Fig 2: Wnt3a enhances radioresistance and autophagy in SCCHN. SCCHN 6‐10B and Tu686 cells were infected with lentivirus‐mediated Wnt3a or control cDNA and then subjected to puromycin screening for 2 weeks. (A) Western blotting was used to examine the expression of proteins associated with the Wnt and autophagy pathways. Radioresistant changes in 6‐10B (B) and Tu686 (C) cells overexpressing Wnt3a or control cDNA were irradiated with the indicated doses of irradiation and survival fraction curves were obtained. Forced expression of Wnt3a in 6‐10B (D) and Tu686 (E) cells led to corresponding changes in γH2AX foci staining. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 3: Wnt3a enhances radioresistance via autophagy in vivo. (A) Schematic flow of the experimental design. Nude mice were subcutaneously injected with 6‐10B cells overexpressing Wnt3a and then subjected to two consecutive 4 Gy irradiation treatments and 3MA treatment when the tumour volume reached 100 mm3. Mice were finally killed at day 23 after tumour injection. (B) Tumour volumes were measured with callipers every 3‐4 days to monitor the growth pattern of mice. (C) The final gross tumours were captured. (D) Average tumour weight in each group was compared. (E) Immunohistochemistry staining was used to examine the expression of Wnt3a and Beclin1. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: Wnt3a enhances radioresistance via autophagy in vitro. SCCHN 6‐10B (A‐C) and Tu686 (D‐F) cells were infected with lentivirus‐mediated Wnt3a or control cDNA and then exposed to 3MA treatment. (A, D) Western blotting assays were used to evaluate the expression LC3B and Beclin1. (B, E) Survival curves of cells in each group were determined by clonogenic assays. (C, F) γH2AX foci were quantified by immunofluorescence staining. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 5: Irradiation activates Wnt signalling pathway and induces autophagy in SCCHN. Western blotting assays were used to evaluate the expression of proteins in the Wnt (A) and autophagy (B) signalling pathways in SCCHN 6‐10B and Tu686 cells exposed to 4 Gy irradiation and examined at different times (0, 12, 24 and 36 hours; Right panel) or subjected to different doses (0, 4 and 8 Gy) of irradiation and examined at 24 hours (Left panel). (C) Immunofluorescence staining was used to confirm the nuclear translocation of β‐catenin in 6‐10B and Tu686 cells at 6 hours post‐irradiation with 4 Gy irradiation. (D) Representative images of autophagosomes (indicated by red arrows) observed by transmission electron microscopy in 6‐10B and Tu686 cells at 3 hours post 4 Gy irradiation exposure. (E) Quantification of autophagosomes in 6‐10B and Tu686 cells. (F) 6‐10B cells were subcutaneously injected into the flanks of nude mice and irradiated with 8 Gy irradiation. Two weeks post‐irradiation, Western blotting assays were conducted to quantify the expression of proteins in the Wnt and autophagy signalling pathways. (G) Expression of Wnt3a, Beclin1 and Survivin in four cases of SCCHN tissues before and after radiotherapy as assayed by Western blotting assay. IR stands for irradiation. **P < 0.01