Fig. 3: ENSA plays a crucial role in cholesterol biosynthesis in TNBC. | Nature Communications

Fig. 3: ENSA plays a crucial role in cholesterol biosynthesis in TNBC.

From: Copy number amplification of ENSA promotes the progression of triple-negative breast cancer via cholesterol biosynthesis

Fig. 3

a GSEA of downregulated pathways after ENSA knockdown in MDA-MB-231 cells. The top 10 pathways (P < 0.05 and FDR q < 0.25) ranked by absolute normalized enrichment scores are shown. NES score and nominal P-value were given by GSEA software. b Enrichment plot of the cholesterol homeostasis pathway after ENSA knockdown in MDA-MB-231 cells. c Enrichment plot of the apoptosis pathway after ENSA knockdown in MDA-MB-231 cells. d Scatter plot showing the correlation of ENSA expression with the cholesterol biosynthesis pathway score in FUSCC TNBC data identified by ‘gsva’ method. Correlation coefficients were calculated using the Pearson test. Two-tailed P-values were given. e qRT-PCR analysis of the relative transcript levels of cholesterol biosynthesis pathway genes after ENSA knockdown in MDA-MB-231 cells. n = 4. Data are presented as mean ± SD. Two-tailed unpaired Student’s t tests. f Western blotting images showing proteins involved in the cholesterol biosynthesis pathway after ENSA knockdown in MDA-MB-231 cells. n = 3 independent experiments. g Heatmap displaying the concentration of cholesterol and intermediates in the cholesterol biosynthesis pathway after ENSA knockdown in MDA-MB-231 cells. n = 4. h Total cellular cholesterol contents in MDA-MB-231 cells were analyzed by LC-MS with normalization to cell quantity. n = 4. The center line corresponded to the median, the lower and upper hinges corresponded to the first and third quartiles, and the upper/lower whisker extends from the hinge to the largest/smallest value no further than 1.5 times interquartile range. Two-tailed unpaired Wilcoxon test. i Filipin III staining showing the cellular free cholesterol content in MDA-MB-231 and BT549 cells with ENSA knockdown. n = 3 independent experiments. j Colony formation of BT549 and MDA-MB-231 cells expressing control or ENSA shRNA after treatment with 2.5 μg/ml exogeneous cholesterol. n = 3. Data are presented as mean ± SD. Two-tailed unpaired Student’s t tests. Source data are provided as a Source Data file. GSEA gene set enrichment analysis, NES normalized enrichment score.

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