Figure 2
Notch-c-Myc signaling contributes to the stromal cell-mediated glycolytic switch. (A) Changes of the c-myc relative gene expression were evaluated in primary CLL cells (n = 5) cocultured with HS-5 cells for 6 days as compared with cells cultured alone (set as 1). (B) Changes of cyclinb1 and cdk4 relative gene expression, both c-Myc target genes, in CLL cells (n = 6) with/without stromal contact as quantified by qPCR. (C) Changes of the relative expression of genes indicative for a canonical (hes-1) and noncanonical (deltex) Notch pathway activation is shown for CLL cells (n = 6) cocultured with HS-5 cells for 6 days as compared with cells cultured alone (set as 1). (D) Upregulation of Notch receptors was evaluated by FACS in nonpermeabilized CLL cells (n = 4) cultured in the presence or absence (set as 1) of HS-5 cells. (E) Relative gene expression of glycolytic enzymes (hk2, ldha, eno1), of c-myc, and of the c-Myc target gene cyclinb1 in primary CLL cells (n = 4-8) cocultured with HS-5 cells ± the GS inhibitor RO4929097 in a noncytotoxic dosage (2.5 µM). Expression levels in untreated cells are set as 100%. (F) The ECAR as a surrogate for glycolysis was measured in CLL cells (n = 4) using an XFe96 flux analyzer under baseline conditions, in response to glucose administration, and upon application of oligomycin, which blocks mitochondrial ATP production. CLL cells were cultured in the presence/absence of HS-5 cells and of the GS inhibitor RO4929097 (RO) as indicated. (G) Primary CLL cells (n = 4) cocultured with HS-5 stromal cells were treated with either fludarabine (Flud) or RO4929097 (RO) or both as indicated. The specific cell death was assessed by FACS. (H) The ECAR, which represents a surrogate for the overall glycolytic flux, was measured in purified primary CLL cells carrying a wild-type (WT) (n = 4) or a mutated (MT) (n = 5) Notch-1 under basal conditions, in response to glucose, and upon blocking the mitochondrial ATP generation by oligomycin. Bars indicate the standard error of the mean. * P < .05; ** P < .005; *** P < .001.

Notch-c-Myc signaling contributes to the stromal cell-mediated glycolytic switch. (A) Changes of the c-myc relative gene expression were evaluated in primary CLL cells (n = 5) cocultured with HS-5 cells for 6 days as compared with cells cultured alone (set as 1). (B) Changes of cyclinb1 and cdk4 relative gene expression, both c-Myc target genes, in CLL cells (n = 6) with/without stromal contact as quantified by qPCR. (C) Changes of the relative expression of genes indicative for a canonical (hes-1) and noncanonical (deltex) Notch pathway activation is shown for CLL cells (n = 6) cocultured with HS-5 cells for 6 days as compared with cells cultured alone (set as 1). (D) Upregulation of Notch receptors was evaluated by FACS in nonpermeabilized CLL cells (n = 4) cultured in the presence or absence (set as 1) of HS-5 cells. (E) Relative gene expression of glycolytic enzymes (hk2, ldha, eno1), of c-myc, and of the c-Myc target gene cyclinb1 in primary CLL cells (n = 4-8) cocultured with HS-5 cells ± the GS inhibitor RO4929097 in a noncytotoxic dosage (2.5 µM). Expression levels in untreated cells are set as 100%. (F) The ECAR as a surrogate for glycolysis was measured in CLL cells (n = 4) using an XFe96 flux analyzer under baseline conditions, in response to glucose administration, and upon application of oligomycin, which blocks mitochondrial ATP production. CLL cells were cultured in the presence/absence of HS-5 cells and of the GS inhibitor RO4929097 (RO) as indicated. (G) Primary CLL cells (n = 4) cocultured with HS-5 stromal cells were treated with either fludarabine (Flud) or RO4929097 (RO) or both as indicated. The specific cell death was assessed by FACS. (H) The ECAR, which represents a surrogate for the overall glycolytic flux, was measured in purified primary CLL cells carrying a wild-type (WT) (n = 4) or a mutated (MT) (n = 5) Notch-1 under basal conditions, in response to glucose, and upon blocking the mitochondrial ATP generation by oligomycin. Bars indicate the standard error of the mean. * P < .05; ** P < .005; *** P < .001.

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