Fig. 2.

Influence of Nintedanib and Regorafenib on autophagy in CRC cells. (A, B) HT-29 and LoVo cells were exposed to the IC₅₀ dose of Nintedanib (light grey columns) or Regorafenib (dark grey columns) for the indicated times, and the induction of autophagy was determined with a fluorescence imaging kit. The values reflect the degree of autophagy in drug-exposed cells compared to the corresponding untreated control. The values represent the average values for 3 experiments, each done in duplicate. Bars, SD (n = 3), data were analyzed by two-way ANOVA test and were compared to the corresponding untreated control; ****p < 0.0001; ***p < 0.001; *p < 0.01. ns < 0.05 vs. non treated. (C, D) HT-29 and LoVo cells were exposed to the IC₅₀ dose of Nintedanib or Regorafenib for the indicated times followed by cytochemistry to reveal cells undergoing autophagy. Cells were marked with antibodies directed against LC3 (red) or p62/Sequestosome1 (green). The nuclei were counterstained with DRACQ (blue). Representative images from one out of 2 experiments. (E, F) HT-29 and LoVo cells were exposed to the IC₅₀ dose of Nintedanib or Regorafenib for the indicated times and the protein levels of the autophagy-associated proteins LC3, p62/Sequestosome1 and Beclin1 were determined by Western blot analysis. The protein levels of β-actin are shown as the loading control. The numbers reflect the expression of the indicated autophagy-associated protein in drug-exposed cells compared to the corresponding untreated control. Representative Western blots from one out of 2 experiments.