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. 2021 May 11;12:2673. doi: 10.1038/s41467-021-22882-x

Fig. 4. In the absence of INPP5A cholesterol is directed towards lysosomes and lipid droplets.

Fig. 4

a Representative images from three independent experiments of HeLa cells treated with control (SCR) or INPP5A siRNA (5AKD) and transfected with a plasmid encoding D4H-mCherry (red) to detect intracellular cholesterol, Blue, DAPI-stained nuclei. Scale bar, 10 μm. b Representative images from three independent experiments of HeLa cells treated with INPP5A siRNA (5AKD), transfected with a plasmid encoding D4H-mCherry (red) to detect intracellular cholesterol, and stained with anti-LAMP-2(green) antibodies to label lysosomes. Scale bar, 10 μm. c Representative images from three independent experiments of HeLa cells treated with INPP5A siRNA (5AKD), transfected with a plasmid encoding D4H-mCherry (red) to detect intracellular cholesterol, and incubated for 45 min post-fixation with 0.05 mg/ml Filipin (cyan). Scale bar, 10 μm. d Representative images from two independent experiments of HeLa cells treated with INPP5A siRNA (5AKD), incubated with Lysotracker (red, 1:1000 dilution) for 60 min before fixation to stain lysosomes, and incubated for 45 min post-fixation with 0.05 mg/ml Filipin (cyan). Scale bar, 10 μm. e Relative BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) levels incubated for 15 min with 2 μM as a measure for lipid droplets in HeLa cells treated with control (SCR) or INPP5A siRNA (5AKD). One sample t-test: 5AKD: p = 0.03, t = 5.642, df = 2. f Representative images from three independent experiments of HeLa cells treated with INPP5A siRNA (5AKD) and incubated for 15 min with 2 μM of BODIPY (green) before fixation and incubated for 45 min post-fixation with 0.05 mg/ml Filipin (cyan) Scale bar, 10 μm. All data represent mean ± SEM from three independent experiments. Numerical source data are reported in the Source Data file.