Figure 4.

DAPGreen, DAPRed, and DALGreen label canonical autophagy

(A) GFP-LC3-transfected WT MEFs were preincubated with DAPRed (0.1 μM) and LysoTracker Deep Red (0.5 μM), treated with 1 μM rapamycin for 4 h, and time-lapse three-color images were acquired. Whole-cell images are shown in Figure S9. ROI #1: On the punctum labeled only with GFP-LC3 (lightgreen arrowheads), a DAPRed signal colocalized at 70 s (yellow arrowheads). ROI #2: On the GFP-LC3/DAPRed-positive punctum (yellow arrowheads), a LysoTracker signal (white arrowheads) colocalized at 90 s. Bars = 1 μm.

(B and C) The indicated MEFs were prestained with DAPGreen (0.25 μM) or DALGreen (1 μM) plus DAPRed (0.1 μM), and then starved for 5 h. Then, the cells were analyzed using fluorescence microscopy. Representative images are shown in (B). Bars = 10 μm. In (C), the fluorescent area of each probe per cell was calculated from the images. Data are shown as the mean ± S.D. The total number of images used for analysis is given as the n. Black and red asterisks indicate statistical significance vs. the value of WT and vs. Ulk1/2-deficient cells, respectively (one-way ANOVA followed by the Tukey post hoc test; ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significant difference).