Fig. 1
Comparative gene expression profiling of the LD+ phenotype in GBM patient tumors and cells. a, GBM cells (U-87 MG) were incubated in serum-free medium at normoxic and hypoxic (1% O2) conditions for 48 h without or with extracellular lipid (LDL, 50 μg/ml) and analyzed for LDs by confocal microscopy; blue, Hoechst nuclear stain; green: LipidTox LD stain. Shown are representative images of at least three independent experiments. Scale bar, 20 μm. b, Immunofluorescence images of GBM patient tumors. Left panel (GLUT1 + LD) shows co-localization of the hypoxia marker GLUT1 (green) and LDs (Nile red; indicated by red arrowheads). Mid (Tumor CellLD-) and right (Tumor CellLD+) panels show typical tumor areas with and without LDs (LipidTox, green). Blue, Hoechst nuclear stain. Scale bar, 100 μm. c, Schematic overview of the experimental set-up of gene expression analyses. Left: GBM cells (U-87 MG) were incubated in serum-free (SF) medium at normoxic and hypoxic (1% O2) conditions for 48 h with no addition of extracellular lipid or in hypoxia with extracellular lipid (LDL, 50 μg/ml), and subsequently analyzed for gene expression with the HumanHT-12v4 Expression Illumina BeadChip (N = 3 independent experiments). Right: GBM tumors (N = 5 patients) were cryosectioned, and LD-negative (Tumor CellLD-) and LD-positive (Tumor CellLD+) tumor areas (N = 8 and 10, respectively) were isolated by laser microdissection for gene expression analysis with Affymetrix Clariom D Pico gene array. d, Heatmap showing the differentially upregulated genes in LD+ (Hypoxia + LDL) vs LD− (Normoxia) GBM cells and how the same set of genes are differentially expressed in Tumor CellLD+ vs Tumor CellLD- areas from GBM patient tumors. e, Biological processes plot generated from the commonly upregulated genes shown in (d) according to Gene Ontology level 4 annotation, P < 0.01. f, Cellular component analysis generated as described in (e). g, Significantly upregulated genes of interest in Tumor CellLD+ vs Tumor CellLD- from patient tumors (gray bars) and LD+ (Hypoxia + LDL) vs LD− (Normoxia) GBM cells (green bars). Data in (d) and (g) are presented as log2 of fold-change (FC)