Maspardin deficiency increases FYCO1 recruitment to lysosomes, resulting in their delocalization to the cell periphery. (A) Analysis of lysosome distribution and colocalization between GFP-RILP and LAMP1 (red) 48 h after GFP-RILP overexpression in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. The graph shows RILP-LAMP1 colocalization levels (Mean Manders’ coefficient ± SD). n = 6 (three 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. Two-tailed unpaired t test. ns, nonsignificant. (B) Analysis of lysosome distribution and colocalization (Manders’ coefficients) between GFP-FYCO1 and LAMP1 (red) in CTRL and SPG21 KO HeLa cells 48 h after transfection with GFP-FYCO1. Scale bar = 10 µm. n = 6 (three independent experiments with two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment, as described in A. Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001. (C) Analysis of colocalization levels between endogenous FYCO1 (green) and RAB7 (red) in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. Mean Manders’ coefficient ± SD. n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each replicate, as described in A. Two-tailed unpaired t test; ***P < 0.001.