Fig. 5

A Cholesterol concentration measured using 24(S)-hydroxycholesterol ELISA kit. Methionine depletion decreased cholesterol concentration in MZGC1 and MZGC2. B Enrichment analysis using GSEA showing that methionine depletion down-regulated the gene set of reactome cholesterol biosynthesis and up-regulated the gene set of gene ontology negative regulation of lipid storage. NES: normalized enrichment score, FDR-q: false discovery ratio. C Top10 genes from GSEA of affected enzyme related cholesterol biosynthesis shown in heat map and scheme of biosynthetic pathway of cholesterol (modified from Sitaula and Burris [5]). Selected genes are displayed in a black box with white text. D Immunoblot analysis of affected key enzymes related cholesterol biosynthesis. Methionine depletion for 5 days reduced HMGCS1 and FDPS. This experiment was performed in triplicate. (Full-length blots/gels are presented in Supplementary Figure S10B). E Differential expression of de novo biosynthesis and efflux of cholesterol hits with gene signature score in infiltrating cells against pseudopalisading cells around necrosis in all Ivy GAP samples. ABCA1 (ATP-binding cassette subfamily A member 1), which acts as a cholesterol efflux pump in the cellular lipid removal pathway, is up-regulated in pseudopalisading cells around necrosis. Genes of cholesterol biosynthesis such as HMGCS1, HMGCR, and FDPS are up-regulated in infiltrating cells. F Simvastatin for 3 days decreased colony formation in GICs. High magnification (left) and low magnification (right). G qPCR showed down-regulated SREBF2 mRNA expression by methionine depletion in MZGC4 and MZGC5. H Enrichment analysis by GSEA showed up-regulated Horton SREBF target gene set in an environment with sufficient methionine. I qPCR showed that short term statin administration up-regulated FOXM1 mRNA expression along with up-regulated SREBF2. J qPCR showed FOXM1 inhibitor FDI-6 administration for 48 h down-regulated SOX4, SOX2, and PROM1 mRNA expression