Journal: The Journal of Cell Biology
Article Title: Maspardin/SPG21 controls lysosome motility and TFEB phosphorylation through RAB7 positioning
doi: 10.1083/jcb.202501135
Figure Lengend Snippet: Calcineurin hyperactivation is not responsible for the decreased pTFEB/TFEB ratio in SPG21 KO HeLa cells. (A) Cholesterol staining (filipin) in CTRL and SPG21 KO HeLa cells 48 h after transfection with a LAMP1-GFP plasmid. Graphs show quantifications of filipin fluorescence intensity per cell (left graph) and the relative fluorescence intensity of filipin in LAMP1-GFP–positive structures (right graph). n = 8 (four independent experiments including two CTRL and two KO clones). 10 cells were analyzed 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, nonsignificant, A.U., arbitrary unit. (B) CTRL and SPG21 KO HeLa cells were transfected with GFP-TFEB for 24 h and treated during the last 3 h with 5 µM of FK506 (FK) and 10 µM of cyclosporin A (CsA) or with DMSO (vehicle). pTFEB/TFEB ratio was then analyzed by western blotting. α-Tubulin was used as a loading control. n = 6 (three independent experiments including two CTRL and two KO clones). Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; ***P < 0.001. Source data are available for this figure: .
Article Snippet: For filipin staining, the cells were fixed in 3% paraformaldehyde diluted in PBS, pH 7.4, at RT and then incubated for 2 h with a 40 μg/μl filipin III solution (Merck) prior to mounting with Mowiol 40–88 (Merck).
Techniques: Staining, Transfection, Plasmid Preparation, Fluorescence, Clone Assay, Two Tailed Test, Western Blot, Control
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