Fig 2: Inhibition of SREBP2 by 25-HC in HaCaT keratinocytes. Gene-expression analysis of the samples treated with HA-atRA or HA-C18:1 combined with 25-hydroxycholesterol (1 µM). The bars represent the mean ± SD of four independent experiments. The decreases in SREBF2, HMGCS1 and LDLR after the addition of 25-HC were significant (* p < 0.05, t-test) compared to respective HA-atRA and HA-C18:1 treatments.

Fig 3: SEMA3C upregulates lipogenic and cholesterogenic enzymes. (A) LNCaP cells treated with recombinant SEMA3C 1 μM in 5% CSS-supplemented phenol red-free RPMI for 24 h. mRNA expression of genes involved in cholesterol pathway by RT-qPCR method. SEMA3C was silenced in (B) C4-2 cells and (C) 22Rv1 cells by siRNA and mRNA expression of cholesterogenic genes was quantified in siSEMA3C-treated cells compared to respective control cells. (D) Increased activity of SREBP-responsive element in transiently transfected luciferase reporter plasmid into LNCaP cells followed by recombinant SEMA3C 1 μM or vehicle treatment (n = 6). (E) LNCaP cells were treated with erlotinib 2 μM approximately 2 h prior to the treatment with recombinant SEMA3C 1 μM. After 24 h, SREBP-responsive promoter activity was assessed by luciferase assay. (F) qPCR was performed to quantify relative mRNA expression of a panel of SREBP targets in LNCaP cells in the presence of erlotinib 2 μM. Only HMGCS1 mRNA expression level was affected. (G) The whole protein of LNCaPSEMA3C and LNCaPvector was immunoblotted to examine the protein level of genes involved in the cholesterol pathway. (mean ± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4: Distinct expression profiles of A549 cells treated by gilteritinib alone or gilteritinib plus 25HC using RNA-seq.A Volcano plot shows the differentially expressed genes between gilteritinib and gilteritinib plus 25HC-treated A549 cells. B Go term analyses of differentially expressed genes between gilteritinib and gilteritinib plus 25HC-treated A549 cells. C The mRNA levels of cholesterol biosynthesis genes in A549 and H1650 cells treated with gilteritinib alone, 25HC alone, or both agents, were measured by qRT-PCR. D Western blotting analysis of the expression of the ABCA1 and cholesterol biosynthesis proteins (HMGCS1, MVK, IDI1, FDFT1, SQLE, LSS) in A549 and H1650 cells treated with gilteritinib alone, 25HC alone, or both agents. E ABCA1 mRNA levels in A549 and H1650 cells treated with gilteritinib alone, 25HC alone, or both agents, were measured by qRT-PCR. F A549 cells were treated with 100 nM gilteritinib alone, or 3 μM 25HC alone, or in combination, filipin staining was used to label free cholesterol. ns, no significant difference, ***P < 0.001.

Fig 5: Overexpression of BHLHE40 promotes tumorigenesis via altering the oncogenic transcriptome in PCa cells. A) Integrated analysis flowchart of RNA‐ and ChIP‐seq in PANC‐1 cells. Downregulated genes after BHLHE40 knockdown with log2 fold change >1.5 times were intersected with binding loci of BHLHE40. B) Bubble plot showing GO enrichment analysis of downregulated genes related to fatty acid metabolism after BHLHE40 knockdown. C) Pie chart of the distributions of BHLHE40‐binding regions. D) Close view of the SREBF1 gene loci and BHLHE40 ChIP‐seq signal in PANC‐1 and BxPC‐3 cells. E) Luciferase reporter assay results showed that BHLHE40 overexpression promoted the luciferase activity of the pGL4.10‐SREBF1 promoter (WT) rather than the mutant promoter. F) Enrichment levels of H3K4me3 at the promoter region of SREBF1 in BHLHE40 knockdown or control PANC‐1 cell were determined via ChIP‐qPCR assay. G) Representative images of SREBF1 IHC staining of PCa tumor and para‐tumor tissues. Scale bar, 200 µm (upper) or 50 µm (bottom). H) High SREBF1 protein levels also correlated with reduced overall survival (OS) outcomes based on the analysis of tissue microarray data from the RJ‐1 dataset. I,J) Expression levels of SREBF1 and subsequent fatty acid metabolism related proteins (HMGCS1, ORP1, LSS, and SCD1) were measured using western blot and qPCR assays in BHLHE40 knockdown or control PANC‐1 cells. K) Representative images of double immunofluorescence staining of SREBF1 (green) and BHLHE40 (red) in PTO1. DAPI (blue) was used for nuclei counterstaining. Data presentation: E,F,J) data were the mean ± s.d. of n = 3 independent experiments. Statistical analysis: one‐sided Fisher's exact test for (B); unpaired two‐sided t‐test for E,F,J); log‐rank test for (H); *P < 0.05, **P < 0.01, ***P < 0.001.