Fig. 5

Validation of increased mitochondria sequestration with autophagic flux capacity. a HL-1 and HeLa cells stably expressing GFP-LC3 were transfected with RFP-Bnip3 WT, 2SA (inactive LIR) and 2SE (active LIR). Imagestream X measurements of cellular autophagosome content in HL-1 and HeLa cell lines were performed at 48 h of expression. The distribution of population measurements is shown as a Violin plot, with median indicated on plot, and mean response listed. b HeLa cells were transfected 24 h with GFP-LC3B and either RFP-Bnip3 WT or 2SE, and treated 6 h with CCCP (20 μM). From acquired Z-stacks, channels were segmented and the co-localization of mitochondria with autophagosomes was calculated on a slice-by-slice basis. The co-localized fraction is reported. c Examples of single-cell, Z-stack projections of mitochondria (red) with co-localized autophagosomes (yellow) for WT and 2SE mutant in MCF7 cells