Dii ldl

HepG2 cells were changed to serum-free media and incubated with 10 μg/ml DiI-LDL (lifetech) for 24 h at 37 °C in the dark. After incubation, the cells were washed with PBS and fixed in the presence of a 4 % paraformaldehyde, and the nuclei were subsequently stained with Hoechst dye. Finally, the cells were examined with fluorescence microcopy (DMI-4000B, Leica).

HeLa-Kyoto cells are a strongly adherent Hela isolate (gift from S. Narumiya, Kyoto University Japan) that, as we demonstrated earlier, enable reliable measurements of LDL-cholesterol uptake dynamics and show lipid homoeostatic mechanisms similar to those described for liver-derived cell models^{19 ,30 (link),31} . DiI-LDL (Life Technologies), DRAQ5 (Biostatus), Dapi (Molecular Probes), 2-hydroxy-propyl-beta-cyclodextrin (HPCD) (Sigma), Lipofectamine 2000 (Invitrogen) and Benzonase (Novagen) were purchased from the respective suppliers.

The amount of LDL absorbed by cells depends on the number of receptors on the cell membrane. To study uptake of LDL in hepatic cells, we carried out the DiI-LDL uptake assay. Briefly, the cells were plated in 24 -well plates and incubated in DMEM medium with 5% LPDS and 20 μg/mL of DiI-LDL (Life technologies, California, Carlsbad, USA) for 24 h at 37 °C in the dark. For the fluorescence microscopy, the cells were fixed in the presence of a 4% paraformaldehyde, and the nuclei were subsequently stained with Hoechst dye. Then the cells were examined with fluorescence microcopy (DMI-4000B, Leica, Männedorf, Switzerland). For the fluorescence quantification, the 10⁵ of cells were scraped and moved into the black 96 -well plate, then 400 μL of isopropanol was added into each well, and 200 μL aliquots were used for the analysis with an infinite M200PRO microplate reader (Tecan, Männedorf, Switzerland, excitation/emission at 530/580 nm). 0.5 mol/L of NaOH was used to lyse the remaining cells and aliquots of 10 μL for protein concentration assay. The ratio of DiI-LDL/protein concentration was used to normalize DiI-LDL uptake value to the cell protein.

The LDLR activity of the HepG2 cells was measured. Briefly, the cells were incubated in MEM medium with 5% lipoprotein‐deficient serum (Sigma‐Aldrich) and 20 μg/ml DiI‐LDL (Life Technologies) for 24 hrs at 37°C in the dark. For the fluorescence microscopy, the cells were fixed in the presence of a 4% paraformaldehyde, and the nuclei were subsequently stained with Hoechst dye. Then the cells were examined with fluorescence microcopy (DMI‐4000B, Leica, Buffalo Grove, Illinois, USA.). For the fluorescence quantification, 400 μl of isopropanol was added into each well, and 200μl aliquots were used for the analysis with a infinite M200PRO microplate reader (Tecan, Männedorf, Switzerland, excitation/emission at 530/580 nm). 0.5 mol/l of NaOH was used to lyse the remaining cells and aliquots of 10 μl for protein concentration assay. The ratio of DiI‐LDL/protein concentration was used to normalize DiI‐LDL uptake value to the cell protein.

HeLa Kyoto cells were grown in DMEM medium (Gibco) supplemented with 10% (w/v) foetal calf serum (FCS)(PAA) and 2 mM L-glutamine (Sigma) at 37 °C with 5% CO₂ and saturated humidity. Cells were plated at a density of 6 × 10⁴ per plate on the cell microarrays for solid-phase siRNA transfection^{60 (link)} and cultivated for 48 h before performing the LDLc uptake assay. For liquid phase transfection-based validation experiments, cells were plated in 12-well plates the day prior to transfection, and siRNA-transfected cells were cultivated for 48 hours. The assays to monitor cellular uptake of fluorescently-labelled LDLc (DiI-LDL) were performed as described also in the previous publications^{19 ,30 (link),31} . Cells cultured in serum-free medium (DMEM/2 mM L-glutamine/0.2% (w/v) BSA) and exposed to 1% 2-hydroxy-propyl-beta-cyclodextrin for 45 min were labelled with 50 µg/ml DiI-LDL (Invitrogen) for 30 min at 4 °C. DiI-LDL uptake was stimulated for 20 min at 37.0 °C. Endocytosis of labelled LDLc was stopped by washing with ice-cold media. We removed non-internalized DiI-LDL from the plasma membrane by acidic wash for 1 min in medium at pH 3.5 performed also at 4 °C. This was followed by fixation, counterstaining for nuclei (Dapi) and cell outlines (DRAQ5). For RNAi-based gene interaction screening, each of the five cell microarrays was assayed in 7–10 biological replicas.