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. 2021 Sep 3;7(36):eabf6033. doi: 10.1126/sciadv.abf6033

Fig. 3. RNA-seq analysis reveals cholesterol depletion in SN-treated GSC.

Fig. 3.

(A) Volcano plot showing the number of genes that are differentially expressed in 5 μM SN- versus DMSO-treated GSC (3 days). (B) ConsensusPathDB analysis of up- and down-regulated genes upon SN treatment. (C) Validation of the up-regulated genes in the cholesterol homeostasis pathway by qRT-PCR analysis (n = 4) (means ± SD). HSP70 and TBP serve as the housekeeping genes. *P < 0.01. (D) Western blot analysis of full-length (FL) and cleaved (Cl) SREBP2 protein after treatment of GSC TS543 with DMSO, 5 μM SN, or 5 μM lovastatin (3 days). Vinculin serves as the loading control. SREBP2 Cl/FL ratios are indicated. (E) Filipin staining of GSC TS543 after treatment with DMSO, 5 μM SN, or 5 μM lovastatin (3 days) (n = 10) (means ± SD). *P < 1 × 10−10. (F) Representative images of (E). Scale bar, 20 μm. (G) Cell viability of GSC TS543 treated with DMSO or 5 μM SN, in the presence exogenous cholesterol (n = 8) (means ± SD). **P < 1 × 10−8; *P < 1 × 10−4. (H) Filipin staining of 5 μM SN-treated GSC TS543 with or without cholesterol (0.75 μg/ml) (3 days) (n = 10) (means ± SD). *P < 1 × 10−7. (I) Representative images of (H). Two-tailed unpaired Student’s t test. Scale bar, 20 μm.