upper panel shows the merged images of PrPSc (green) and nuclei (blue). The bottom panel shows the merged images of PrPSc (green), Lamp1 (red) and nuclei (blue). Scale bars: 10 µm.
Figure 12.
Degradation of LDL in cells treated with anti-prion compounds.
ScN2a-3-22L cells were incubated with Alexa Fluor 488-conjugated LDL (4 µg/ml) for 6 h. After the incubation, the cells were cultured in the presence or absence of 0.4 µg/ml mAb 44B1, 0.1 µg/ml PPS, 10 µg/ml CPZ, or 5 µM U18666A for 6–48 h. The cells were subjected to immunostaining of Lamp1 and staining of the cell nuclei 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 area. (A) Localization of LDL. The panel shows the representative images of the signals of LDL (green), Lamp1 (red) and nuclei (blue) in cells treated with the indicated anti-prion compound for 24 h. The merged images of LDL and nuclei are shown on the left, those of Lamp1 and nuclei are shown in the middle, and those of LDL, Lamp1, and nuclei are shown on the right. The rightmost column presents the higher-magnification images of the boxed regions in the second right column. Scale bars: 10 µm. (B) Intensity of LDL in Lamp1-positive vesicles. The graph shows the values of the fluorescent intensities of LDL in Lamp1-positive vesicles per cell relative to those of LDL in Lamp1-positive vesicles per cell in mock-treated control cells for 6 h. The means and SDs of the value acquired in five view fields are shown. Asterisks indicate a significant difference compared with the control at the same time point (Student’s t-test, p<0.05).