Fig. 2: Blocking AURKA stalls GBM growth by reducing glycolysis.

a SF188 and GBM22 cells were cultured in galactose or glucose media for a week, treated with increasing concentrations of alisertib for 72 h, and cellular viability was analyzed (n = 4 independent samples). b SF188 GBM cells either transduced with a non-targeting (sgNT) or two different AURKA (sgAURKA) sgRNAs were cultured in galactose or glucose media for a week and treated with increasing concentration of alisertib. Cellular growth over time was determined (n = 4 independent samples). For c, d SF188 and GBM22 cells were treated with DMSO or alisertib and analyzed in the context of a glycolysis stress assay on a Seahorse XFe24 extracellular flux analyzer. Extracellular acidification rate (ECAR) is recorded at baseline, after injection of Glucose (G), Oligomycin (OM), and 2-DG. Quantification of glycolysis is shown in (d) (n = 5 independent samples). e Real-time PCR analysis of the mRNA level of genes related to glycolysis in SF188 and GBM22 cells treated with DMSO or alisertib for 24 h (n = 8 in SF188 + DMSO and GBM22 + DMSO, n = 4 in SF188 + Ali 0.1 μM and GBM22 + Ali 1 μM, independent samples). (HK2: SF188 ***p = 0.0002, GBM22: ***p = 0.0003; **p = 0.0082, ****p < 0.0001). 18S: internal control. FC: fold change. f SF188 and GBM22 cells were treated with DMSO or alisertib for 24 h and the whole-cell protein lysates were subjected to protein capillary electrophoresis. Vinculin is used as a loading control. g SF188 cells were transfected with non-targeting siRNA or specific AURKA siRNAs (single or pool) and the whole-cell lysates were analyzed by protein capillary electrophoresis for the indicated proteins. h Real-time PCR analysis of the mRNA level of genes related to glycolysis in SF188 transfected with non-targeting siRNA or specific siRNA targeting AURKA (n = 4 independent samples) (*p = 0.0179, ***p = 0.0001). i Shown are the ATP levels measured by polar LC/MS of SF188 cells treated with DMSO or 100 nM alisertib (n = 5 independent samples) (*p = 0.0221). For j, k SF188 and GBM22 cells were transfected with non-targeting or specific siRNA targeting AURKA and were analyzed in the context of a glycolysis stress assay on a Seahorse XFe24 extracellular flux analyzer. The graphs show glycolysis level in (j) (SF188: n = 2 in siNT, n = 3 in siAURKA; GBM22: n = 3 independent samples) (**p = 0.0032) or OCR/ECAR levels in (k) (SF188: n = 3; GBM22: n = 3 in siNT, n = 2 in siAURKA independent samples) (***p = 0.0001). l SF188 cells were transfected with c-Myc-WT and c-Myc mutant (T58A), treated with increasing concentrations of alisertib for 24 h, and the whole-cell lysates were analyzed by protein capillary electrophoresis. m SF188 cells were transfected with c-Myc-WT and c-Myc mutant (T58A), treated with 50 nM alisertib for 24 h, and analyzed on a Seahorse XFe24 extracellular flux analyzer. Shown is the quantification of ECAR level (n = 5 independent samples) (**p = 0.0073, n.s not significant). n SF188 and GBM22 cells were treated with alisertib for 24 h and were subjected to CHIP with an IgG as a negative control or a c-Myc specific antibody. The HK2 region was amplified by PCR (n = 3 independent samples). Statistical significance was assessed by a two-tailed student’s t-test. Data are shown as mean ± SD in (a−e, h−k, m, n). Source data are provided as a Source Data file.