Fig. 5.

Influence of autophagy modulation on the cytotoxicity of Nintedanib and Regorafenib. Autophagy induction was monitored by expression of LC3II in HT-29 (A) and LoVo cells (B) following 24 h exposure to Nintedanib or Regorafenib in the absence or presence of 3-MA. The protein levels of β-actin are shown as the loading control. The numbers reflect the expression of total LC3 in drug-treated cells compared to the untreated HT-29 cells. HT-29 cells (C) and LoVo cells (D) were exposed to Nintedanib (left) or Regorafenib (right) for 120 h in the absence or presence of 3-MA (2.5 mM) and the viability was determined by the MTT viability assay. The curves represent the average of 3 independent experiments, each done in triplicate. Bars, SD (n = 3). (E) HT-29 cells were transfected with shRNA toward Beclin1 or, as control, empty vector. Expression of BECN1 mRNA in parental, control or Beclin1-transfected cells. (F) Expression of Beclin1 protein in parental, control or shBeclin1-transfected cells. The protein levels of β-actin are shown as the loading control. The numbers reflect the expression of Beclin1 in transfected cells compared to the parental cells. (G, H) Autophagy in parental, control and shBeclin1 cells following 24 h exposure to Nintedanib or Regorafenib. Autophagy induction was monitored by expression of LC3II. The protein levels of β-actin are shown as the loading control. The numbers reflect the expression of total LC3 in drug-treated cells compared to the corresponding untreated cells. (I, J) The cytotoxicity of Nintedanib or Regorafenib was determined by colony formation of parental, control and shBeclin1 cells after 14 days continued drug exposure. The curves represent the average of 3 independent experiments, each done in triplicate. Bars, SD (n = 3). Representative Western blots from one out of 2 experiments.