Journal: bioRxiv

Article Title: Cholesterol remodels the endoplasmic reticulum to control myofibroblastic CAF function

doi: 10.64898/2026.02.16.706237

Figure Lengend Snippet: (A) Measurement of the relative incorporation of [U- 14 C]glucose into protein, non-polar, RNA, DNA and polar fractions following 24 hours of labelling, n=3. (B) Relative incorporation of [1- 14 C]acetate into the lipid fraction of NFs and CAFs, n=3. (C) Gene Set Enrichment Analysis (GSEA) plot derived from RNA-Seq analysis of CAFs versus NFs, showing enrichment of a “lipid metabolic process” signature (GO:0006629). (D) Heatmap of cholesterol biosynthesis gene expression in NFs and CAFs, as determined by RNA-Seq analysis, n=3. (E) Quantification of relative total cholesterol levels in NFs and CAFs, n=3. (F) UMAP visualisation of 19,601 patient-derived breast tumour stromal cells analysed by single cell RNA-Seq (scRNA-Seq) from the Human Breast Cancer Atlas . Stromal clusters corresponding to immunomodulatory CAFs (iCAFs), myCAFs, perivascular (PVL) and endothelial cell subsets are indicated by colour (top). Feature plots illustrating expression of SQLE (bottom left) and SC5D (bottom right) in breast tumour stromal cell clusters. Gene expression is represented by log-normalised expression values. The myCAF cluster is delineated by a dashed line. (G) Representative filipin staining (green) of free cholesterol in NFs and CAFs. Nuclei are stained with Draq5 (blue). Dashed boxes indicate magnified regions shown to the right. White scale bar represents 10 µm, yellow scale bar represents 5 µm. (H) Quantification of relative cholesterol levels in the ER-enriched fraction isolated from NFs and CAFs, n=3.

Article Snippet: Filipin was removed, and cells were washed and imaged in PBS containing 5 μM Draq5 staining solution (Miltenyi Biotec) using a Zeiss Elyra PS.1 confocal microscope (Zeiss) equipped with a 63X/1.4 oil DIC objective.

Techniques: Derivative Assay, RNA Sequencing, Gene Expression, Single Cell, Expressing, Staining, Isolation