Figure 6.
Removal of RAB7 from lysosomal membranes rescues TFEB phosphorylation level and localization. (A) Colocalization analysis between mCherry-RAB7 WT and LAMP1 (green) in SPG21 KO HeLa cells 48 h after transfection with an empty plasmid or TBC1D5-HA (blue). Scale bar = 10 µm. The graphs show the quantifications (Manders’ coefficient) of three independent experiments with two different KO clones (n = 6). 10 cells were analyzed per clone in each experiment (shown in light gray). The average values for each clone are shown as follows: black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. **P < 0.01. (B) Western blotting detection of the pTFEB/TFEB ratio in CTRL and SPG21 KO HeLa cells 48 h after the co-transfection of GFP-TFEB with an empty plasmid or a TBC1D5-HA construct (detected with an anti-HA antibody). The graph shows mean fold changes in pTFEB/TFEB ratio ± SD. n = 8 (four independent experiments with two CTRL and two KO clones). Two-tailed unpaired t test. *P < 0.05. (C) Analysis of GFP-TFEB localization by confocal microscopy in CTRL and SPG21 KO HeLa cells 72 h after co-transfection with an empty plasmid or a TBC1D5-HA (red) construct. Scale bar = 10 µm. The graphs show the percentage of cells with predominant nuclear TFEB (left graph) or the TFEB nuclear/cytoplasmic signal ratio (right graph). n = 6 (three independent experiments including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment as described in A. Open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F6.

Removal of RAB7 from lysosomal membranes rescues TFEB phosphorylation level and localization. (A) Colocalization analysis between mCherry-RAB7 WT and LAMP1 (green) in SPG21 KO HeLa cells 48 h after transfection with an empty plasmid or TBC1D5-HA (blue). Scale bar = 10 µm. The graphs show the quantifications (Manders’ coefficient) of three independent experiments with two different KO clones (n = 6). 10 cells were analyzed per clone in each experiment (shown in light gray). The average values for each clone are shown as follows: black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. **P < 0.01. (B) Western blotting detection of the pTFEB/TFEB ratio in CTRL and SPG21 KO HeLa cells 48 h after the co-transfection of GFP-TFEB with an empty plasmid or a TBC1D5-HA construct (detected with an anti-HA antibody). The graph shows mean fold changes in pTFEB/TFEB ratio ± SD. n = 8 (four independent experiments with two CTRL and two KO clones). Two-tailed unpaired t test. *P < 0.05. (C) Analysis of GFP-TFEB localization by confocal microscopy in CTRL and SPG21 KO HeLa cells 72 h after co-transfection with an empty plasmid or a TBC1D5-HA (red) construct. Scale bar = 10 µm. The graphs show the percentage of cells with predominant nuclear TFEB (left graph) or the TFEB nuclear/cytoplasmic signal ratio (right graph). n = 6 (three independent experiments including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment as described in A. Open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal