Figure 4(A Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green and (A) TOMM20 (mitochondria), in red with xy- and xz-axis orthogonal views framing the corresponding image. (B-C Manders' coefficients M1 representing the proportion of Filipin III overlapping with (B) TOMM20 M2 representing the proportion of (C) TOMM20 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm TOMM20: translocase of the outer mitochondrial membrane complex subunit 20.D, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (D) ER-RFP in red with xy- and xz-axis orthogonal views framing the corresponding image. E-F Manders' coefficients M1 representing the proportion of Filipin III overlapping with (E) ER-RFP M2 representing the proportion of (F) ER-RFP overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm ER-RFP: endoplasmic reticulum red fluorescent proteinG, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (G) GM130 (Golgi apparatus) in red with xy- and xz-axis orthogonal views framing the corresponding image. H-I, Manders' coefficients M1 representing the proportion of Filipin III overlapping with (H) GM130 M2 representing the proportion of (I) GM130 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm GM130: Golgi matrix protein 130J, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green , (J) EEA1 (endosomes) in red with xy- and xz-axis orthogonal views framing the corresponding image. Manders' coefficients M1 representing the proportion of Filipin III overlapping with (K) EEA1 M2 representing the proportion of (L) EEA1 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm EEA1. early endosome antigen 1M, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (M) LipidSpot (lipid droplets) in red with xy- and xz-axis orthogonal views framing the corresponding image. N-O (N) LipidSpot M2 representing the proportion of (O) LipidSpot overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µmP) Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (P) M6PR (lysosomes) in red with xy- and xz-axis orthogonal views framing the corresponding image. Q-R) Manders' coefficients M1 representing the proportion of Filipin III overlapping with (Q) M6PR, and M2 representing the proportion of (R) M6PR overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm