Fig. 4: The unconventional autophagic response generates Cherry-LC3/quinacrine double-positive vesicles and inhibits ATP release during cell death.
From: An unconventional autophagic pathway that inhibits ATP secretion during apoptotic cell death
a–d Induction of Cherry-LC3/quinacrine double-positive vesicles in Bax-/- cells expressing full-length ATG16L1 but not in those expressing ATG16L1-ΔWDD. a Bax-/- MEFs engineered for ATG16L1 expression and expressing Cherry-LC3 were transduced with the indicated BH3-only molecules and treated with 25 μM zVAD.fmk 7 h later. Cells were stained with quinacrine 17 h post-transduction and analysed in vivo by confocal microscopy. Shown are representative confocal pictures (Ch-LC3: Cherry-LC3 (magenta); Quin.: quinacrine (green)). Scale bars represent 10 μm. b–d Quantification of the phenotypes shown in (a). Graphs display the number of Cherry-LC3-positive puncta per cell (b), the number of quinacrine-positive puncta per cell (c), and the percentage of Cherry-LC3-positive signal that colocalizes with the quinacrine-positive signal (d). Data are presented as box-plots where the central line represents the median value, the box shows percentiles 25-75 and the whiskers include the most extreme values (n = 25 cells; ****P < 0.0001 two-tailed Student’s t-test). Numeric P-values are shown. e Increased levels of ATP released by apoptotic Bak-/- MEFs. The shown MEF strains were transduced with the indicated BH3-only molecules for 22 h (left), or treated with MTX (2 μM) for 24 h (right), and the levels of extracellular ATP measured at 14 and 22 h (BH3-only molecules) or 16 and 24 h (MTX). Graphs show mean values −/+ s.d. of cumulative data resulting from the sum of the luciferase activity units obtained at both time points from triplicate experimental points (n = 3 biological replicas; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 two-tailed Student’s t-test). Numeric P-values are shown. f Increased levels of ATP released by apoptotic Bak-/- MEFs expressing ATG16L1-ΔWDD. Bax-/- MEFs engineered for ATG16L1 expression were transduced with the shown BH3-only molecules (left) or treated with MTX (right), and the levels of extracellular ATP measured and displayed as in (e). Source data are provided as a Source Data file.
