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. 2019 May 31;9:463. doi: 10.3389/fonc.2019.00463

Figure 1.

Figure 1

HIF-1α activity modulation and consequences on the apoptotic pattern. ELISA-based HIF-1α activity quantification after 48 h of 25 μM TMZ, 10 mM MET, or COMBO treatment under normoxic and hypoxic conditions in U251 (A) and T98 (B) cells. The data are expressed as absorbance at 450 nm. *p < 0.05, ***p < 0.001 vs. control under normoxic conditions; #p < 0.05, ##p < 0.001 vs. control under hypoxic conditions. The evaluation of HIF-1 target gene, VEGF, was performed by the ELISA of the VEGF released by U251 (C) and T98 (D) glioma cells in cell medium after 25 μM TMZ, 10 mM MET or COMBO treatment. *p < 0.05, ***p < 0.001 vs. control under normoxic conditions; #p < 0.05, ##p < 0.001 vs. control under hypoxic conditions. Evaluation of time-response viability of responsive and resistant cells after TMZ or COMBO. Cell viability was assessed by means of a Trypan blue exclusion test and expressed as the percentage of viable cells after 24, 48, or 72 h of treatment under hypoxic condition in U251 (E) and T98 (F) cells. **p < 0.01; ***p < 0.001 vs. control cells. The induction of pro-apoptotic (Bad and Bax) and anti-apoptotic genes (Bcl-2) was analyzed by means of real-time PCR in glioma cells treated with TMZ under hypoxic condition (G,H). The data were normalized to β-actin, and the ΔΔct values were expressed as the ratio between the mean values in the responsive and resistant cells [Fold of Induction (FOI)]. *p < 0.05; ***p < 0.001 treated vs. control cells. Mean values ± SD of three independent experiments.