Fig. 6.

The verification of autophagy level in vitro (HOS) and in vivo. Autophagy-related genes expression were determined by RT-qPCR and compared between 3D models and 2D or CSC models (A). The data are expressed as mean ± SD (n = 4). **p < 0.01 indicate statistical significance respectively. Representative immunohistochemistry (B) and immunofluorescence (C) photos of LC3-high and LC3-low osteosarcoma biopsies. Scale bar = 200 μm. Immunofluorescence staining of autophagy markers in 2D, CSC and 3D osteosarcoma models. ULK1 (D), TSC2 (E) and LC3 (F) are shown in green, while nuclei are shown in blue. Scale bar = 100 μm. Kaplan-Meier plot of the overall survival for 88 patients according to ULK1 (G), TSC2 (H) and LC3B (I) expression defined by cut-offs of 163.6 (G), 290.8 (H) and 662.9(I).