Figure EV1. TMEM135 depletion results in lysosomal cholesterol accumulation without affecting peroxisome and lysosome abundance.

• A–D
  RPE1 cells were transfected with either scramble or TMEM135 siRNAs and immunostained for LAMP1 as a lysosome marker (red) and PMP70 as a peroxisome marker (green). (A, B) Fluorescent intensity of lysosome and peroxisome, respectively, within an area of cell was separately measured by ImageJ software. A. U means arbitrary unit. Data represent mean ± SD (n = 3 experiments), and 50 cells were scored per condition per experiment. NS, not significant; P > 0.05, Student's t‐test. (C, D) Lysosome and peroxisome puncta were determined within an area of cell. Data represent mean ± SD (n = 3 experiments), and 50 cells were scored per condition per experiment. NS, not significant; P > 0.05, Student's t‐test.
• E
  RPE1 cells were transfected with siRNAs, followed by sequential staining with lysotracker (red) and filipin (blue). Scale bar, 20 μm.