A1 mp confocal imaging system

For mitochondria staining, AML12 cells were cultivated on poly-L-lysine-coated microscopy glasses. Both TMRE (tetramethylrhodamine, ethyl ester) (Thermo Fisher Scientific, Waltham, MA, USA) and MitoTracker Green FM (Thermo Fisher Scientific, Waltham, MA, USA) were used at day 2 after LL35 knockdown according to manufacturer’s protocol. Imaging was performed by Nikon A1+MP confocal imaging system (Nikon, Minato City, Japan).

Autophagy was assessed on a panel of cell lines stably expressing mCherry-GFP-LC3 reporter frame. Treatment with Abisil was carried out in concentration 50 μg/ml for 16 hours. Rapamycin (R0395, Sigma-Aldrich) was used as a positive control.
To detect mitophagy in MRC5-SV40 and LECH-4 cells, concentrations of 50 μg/ml of Abisil were used for treatment for 24 hours. After incubation, the cells were stained with 200 nM of MitoTracker Red CMXRos (Thermo Fisher Scientific) and 100 nM of LysoTracker Green (Thermo Fisher Scientific) for 20 minutes at 37° C.
Confocal microscopy was performed on the Nikon A1+MP confocal imaging system using an Apo TIRF 60x/1.49 oil DIC objective (numerical aperture=1.49; Nikon Japan), Apo LWD 40x/1.15 S water immersion objective (numerical aperture=1.15; Nikon Japan). Images were scanned sequentially using 488- and 561-nm diode lasers in combination with a DM405/488/561/633-nm dichroic beam splitter. Differential Interference Contrast Imaging (DIC) microscopy was used to visualize cell contours. The images were analyzed with NIS-elements AR (Nikon Japan).

AML12 cells were cultivated on 35-mm confocal dishes (VWR International, Radnor, PA, USA) and Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) staining was performed 48 h after ASO-mediated LL35 knockdown or control Luc knockdown. Briefly, Oil Red O powder was dissolved in 100% isopropanol to obtain a 30% solution. Then 3 parts of 30% Oil Red O solution were added to 2 parts of water, mixed and filtered to obtain a working solution. Cells were washed twice with 1× PBS and fixed for 30 min in 4% formaldehyde. After two washes with water and 5 min incubation in 60% isopropanol, cells were stained with Oil Red O working solution for 20 min followed by staining of cell nuclei with DAPI (Invitrogen, Waltham, MA, USA). After washing off the DAPI, cell imaging was performed by Nikon A1+MP confocal imaging system (Nikon, Japan).