Fig. 5
Downregulation of Claudin5 promotes malignant progression and radioresistance through Beclin1-mediated autophagy. A Western blotting was performed to detect LC3 I, LC3 II and Beclin1 protein expression in TE1 cells transfected with Claudin5 siRNA, Beclin1 shRNA or treated with 3-MA for 4 h. GAPDH served as the loading control. B TEM images of autophagosomes in TE1 cells transfected with Claudin5 siRNA, Beclin1 shRNA or treated with 3-MA for 4 h. C Laser confocal fluorescence microscopy was used to analyze autophagosomes and autolysosomes in TE1 cells transfected with Claudin5 siRNA, Beclin1 shRNA or treated with 3-MA for 4 h. Merged image shows yellow puncta for autophagosomes and red puncta for autolysosomes. The puncta were analyzed by ImageJ software. D–E The ability of proliferation (D), invasion (E, magnification, × 40) and migration (F, magnification, × 200) in TE1 cells transfected with Claudin5 siRNA, Beclin1 shRNA or treated with 3-MA for 4 h was assessed by CCK-8 assay, transwell invasion assay and wound healing assay. G The radiosensitivity in TE1 cells transfected with Claudin5 siRNA, Beclin1 shRNA or treated with 3-MA for 4 h was assessed by clonogenic survival assay. The survival curves were plotted using GraphPad Prism Version 9.5.1 according to the single-hit multitarget model (SF = 1 − (1 − e^ [− kD])^N). Associated radiobiological parameters (D0, Dq, N, and SF2) were calculated and shown in Table 2. The statistical difference was assessed with the two-tailed unpaired Student t test in B, C, D, E, F and G. Error bars show the SD from three independent experiments. *p < 0.05