Figure S5.
Validation of lysosomal phenotypes with additional independent shRNA targeting VPS13C in iPSC-derived dopaminergic neurons. (A and B) Representative immunoblot and quantification of VPS13C KD (KD-2) efficiency in hiPSC-derived dopaminergic neurons (day 70) after 14 days of control and VPS13C shRNA treatment (N = 3). (C) Representative live-cell confocal images of LAMP1-mGFP (green) -positive vesicles in control (upper) and VPS13C KD-2 (lower) neurons with insets showing enlarged lysosomes (yellow arrow) in VPS13C KD-2 condition in comparison with lysosomes in control condition (white arrow) (scale bar: 10 µm, inset: 1 µm). (D) Quantification of the average lysosomal size (N = 4, from n = 17 cells). (E) Time-lapse images of L-L contacts showing tethered lysosomes (yellow arrows) that untether at 37 s (white arrows) in control condition but remain tethered in VPS13C KD-2 neurons until 140 s (yellow arrow) (scale bar: 1 µm). (F) Quantification of the percentage of lysosomes in stable L-L contacts (≥10 s). (G) Quantification of the minimum duration of L-L contacts (N = 4, from n = 12 cells). (H) Representative live-cell confocal images showing LAMP1-positive vesicles (green) and examples of lysosomal motility demonstrated by kymographs (bottom panels) in control and VPS13C KD-2 neurons (scale bar: 10 µm, kymograph scale bar: x = 1 µm, y = 20 s). (I and J) Quantification of the average distance traveled by lysosomes in 60 s and the average lysosomal motility N = 4, from n = 11 cells (control) and n = 13 cells (VPS13C KD-2). (K) Representative live-cell confocal images of Magic Red cathepsin B staining in control neurons showing stronger cathepsin B intensity (white arrow) and in VPS13C KD-2 neurons showing reduced cathepsin B intensity (yellow arrow) Scale bar: 10 µm, inset: 1 µm. (L and M) Quantification of the number of Magic Red cathepsin B-positive puncta per cell area (L) and the mean fluorescence intensity of Magic Red cathepsin B-positive puncta (M) (N = 4, from n = 22 cells). (N) Representative live-cell confocal images of LysoTracker Red DND-99 staining in control neurons showing stronger LysoTracker intensity (white arrow) and in VPS13C KD-2 neurons showing reduced LysoTracker intensity (yellow arrows). Scale bar: 10 µm, inset: 1 µm. (O and P) Quantification of the number of LysoTracker Red-positive puncta per cell area (O) and the mean fluorescence intensity of LysoTracker Red-positive puncta (P) (N = 4, from n = 19 cells). (Q–T) Representative immunoblots and quantifications of phospho-Rab10 (pRab10) and total Rab10 protein levels from whole cell HEK-293 FT lysates (N = 4). Dashed lines in C, H, K, and N represent the outline of the cell. Data collected for the non-targeting control condition were used for the comparison between control and KD-1 in Figs. 1, 2, 3, 6, and 7, and for the comparison between control and KD-2 in Fig. S5. Data represented as mean ± SEM; unpaired two-tailed t test (B, D, F, G, I, J, L, M, O, P, S, and T); ns: not significant (T), *P < 0.05 (D, I, J, and S), **P < 0.01 (B), ***P < 0.001 (F), ****P < 0.0001 (G, L, M, O, and P). Source data are available for this figure: SourceData FS5.

Validation of lysosomal phenotypes with additional independent shRNA targeting VPS13C in iPSC-derived dopaminergic neurons. (A and B) Representative immunoblot and quantification of VPS13C KD (KD-2) efficiency in hiPSC-derived dopaminergic neurons (day 70) after 14 days of control and VPS13C shRNA treatment (N = 3). (C) Representative live-cell confocal images of LAMP1-mGFP (green) -positive vesicles in control (upper) and VPS13C KD-2 (lower) neurons with insets showing enlarged lysosomes (yellow arrow) in VPS13C KD-2 condition in comparison with lysosomes in control condition (white arrow) (scale bar: 10 µm, inset: 1 µm). (D) Quantification of the average lysosomal size (N = 4, from n = 17 cells). (E) Time-lapse images of L-L contacts showing tethered lysosomes (yellow arrows) that untether at 37 s (white arrows) in control condition but remain tethered in VPS13C KD-2 neurons until 140 s (yellow arrow) (scale bar: 1 µm). (F) Quantification of the percentage of lysosomes in stable L-L contacts (≥10 s). (G) Quantification of the minimum duration of L-L contacts (N = 4, from n = 12 cells). (H) Representative live-cell confocal images showing LAMP1-positive vesicles (green) and examples of lysosomal motility demonstrated by kymographs (bottom panels) in control and VPS13C KD-2 neurons (scale bar: 10 µm, kymograph scale bar: x = 1 µm, y = 20 s). (I and J) Quantification of the average distance traveled by lysosomes in 60 s and the average lysosomal motility N = 4, from n = 11 cells (control) and n = 13 cells (VPS13C KD-2). (K) Representative live-cell confocal images of Magic Red cathepsin B staining in control neurons showing stronger cathepsin B intensity (white arrow) and in VPS13C KD-2 neurons showing reduced cathepsin B intensity (yellow arrow) Scale bar: 10 µm, inset: 1 µm. (L and M) Quantification of the number of Magic Red cathepsin B-positive puncta per cell area (L) and the mean fluorescence intensity of Magic Red cathepsin B-positive puncta (M) (N = 4, from n = 22 cells). (N) Representative live-cell confocal images of LysoTracker Red DND-99 staining in control neurons showing stronger LysoTracker intensity (white arrow) and in VPS13C KD-2 neurons showing reduced LysoTracker intensity (yellow arrows). Scale bar: 10 µm, inset: 1 µm. (O and P) Quantification of the number of LysoTracker Red-positive puncta per cell area (O) and the mean fluorescence intensity of LysoTracker Red-positive puncta (P) (N = 4, from n = 19 cells). (Q–T) Representative immunoblots and quantifications of phospho-Rab10 (pRab10) and total Rab10 protein levels from whole cell HEK-293 FT lysates (N = 4). Dashed lines in C, H, K, and N represent the outline of the cell. Data collected for the non-targeting control condition were used for the comparison between control and KD-1 in Figs. 1, 2, 3, 6, and 7, and for the comparison between control and KD-2 in Fig. S5. Data represented as mean ± SEM; unpaired two-tailed t test (B, D, F, G, I, J, L, M, O, P, S, and T); ns: not significant (T), *P < 0.05 (D, I, J, and S), **P < 0.01 (B), ***P < 0.001 (F), ****P < 0.0001 (G, L, M, O, and P). Source data are available for this figure: SourceData FS5.

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