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. 2012 Jun 25;32(18):2261–2272. doi: 10.1038/onc.2012.252

Figure 1.

Figure 1

Autophagic flux in mammospheres and adherent cells. (a) Immunoblot analysis of LC3 I and LC3 II levels in MCF-7 adherent cells (left) and mammospheres (right). Cells were incubated in complete medium (CM) or with EBSS for the time indicated in the presence or absence of Baf A1 (final concentration: 100 nℳ) for 2 h. (b) Immunoblot analysis of LC3 I and LC3 II levels in primary adherent cells (left) and primary mammospheres (right). Cells were incubated in CM or with EBSS for 4 h in the presence or absence of Baf A1 (final concentration: 100 nℳ) for 2 h. Actin immunoblotting was used as a loading control. The LC3 II/actin ratio was determined using the Bio1D software. Results are representative of three independent experiments. Control was set as 1 in adherent untreated cells. The autophagic flux determined as the ratio between the LC3 II levels with Baf A1 and without Baf A1 in (a) and (b) (bottom) is expressed in arbitrary units. (c) MCF-7-mCherry-GFP-LC3 adherent cells and mammospheres were incubated in CM or with EBSS for the times indicated, fixed and then visualized by confocal microscopy. Bars: 15 μm. (d) The number of GFP+/mCherry+(yellow) and GFP−/mCherry-LC3+ (red) dots was scored on ∼50 to 100 cells. The data are presented as means±s.d. from three independent experiments and analyzed using Student's t-test (*P<0.05, **P<0.01).