Research Paper Volume 16, Issue 11 pp 9334—9349

Figure 1. Generation of MCF7 cells containing a BNIP3(L)-eGFP reporter, permits the selection of a sub-population of cells with high mitophagy activity, which show increased levels of lysosomes and basal mitophagy. (A) Diagram of BNIP3-eGFP-Puro^R and BNIP3L-eGFP-Puro^R constructs. MCF7 cells stably-transduced with the BNIP3(L)-GFP constructs were subjected to FACS sorting, to isolate the 5% highest GFP (GFP-high) and the 5% lowest GFP (GFP-low) sub-populations. (B) BNIP3 and BNIP3L protein levels in GFP-high and GFP-low sub-populations were assessed by Western blotting. Vinculin was used as a protein loading control. (C) Lysosomal mass was measured using Lysotracker Deep Red by flow cytometry. Data are shown as the mean ± standard deviation (SD) (n = 3). Statistical significance was determined using an unpaired Student’s t-test, * p ≤ 0.05, ** p ≤ 0.01. (D) Mitophagy levels were assessed using the Mitophagy Red Probe by flow cytometry. Data are shown as the mean ± standard deviation (SD) (n = 4). Statistical significance was determined using an unpaired Student’s t-test, * p ≤ 0.05.