Figure 2

Robust autophagic flux in mammospheres is correlated with the CSC/progenitor phenotype. (a) Immunoblot analysis of LC3 I and LC3 II levels in MCF-7 mammospheres. Cells were incubated in complete mammosphere medium or adherent medium for 16 h in the presence or absence of Baf A1 (final concentration: 100 nM) for 2 h. (b) The LC3 II/actin ratio and autophagy flux were determined as described in Figure 1c. ALDH1⁺ cells were isolated by fluorescence-activated cell sorting. ALDH1 activity in an MCF-7 mammosphere was measured by flow cytometry using the ALDEFLUOR reagent in the presence or absence of the ALDH1 inhibitor DEAB. Cells expressing ALDH1 were sorted using the ALDH1⁺ gate based on cells treated with the ALDH1 inhibitor, DEAB. ALDH1⁺ cells are collected, and then incubated in complete adherent medium. (d) Immunoblot analysis of p62, LC3 I and LC3 II levels in ALDH1⁺ cells (left) and bulk population mammospheres (right) were incubated in CM or with EBSS for the times indicated in the presence or absence of Baf A1 (final concentration: 100 nM) for 2 h. (e) The LC3 II/actin ratio and autophagy flux were determined as described in Figure 1.