Figure 1.

Effect of tyrosine kinase receptor inhibitor (K252a) on TrkB/BDNF expression and autophagy induction. SW480 and SW620 were treated with 100 nM of K252a for 3 hrs, recovered, washed and total RNA and proteins were extracted (see Materials and methods). (A) BDNF, Full Length (FL) and Truncated (Tr) TrkB, transcripts expression was assessed by qPCR. Histograms are representative of three independent experiments. The fold expression was obtained by the comparative cycle threshold method using the GAPDH RNA expression level as internal control. (B) BDNF, FL TrkB, Tr TrkB, phospho‐TrkB (P TrkB), AKT, phospho‐AKT (P AKT), mTOR and phospho‐mTOR (P mTOR), proteins expression were assessed by Western blotting. The density of each band representative for protein expression was calculated with ImageJ software. Images show representative results of three experiments. Statistically results are explained in comparison with control cells, normalized at 1 (*P < 0.05; **P < 0.01; ***P < 0.001). (C) Staining of SW480 and SW620 was realized with anti‐BDNF antibody (green), anti‐TrkB antibody (red) and DAPI (blue). Images were obtained through confocal microscopy (magnification ×1000). (D) Beclin1, ATG5 and LC3 proteins expression was assessed by Western blotting after treatment with either K252a (100 nM, 3 hrs) or with CQ (25 μM, 3 hrs). The density of each band representative for protein expression was calculated with ImageJ software. Images show representative results of three experiments. Statistically results are explained in comparison with control cells, normalized at 1 (*P < 0.05; **P < 0.01; ***P < 0.001). (E) Staining of SW480 and SW620 was realized with anti‐LC3 antibody (green) and DAPI (blue) through indirect immunofluorescence. Rapamycin (20 nM, 3 hrs) was used as a positive control for autophagy induction. Relative fluorescence quantification was accomplished by using a green surface plot with the ImageJ software application.