FIG 5.

S-phase-arrested NIH 3T3 cells display increased numbers and dispersed localization of lipid droplets. (A) Experimental scheme of NIH 3T3 cell cycle arrest by thymidine administration. (B) Cell cycle analysis was performed on NIH 3T3 cells after synchronization by confluence and serum starvation (left) or after thymidine incubation (right). The percentage of cells in each stage of the cell cycle (G₀/G₁, S, and G₂/M) is shown. (C and D) Lipid droplet quantification through flow cytometry. G₀/G₁- or S-phase-arrested NIH 3T3 cells were stained with Bodipy and were analyzed by flow cytometry. Data are shown in a fluorescence intensity overlay graph (C) or are expressed as the mean fluorescence emission intensity (MFI) (D). Data are means; error bars, standard deviations. An asterisk indicates a significant difference in values (*, P < 0.05). (E) Lipid droplet quantification by Oil Red O staining. G₀/G₁- or S-phase-arrested NIH 3T3 cells were fixed on coverslips and stained with the fluorescent dye Oil Red O. The number of lipid droplets per cell was determined using ImageQuant TL software (GE Healthcare). Data are means ± standard deviations from three independent experiments. An asterisk indicates a significant difference in values (P < 0.05). (F) Subcellular localization of lipid droplets. G₀/G₁- or S-phase-arrested NIH 3T3 cells were fixed on coverslips and were treated with a polyclonal anti-α-tubulin antibody for the detection of microtubules (red), with the hydrophobic fluorescent probe Bodipy for lipid droplet staining (green), and with DAPI for nuclear staining (blue). Fluorescence microscopy analysis shows cellular morphology and the subcellular localization of lipid droplets in G₀/G₁- or S-phase-arrested NIH 3T3 cells. All data are representative of the results of at least three independent experiments.