Microsocopic Images acquired in the 1st and the 2nd screening

The accumulation of damaged mitochondria in the heart is associated with heart failure. Mitophagy is an autophagic degradation system that specifically targets damaged mitochondria. We previously reported that Bcl2-like protein 13 (Bcl2-L-13) mediates mitophagy and mitochondrial fission in mammalian cells. However, the in vivo function of Bcl2-L-13 remains unclear. Here, we demonstrate that Bcl2-L-13-deficient mice and knock-in mice, in which the phosphorylation site (Ser272) on Bcl2-L-13 was changed to Ala, showed left ventricular dysfunction in response to pressure overload. Attenuation of mitochondrial fission and mitophagy led to impairment of ATP production in these mouse hearts. In addition, we identified AMPKα2 as the kinase responsible for the phosphorylation of Bcl2-L-13 at Ser272. These results indicate that Bcl2-L-13 and its phosphorylation play an important role in maintaining cardiac function. Furthermore, the amplitude of stress-stimulated mitophagic activity could be modulated by AMPKα2.

Methods
For the primary screen, the Silencer™ Human Kinase siRNA Library (three siRNAs per gene) targeting 708 genes (ThermoFisher Scientific, A30079) was used. 96-well tissue culture plates were prearrayed with 3.6 pmol of siRNA and 0.24 mL of Lipofectamine RNAiMAX (Invitrogen) per well. Reverse transfection of 3,000 HEK293A cells stably expressing HA-Bcl2-L-13 was performed with a 30 nM final concentration of siRNAs. DMSO-treatated samples were used as the positive control.

Seventy-two hours post-transfection, 15 mM CCCP was added to induce mitophagy together with 100 nM bafilomycin A1 for four hours. Cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS. Cells were then incubated with the anti-phospho-Bcl2-L-13 (Ser272) antibody overnight at 4°C, followed by incubation with anti-goat Alexa 568 for one hour at RT. After washing, images were obtained by automated scanning using a fluorescence microscope (BZ-X700, Keyence; x20 objective lens, x2 digital zoom, nine view fields per well). For quantification of phospho-Bcl2-L-13 (Ser272) puncta, local maxima were determined using the “find maxima” function of the ImageJ software package. We chose target genes that demonstrated an over 60% reduction compared with positive control well in at least one out of three siRNAs or an over 40% reduction in at least two siRNAs for the secondary screen. In addition, we used Z scoring for candidate selection. We selected genes with a Z score greater than 1.5 as hits. We carried forward genes which fulfilled each of those criteria. 

For the secondary screen, HEK293A cells stably expressing HA-Bcl2-L-13 were transfected with candidate siRNA using RNAiMAX. A non-targeting siRNA control was used as the positive control. Seventy-two hours after the transfection, cells were treated with 15 mM CCCP and 100 nM bafilomycin A1 for four hours. Cells were fixed and permeabilized with methanol for ten minutes at -20°C. Cells were then incubated with rabbit anti-LC3B antibody and mouse anti-ATP synthase antibody overnight at 4°C, followed by incubation with secondary antibodies for one hour at RT. After washing, cells were mounted with ProLong Gold Antifade Mountant and analyzed using a Nikon Ti-Eclipse inverted microscope (Nikon). Mitophagy was manually evaluated by counting the number of ATP synthase dots colocalized with LC3B dots. At least 20 cells were quantified. Target genes that demonstrated an over 40% reduction compared with the positive control were selected for the in vitro kinase assay. We also evaluated mitophagy using the JACoP plugin of ImageJ for colocalization analysis. We chose genes with a Z score greater than 1.0 as hits.  We carried forward all genes which were selected under either of those two evaluation methods.