Figure 7.
FABP3 and FABP8 are critical for lysosomal cholesterol egress. (A and B) SV589 fibroblasts were infected with lentivirus expressing indicated shRNAs, fixed and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the bottom. Scale bars, 10 µm (main), 1 µm (inset). The relative fluorescence intensity of filipin was quantified in B. The filipin fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 885 cells from three independent experiments). Mann–Whitney U test. (C and D) WT and FABP7 KO2 SV589 fibroblasts infected with lentivirus expressing indicated shRNAs were fixed and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the bottom. Scale bars, 10 µm (main), 1 µm (inset). The relative fluorescence intensity of filipin was quantified in D. The filipin fluorescence intensity of wild-type (WT) cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference, except for FABP7 KO2 cells infected with lentivirus expressing shRNA targeting FABP3 and FABP8. The filipin fluorescence intensity of FABP7 KO2 cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is the reference when comparing FABP7 KO2 cell groups. Data are presented as median with interquartile range (n = 728 cells from three independent experiments). Mann–Whitney U test. (E) Indicated cells were treated as in C, and lysosomes were purified and subjected to immunoblotting analysis. (F) Relative cholesterol levels of purified lysosomes normalized to protein amounts. The lysosomal cholesterol levels in WT cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference, except for FABP7 KO2 cells infected with lentivirus expressing shRNA targeting FABP3 and FABP8. The lysosomal cholesterol levels of FABP7 KO2 cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) are the reference when comparing FABP7 KO2 cell groups. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Source data are available for this figure: SourceData F7.

FABP3 and FABP8 are critical for lysosomal cholesterol egress. (A and B) SV589 fibroblasts were infected with lentivirus expressing indicated shRNAs, fixed and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the bottom. Scale bars, 10 µm (main), 1 µm (inset). The relative fluorescence intensity of filipin was quantified in B. The filipin fluorescence intensity of cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 885 cells from three independent experiments). Mann–Whitney U test. (C and D) WT and FABP7 KO2 SV589 fibroblasts infected with lentivirus expressing indicated shRNAs were fixed and stained with filipin (magenta) and the anti-LAMP1 antibody (green). Boxed areas are shown at a higher magnification on the bottom. Scale bars, 10 µm (main), 1 µm (inset). The relative fluorescence intensity of filipin was quantified in D. The filipin fluorescence intensity of wild-type (WT) cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference, except for FABP7 KO2 cells infected with lentivirus expressing shRNA targeting FABP3 and FABP8. The filipin fluorescence intensity of FABP7 KO2 cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is the reference when comparing FABP7 KO2 cell groups. Data are presented as median with interquartile range (n = 728 cells from three independent experiments). Mann–Whitney U test. (E) Indicated cells were treated as in C, and lysosomes were purified and subjected to immunoblotting analysis. (F) Relative cholesterol levels of purified lysosomes normalized to protein amounts. The lysosomal cholesterol levels in WT cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) is defined as 1 and used as the reference, except for FABP7 KO2 cells infected with lentivirus expressing shRNA targeting FABP3 and FABP8. The lysosomal cholesterol levels of FABP7 KO2 cells infected with lentivirus expressing scrambled control shRNA (NC, negative control) are the reference when comparing FABP7 KO2 cell groups. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Source data are available for this figure: SourceData F7.

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