Fig. 5
Cholesterol regulates FACI transport from the PM to ERC in a clathrin-dependent manner. A Cholesterol loading assay. The AML12-mRuby2-FACI cell line, which stably expresses mRuby2-FACI in a tetracycline-inducible manner, was used in cholesterol (Chol) loading assay. Before the day of the experiment, doxycycline (dox) was added into the medium of AML12-mRuby2-FACI cells to induce mRuby2-FACI expression. For cholesterol loading, cells were treated with 40 µg/mL water-soluble cholesterol for 1 h. Z-stack scans were taken by spinning disc confocal microscopy (SDCM) before and after the cholesterol treatment for the same cells. Representative xy and Z-stack xz images were shown. Scale bar, 10 µm. B Time-lapse confocal live-cell imaging. AML12-mRuby2-FACI cells were treated with 40 µg/mL water-soluble cholesterol together with 2 ng/mL TopFluor cholesterol (TF-chol). Images were acquired with a 10 s time interval. The co-translocation of mRuby2-FACI and TopFluor cholesterol from the PM to cytosol was tracked (white arrowhead). Green: TopFluor cholesterol; Magenta: mRuby2-FACI. N nucleus. C cytoplasm. Scale bar, 5 µm. C AML12-mRuby2-FACI cells were treated with 40 µg/mL water-soluble cholesterol together with 2 ng/mL TopFluor cholesterol for 1 h. Images of cells before and after cholesterol treatment were shown. Scale bar, 10 µm. D CME blockade assay. AML12-mRuby2-FACI cells were pretreated with Pitstop-2 for 15 min and then incubated with water-soluble cholesterol for 1 h. Images of cells before and after cholesterol treatment were shown. Green: TopFluor cholesterol; Magenta: mRuby2-FACI. Scale bar, 10 µm. E Cholesterol depletion assay. Cells were treated with a cholesterol-depleting medium (with 5 mM MβCD) for 2 h. Cells with mock treatment were used as control. Cells were fixed and examined by confocal microscopy. Scale bar, 10 µm. F Percentages of intracellular FACI signals against whole cell FACI signals were quantified. n = 30. Scale bar, 10 µm. Ctrl: control. Statistical significance was evaluated by two-tailed unpaired Student’s t test. ***: P < 0.001. G Disruption of cytoskeleton impairs FACI trafficking from the PM to ERC. The scheme of drug treatment. Cells were first treated with 2 µM nocodazole (NDZ) or 2 µM cytochalasin D (CytD) for 30 min. Water-soluble cholesterol (40 µg/mL) was then added into the culture media for another 60 min. Images were acquired at the indicated time points (red arrow). H Cells were treated as shown in (G). Cells were kept on the TOKAI HIT stage-top incubator with 5% CO2 at 37 °C and imaged by SDCM at the indicated time points. Scale bar, 10 µm. I Quantification of the intracellular mRuby2-FACI fluorescence intensity relative to the intensity of the whole cell. n = 25 ~ 35. Statistical significance was evaluated by one-way ANOVA with Tukey's post hoc tests. *: P < 0.05. ***: P < 0.001. ns: not significant