Fig. 8.

Lipoproteins in media affect the expression of FA metabolic genes and abundance of LDs in MDA-MB-231 BC cells. A–C: qRT-PCR was used to assess the impact of media lipoproteins on the expression of FA metabolism-related genes. Gene expression is displayed as 2^−ΔΔCT (relative to the control). Data are mean ± SEM of >3 experiments. Two-tailed unpaired t-tests: P ≥ 0.05 (ns), *P < 0.05, **P < 0.01, and ***P < 0.001. A: MDA-MB-231 BC cells cultured in LPDS media for 96 h displayed increased expression of FA synthesis and cholesterol metabolism genes and significantly decreased the expression of FA storage genes (DGAT1, PLIN2) compared with cells grown in matched-control media. The expression of FFA/lipoprotein uptake genes was variably affected by lipoprotein depletion. B: BC cells were grown for 96 h in matched or LPDS media ± VLDLs (100 µg/ml) to assess the impact of “addback” on the expression of FA synthesis genes. Data are normalized to the matched media control. Culturing cells in LPDS media resulted in upregulation of ACLY, FASN, and SCD (as shown in A). The addition of VLDLs (100 μg/ml) reduced the expression of these genes back to or below the matched-control media baseline. C: qRT-PCR of cells grown in standard media ± VLDLs (100 μg/ml, 72 h). VLDL supplementation increased the expression of FFA/lipoprotein uptake and FA storage genes; there were no significant changes or decreases in FA synthesis and cholesterol metabolism genes. D: Increased size and abundance of LDs observed in cells grown in media supplemented with VLDLs (100 μg/ml, 72 h) compared with the media alone control. Cells were stained with Hoechst 33342 nuclear stain (blue) and LipidTOX Red Neutral Lipid Stain (green) and visualized using fluorescence confocal microscopy.