Dm600 microscope

Autophagic flux was evaluated in RFP-GFP-LC3-transduced cells. Representative images were captured with an epifluorescence microscope (DM600 microscope, Leica) and quantification of yellow puncta (autophagosomes) and red puncta (autolysosomes) was performed using Image J software.

H9C2 cells were fixed with 4% PFA, permeabilized with 0.5% Triton, blocked with 3% BSA and incubated with anti‐vinculin (Sigma) or anti‐pH2A.X (Millipore) overnight at 4°C. The secondary antibody was Alexa‐Green‐488 goat anti‐mouse (Invitrogen). Images were acquired using epifluorescence microscopy (DM600 microscope; Leica). For mitophagy detection, EGFP‐LC3 plasmid and MitoID (Enzo) were used to stain autophagosomes and mitochondria, respectively. Image acquisition was performed with an LSM780 laser scanning confocal microscope (Carl Zeiss). For mitochondrial 8‐OH‐dG detection, cells were fixed with methanol for 30 min at −20°C, permeabilized with 0.2% Triton and treated with RNase A at 37°C for 1 hr, followed by denaturation with ice‐cold 25 mM NaOH in 50% ethanol, as previously described (Ohno, Oka, & Nakabeppu, 2009). After blocking with 10% BSA, the cells were incubated with mouse anti‐8‐OH‐dG (clone 483.15; Millipore) overnight at 4°C. The secondary antibody was goat anti‐mouse Alexa 488.

Acridine Orange (AO): Acridine Orange (Sigma Aldrich), a weak base that accumulates in acidic organelles, was used to label the lysosomes. After treatments, cells were incubated with 5 µM AO for 10 min at 37˚C and washed twice with PBS. Red fluorescence (corresponding to acidic vesicle staining) was measured using a Varioskan Flash Multimode Microplate Reader with excitation/emission at 480/630 nm. Representative pictures were taken with an epifluorescence microscope (DM600 microscope, Leica).
Lysosensor: Lysosomal acidification was measured in cells loaded with 2µM LysoSensor yellow/blue DND-160 (Invitrogen) for 15 min at 37˚C. The LysoSensor dye is a ratiometric probe that produces yellow fluorescence in acidic environments but changes to blue fluorescence in more neutral environments. pH calibration was performed following the protocol previously established by Diwu et al 45 (link). Briefly, cells were incubated in MES buffer (5 mM NaCl, 115 mM KCl, 1.3 mM MgSO₄, 25 mM MES) supplemented with 10 µM nigericin and 10 µM monensin, for 10 min, with pH adjusted within 3 to 7. The samples were read in a Varioskan Flash Multimode Microplate Reader with excitation at 360 nm. The emission 440/540 nm ratio was then calculated for each sample.