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 modu..., 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 digita..., , # Microsocopic Images acquired in the 1st and the 2nd screening

[https://doi.org/10.5061/dryad.9cnp5hqsx](https://doi.org/10.5061/dryad.9cnp5hqsx)

## Description of the data and file structure

For the primary screen, siRNA Library (three siRNAs per gene) targeting 708 genes was used. CCCP was added to induce mitophagy. Candidates that were able to reduce the number of phospho-Bcl2-L-13 (Ser272)-positive dots induced by CCCP treatment were selected. To narrow down the candidates, we conducted a secondary screen. We evaluated mitophagy induced by CCCP via fluorescent immunocytochemistry using anti-ATP synthase and anti-LC3B antibodies after the knockdown of the candidate genes. As the knockdown of the responsible kinase should reduce Bcl2-L-13-mediated mitophagy, candidates that were able to reduce the number of mitophagy induced by CCCP treatment were selected.

### Files and variables

The target genes of the each plate were listed in Contents.xlsx.

#### File: 2nd\_scr\_plate\_1\_1-...