Figure 5. Influence of bevacizumab and nintedanib on CRC cells under normoxia and hypoxia.
(A) Western blot analysis of HIF-1alpha, HIF-2alpha and beta-actin in DLD-1 and HT-29 cells after 24 hrs exposure to nintedanib (0,01, 0,1 or 1 μM) or bevacizumab (250 μg/ml) under normoxia or hypoxia (1% O2). (B) Induction of VEGF mRNA in DLD-1 (grey columns) and HT-29 (black columns) cells following 24 hrs exposure to nintedanib (0,01, 0,1 or 1μM) or bevacizumab (250 μg/ml) under normoxia or hypoxia. VEGF expression was normalized with 36B4 mRNA and is the average of three independent experiments. The statistical analysis of experimental data was performed using a Student's paired t-test comparing the treatment group with the vehicle control. Bars, SD; * p < 0,05; ** p < 0,01; *** p < 0,001. (C) Hypoxia-induced apoptosis in DLD-1 (grey columns) and HT-29 (black columns) cells after hypoxia (1% O2) exposure for the indicated times as measured by the TUNEL assay (left) or by the formation of catalytically active cleaved caspase 3 (right). Values are the averages of at least two independent experiments done in duplicate. The statistical analysis of experimental data was performed using a Student's paired t-test comparing the treatment group with the vehicle control. Bars, SD; * p < 0,05; ** p < 0,01; *** p < 0,001. (D) Viability of DLD-1 cells (left) or HT-29 cells (right) after 24 hrs exposure to the indicated concentrations of nintedanib under normoxia (grey circles) or hypoxia (black circles) for 24 hrs followed by 96 hrs post-incubation under normoxia and MTT determination. Bars indicate SD and are shown when they exceed symbol size. The data represents three independent experiments each done in duplicate.