Figure 4.

Autophagy promotes quiescent ovarian cancer spheroid cells to reenter the cell cycle. (a) Immunostaining of Oct-4 and Ki-67 in A2780 spheroid cells. Cells were cultured under spheroid culture condition for 48 h, trypsinized, and seeded back to attach to the plate for 4 h, fixed, stained with anti-Oct-4 antibody (green) and anti-Ki-67 antibody (red), counterstained with DAPI (blue), and observed with a fluorescence microscope. (b) Cell sorting of PY^Low and PY^High cells. A2780 spheroid cells were trypsinized, stained with Hoechst 33342 and pyronin Y, and sorted by flow cytometry. (c) Colony formation of sorted PY^Low and PY^High cells on soft agar. Sorted PY^Low and PY^High cells described in (b) were grown on soft agar for 28 d to form colonies. (d) Western blot analysis of the protein levels of Oct-4, NOTCH1, vimentin, ATG5, and LC3-I/II from extracts of freshly sorted PY^Low and PY^High cells or colonies formed by sorted PY^Low and PY^High cells. The colonies described in (c) were collected, resolved in SDS loading buffer, and subjected to Western blot analysis. The relative intensity of indicated proteins normalized to housekeeping protein was shown. LC3-II/LC3-I ratios were calculated. (e) Cell cycle analysis of Nc and ATG5 shRNA A2780 cell strains. The cells were trypsinized, fixed, digested with RNase A, stained with propidium iodide, and subjected to cell cytometry analysis (mean ± SEM, n = 3). (f) Colony formation of PY^Low and PY^High cells with or without rapamycin (Rap, 1 μM) on soft agar. 1.5 × 10⁴ of PY^Low and PY^High cells were cultured on soft agar with or without Rap for 14 d. The images were taken with a gel imaging system, and the colonies were counted with ImageJ software (mean ± SEM, n = 2).