Figure 3.
FABP7 promotes LDL cholesterol transport to the PM. (A–C) WT and FABP7 KO2 SV589 cells were incubated in the cholesterol (Chol)-depleting medium for 16 h and refed with cholesterol-depleting medium containing 20 µg/ml LDL for the indicated periods as shown in A. Cells were then stained with the His6-EGFP-D4H probe and subjected to flow cytometry for EGFP fluorescence measurement (B) or harvested for biochemical analysis of cholesterol levels (C). The EGFP fluorescence intensity of WT and FABP7 KO2 cells cultured in normal medium is defined as 1. Data are presented as mean ± SEM (from three biological replicates). Student’s unpaired two-tailed t test. Compared with WT cells refed with LDL for the same periods of time. (D and E) WT and FABP7 KO2 SV589 cells were depleted of cholesterol as in A, and in the last 10 min of 16 h cholesterol depletion was treated with 1.5% 2-hydroxypropyl-β-cyclodextrin (HPCD). Cells were then refed with cholesterol-depleting medium containing 20 µg/ml LDL for the indicated periods as shown in D, and stained with the His6-EGFP-D4H probe and subjected to flow cytometry for EGFP fluorescence measurement. The EGFP fluorescence intensity of WT and FABP7 KO2 cells cultured in normal medium is defined as 1. Data are presented as mean ± SEM (from three biological replicates). Student’s unpaired two-tailed t test. Compared with WT cells refed with LDL for the same periods of time. (F) SV589 cells infected with lentivirus (Lenti-) expressing negative control (NC) or FABP7 were treated as in D, and EGFP fluorescence intensity was quantified. The EGFP fluorescence intensity of WT and FABP7 KO2 cells cultured in normal medium is defined as 1. Data are presented as mean ± SEM (from three biological replicates). Student’s unpaired two-tailed t test. Compared with cells infected with lentivirus expressing negative control (NC) and refed with LDL for the same periods of time. (G and H) WT and FABP7 KO2 cells were depleted of cholesterol for 16 h and treated with the indicated concentrations of LDL for 5 h at 37°C. Cells were subjected to SREBP2 cleavage analysis. (G) Representative immunoblots showing SREBP2 cleavage. P, the precursor form of SREBP2; N, the nuclear form of SREBP2. (H) The ratio of the densitometry of the nuclear SREBP2 over that of both nuclear and precursor forms of SREBP2. The ratio of cells without LDL repletion was defined as 1. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with WT cells refed with LDL at the same concentrations. Source data are available for this figure: SourceData F3.

FABP7 promotes LDL cholesterol transport to the PM. (A–C) WT and FABP7 KO2 SV589 cells were incubated in the cholesterol (Chol)-depleting medium for 16 h and refed with cholesterol-depleting medium containing 20 µg/ml LDL for the indicated periods as shown in A. Cells were then stained with the His6-EGFP-D4H probe and subjected to flow cytometry for EGFP fluorescence measurement (B) or harvested for biochemical analysis of cholesterol levels (C). The EGFP fluorescence intensity of WT and FABP7 KO2 cells cultured in normal medium is defined as 1. Data are presented as mean ± SEM (from three biological replicates). Student’s unpaired two-tailed t test. Compared with WT cells refed with LDL for the same periods of time. (D and E) WT and FABP7 KO2 SV589 cells were depleted of cholesterol as in A, and in the last 10 min of 16 h cholesterol depletion was treated with 1.5% 2-hydroxypropyl-β-cyclodextrin (HPCD). Cells were then refed with cholesterol-depleting medium containing 20 µg/ml LDL for the indicated periods as shown in D, and stained with the His6-EGFP-D4H probe and subjected to flow cytometry for EGFP fluorescence measurement. The EGFP fluorescence intensity of WT and FABP7 KO2 cells cultured in normal medium is defined as 1. Data are presented as mean ± SEM (from three biological replicates). Student’s unpaired two-tailed t test. Compared with WT cells refed with LDL for the same periods of time. (F) SV589 cells infected with lentivirus (Lenti-) expressing negative control (NC) or FABP7 were treated as in D, and EGFP fluorescence intensity was quantified. The EGFP fluorescence intensity of WT and FABP7 KO2 cells cultured in normal medium is defined as 1. Data are presented as mean ± SEM (from three biological replicates). Student’s unpaired two-tailed t test. Compared with cells infected with lentivirus expressing negative control (NC) and refed with LDL for the same periods of time. (G and H) WT and FABP7 KO2 cells were depleted of cholesterol for 16 h and treated with the indicated concentrations of LDL for 5 h at 37°C. Cells were subjected to SREBP2 cleavage analysis. (G) Representative immunoblots showing SREBP2 cleavage. P, the precursor form of SREBP2; N, the nuclear form of SREBP2. (H) The ratio of the densitometry of the nuclear SREBP2 over that of both nuclear and precursor forms of SREBP2. The ratio of cells without LDL repletion was defined as 1. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with WT cells refed with LDL at the same concentrations. Source data are available for this figure: SourceData F3.

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