Figure 1.

Panel (a): Proliferation curve of SAOS- and SAOS400-derived radioresistant cells. Cells were counted in duplicate at the indicated time points with Trypan blue stain using an automatic cell counter, as described. The mean of three experiments is shown ± SD. * p < 0.05 SAOS vs. SAOS400, Student’s t test. Panels (b,c): Comparison of the expression of autophagy markers in SAOS vs. SAOS400 cells. Cells were cultured for 96 h after autophagosome staining with Cyto-ID selective dye (FITC). The nuclei were stained with Hoechst dye, and the intracellular number was expressed as the FITC/DAPI ratio. The bar graphs (b) represent the mean ± SD, and symbols indicate significance: *** p < 0.001, with respect to SAOS. Representative photographs of untreated cells after Cyto-ID staining are reported in (c). Panels (d,e): Immunoblot analysis of the autophagy marker LC3I/II in SAOS and SAOS400 cells cultured at the indicated times. Densitometric analysis (numbers between panels indicate mean ± SD) was calculated by normalizing the expression of LC3-II with α-tubulin, as described in the Materials and Methods section (Section 4). Symbols indicate significance: ** p < 0.01 SAOS400 with respect to SAOS cells.