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. 2019 Oct 29;9:15458. doi: 10.1038/s41598-019-51865-8

Figure 2.

Figure 2

Higher density leads to activation of LXRβ in glioma TS cells. (A) Hypothetical mechanism by which LXRβ maintains the viability of densely plated glioma cells. High cell density leads to increased intracellular cholesterol levels through activation of the mevalonate pathway. Oxysterols, activating ligands for LXRβ, are generated through intracellular oxidation of cholesterol. Activated LXRβ turns on the expression of genes that lower intracellular cholesterol, including the cholesterol efflux transporter, ABCA1. Cells remain viable due to the decreased levels of cholesterol and oxysterols. (B) Fold change in 24-Hydroxycholesterol (24-OHC) levels in TS543, TS576 and TS616 glioma cells. Levels were normalized to sparse for each cell line. Error bars indicates SEM for at least 3 experiments. *p < 0.05; ****p < 0.0001 for One Way ANOVA of sparse vs. dense. (C) Fold change in 7-HOCA levels in TS543 and TS616 glioma cells. Each x or closed circle represents an independent measurement. **** =  < 0.0001 for paired t-test of sparse vs. dense (D) Quantitative real time PCR of ABCA1, NR1H2 (LXRβ), and NR1H3 (LXRα) in TS543 and TS576 cells treated for 24 hrs with 5 μM GW3965 or DMSO control. Data are the average of 3 biological replicates and are normalized to GAPDH and untreated for each gene and cell line. Bars = SEM. (E) Quantitative real time PCR of ABCA1, NR1H2, and NR1H3 in TS543 and TS576 cells treated for 24 hrs with 5 μM SR9243 or DMSO control. Data are the average of 2 biological replicates and are normalized to GAPDH and untreated for each gene and cell line. Bars = SEM. (F) Western blot analysis of ABCA1 in TS543, TS576 and TS600 LXRBβ CRISPR knockout cells comparing sparse vs dense conditions in fractions of cytosolic vs membrane components. Data shown are representative of at least 3 biological replicates.