Figure S3.
Basal expression levels of RAB7, TBC1D5, and VPS35 in CTRL and SPG21 KO cells; coprecipitation assay of CCZ1 with RAB7; colocalization analyses between TBC1D5 and VPS26, VPS35 and LAMP1, maspardin-myc, VPS35 and RAB7, and FYCO1 and LAMP1; and assessment of the presence of RAB7 on lysosomes when FYCO1 is overexpressed. (A and E) Western blotting detection of endogenously expressed RAB7, TBC1D5, and VPS35 in CTRL and SPG21 KO cells. α-Tubulin or GAPDH was used as a loading control. Of note, RAB7 and GAPDH signals are also presented in Fig. 5 B. The graphs show the mean fold change ± SD. For RAB7 expression experiment, n = 8 (four independent experiments, including two CTRL [open circles: CTRL1; open squares: CTRL2] and two KO clones [black triangles: KO1; black diamonds: KO2]); for TBC1D5 and VPS35 expression experiments, n = 6 (three independent experiments, including two CTRL and two KO clones). Two-tailed unpaired t test. ns, nonsignificant; P = 0.06. (B) Expression of WT mCherry RAB7 or mCherry alone in CTRL and SPG21 KO cells followed by their precipitation using RFP-trap beads. The presence of endogenous CCZ1 proteins among the coprecipitated proteins was detected by western blotting. n = 6 (three independent experiments, including two CTRL and two KO clone). The graph shows mean fold changes ± SD of precipitated CCZ1 signals normalized to the amount of precipitated bait proteins. Two-tailed unpaired t test. ns, nonsignificant. (C, D, F and G) Colocalization analysis between endogenously expressed TBC1D5 (green) and VPS26 (red), between VPS35 (green) and LAMP1 (red), between maspardin-myc (green), VPS35 (red) and RAB7 (blue), and between FYCO1 (green) and LAMP1 (red) in CTRL and KO cells (micrographies of individual channels corresponding to the triple labellings shown in Fig. 7 F). Scale bar = 10 µm. The graph shows the Manders’ coefficient. n = 6 (three independent experiments, including two CTRL and two KO clones) except for the triple labelling, where n = 5 independent experiments. 10 cells were quantified per clone in each experiment. These values are shown in light gray. Their averages are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. ns, non-significant; ****P < 0.0001. Scale bar = 10 µm except for the triple labelling (scale bar = 5 µm). (H) Analysis of the colocalization level (48 h after transfection) between mCherry-RAB7 WT and LAMP1 (blue) after transfection of GFP-FYCO1 or of an empty plasmid. Scale bar = 10 µm. The graph shows the quantifications of n = 3 independent experiments. 10 cells were analyzed per condition in each experiment, as described above. Mean ± SD. Two-tailed unpaired t test. ****P < 0.0001. RFP, red fluorescent protein. Source data are available for this figure: SourceData FS3.

Basal expression levels of RAB7, TBC1D5, and VPS35 in CTRL and SPG21 KO cells; coprecipitation assay of CCZ1 with RAB7; colocalization analyses between TBC1D5 and VPS26, VPS35 and LAMP1, maspardin-myc, VPS35 and RAB7, and FYCO1 and LAMP1; and assessment of the presence of RAB7 on lysosomes when FYCO1 is overexpressed. (A and E) Western blotting detection of endogenously expressed RAB7, TBC1D5, and VPS35 in CTRL and SPG21 KO cells. α-Tubulin or GAPDH was used as a loading control. Of note, RAB7 and GAPDH signals are also presented in Fig. 5 B. The graphs show the mean fold change ± SD. For RAB7 expression experiment, n = 8 (four independent experiments, including two CTRL [open circles: CTRL1; open squares: CTRL2] and two KO clones [black triangles: KO1; black diamonds: KO2]); for TBC1D5 and VPS35 expression experiments, n = 6 (three independent experiments, including two CTRL and two KO clones). Two-tailed unpaired t test. ns, nonsignificant; P = 0.06. (B) Expression of WT mCherry RAB7 or mCherry alone in CTRL and SPG21 KO cells followed by their precipitation using RFP-trap beads. The presence of endogenous CCZ1 proteins among the coprecipitated proteins was detected by western blotting. n = 6 (three independent experiments, including two CTRL and two KO clone). The graph shows mean fold changes ± SD of precipitated CCZ1 signals normalized to the amount of precipitated bait proteins. Two-tailed unpaired t test. ns, nonsignificant. (C, D, F and G) Colocalization analysis between endogenously expressed TBC1D5 (green) and VPS26 (red), between VPS35 (green) and LAMP1 (red), between maspardin-myc (green), VPS35 (red) and RAB7 (blue), and between FYCO1 (green) and LAMP1 (red) in CTRL and KO cells (micrographies of individual channels corresponding to the triple labellings shown in Fig. 7 F). Scale bar = 10 µm. The graph shows the Manders’ coefficient. n = 6 (three independent experiments, including two CTRL and two KO clones) except for the triple labelling, where n = 5 independent experiments. 10 cells were quantified per clone in each experiment. These values are shown in light gray. Their averages are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. ns, non-significant; ****P < 0.0001. Scale bar = 10 µm except for the triple labelling (scale bar = 5 µm). (H) Analysis of the colocalization level (48 h after transfection) between mCherry-RAB7 WT and LAMP1 (blue) after transfection of GFP-FYCO1 or of an empty plasmid. Scale bar = 10 µm. The graph shows the quantifications of n = 3 independent experiments. 10 cells were analyzed per condition in each experiment, as described above. Mean ± SD. Two-tailed unpaired t test. ****P < 0.0001. RFP, red fluorescent protein. Source data are available for this figure: SourceData FS3.

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