Figure 6.
DAPGreen, DALGreen, and DAPRed also detect alternative autophagy stimulated by genotoxic stress
(A and B) The indicated MEFs were prestained with DALGreen (1 μM) and DAPRed (0.1 μM) and were treated with 10 μM etoposide for 10 h. Then, the cells were analyzed using fluorescence microscopy (A). Bars = 10 μm. From the images, the fluorescent area of DAPRed and DALGreen per cell was calculated (B). 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 (Mann-Whitney U test; ∗∗p < 0.01, ∗p < 0.05; ns, no significant difference).
(C) Staining of alternative autophagic structures with DAPRed. Atg5-deficient MEFs stably expressing with LAMP1-GFP were preincubated with DAPRed (0.1 μM) and treated with etoposide (10 μM) for 10 h. Then, cells were fixed with 0.75% paraformaldehyde/1.5% glutaraldehyde, and their images were observed by fluorescence microscopy. Cells were subsequently fixed with 1% OsO4 and observed by EM. Bars = 5 μm. ROIs are indicated by the blue and yellow squares, and magnified images are shown in the middle and bottom panels, respectively. Bars = 10 μm in the top panels, and 500 nm in the middle and bottom panels. Fluorescent signals are indicated by the dashed circles in the merged image. Arrows indicate phagophores. “G” indicates the Golgi apparatus. CLEM analysis demonstrated that DAPRed-positive/LAMP1-negative puncta were merged with phagophores. In the middle panel, multiple Golgi-derived vesicles that are not labeled by DAPRed were observed close to the Golgi apparatus.
(D) Quantitative time course analysis of fluorescence intensities of DAPRed and DALGreen. Atg5-deficient MEFs were prestained with DAPRed (0.1 μM) and DALGreen (1 μM), treated with etoposide (10 μM), and images were obtained using a fluorescence microscope. Then, the ratio of fluorescent area to total cell area was calculated using the application “BZ-H4C hybrid cell count” (Keyence). Data are shown as the mean ± S.D. (n = 7). ∗p < 0.05, ∗∗p < 0.01 (Student’s t test for comparison to the no treatment control).