Figure 1

FACS analysis of the autophagy activity in lentiviral RFP-GFP-LC3B-transduced human mammary cells. (A) Design of the lentiviral RFP-GFP-LC3B reporter construct. Dual RFP⁺GFP⁺ cells indicate the presence of phagophores and autophagosomes. RFP^highGFP^low fluorescence is indicative of the presence of autolysosomes. (B) Confocal determination of RFP⁺GFP⁺LC3B puncta in sorted fractions of G⁺ and R⁺ cells within stably RFP-GFP-LC3B-transduced MCF10A cells. (C,D) Effect of a 3-h incubation of RFP^-GFP^-LC3B-transduced MCF10A cells in SF7 medium with 1 µM rapamycin or the equivalent concentration of DMSO (0.5%). Bar graph (D) showing the R⁺ and G⁺ cell content of RFP-GFP-LC3B-transduced MCF10A cells determined after a 3-h incubation in DMEM/F12 media with 1 µM rapamycin or the equivalent concentration of DMSO (0.5%). (E) Validation of the FACS-based quantification of R⁺ and G⁺ MCF10A cells to detect changes in their LC3B-I and LC3B-II protein content determined by WB analysis. Statistical analysis: P values were determined using a paired t-test test (B) unpaired t-test (D,E). *P < 0.05; ***P < 0.001.