DOI: 10.5061/dryad.9cnp5hqsx
title: Microsocopic Images acquired in the 1st and the 2nd screening

description: 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.
description: 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
 <em>in vitro</em> 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.
description: # 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\_1-10.zip **Description:** Images from plate 1 in the secondary screening. Images for the target genes No.1 - No.10 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_2\_11-20.zip **Description:** Images from plate 2 in the secondary screening. Images for the target genes No.11 - No.20 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_3\_21-30.zip **Description:** Images from plate 3 in the secondary screening.  Images for the target genes No.21 - No.30 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_4\_31-35.zip **Description:** Images from plate 4 in the secondary screening. Images for the target genes No.31 - No.35 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_5\_36-43.zip **Description:** Images from plate 5 in the secondary screening. Images for the target genes No.36 - No.43 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_6\_44-51.zip **Description:** Images from plate 6 in the secondary screening. Images for the target genes No.44 - No.51 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_7\_52-59.zip **Description:**  Images from plate 7 in the secondary screening. Images for the target genes No.52 - No.59 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_8\_60-64.zip **Description:**  Images from plate 8 in the secondary screening. Images for the target genes No.60 - No.64 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_9\_65-70.zip **Description:**  Images from plate 9 in the secondary screening. Images for the target genes No.65 - No.70 shown in Contents.xlsx and positive and negative controls are included. #### File: 2nd\_scr\_plate\_10\_71-78.zip **Description:**  Images from plate 10 in the secondary screening. Images for the target genes No.71 - No.78 shown in Contents.xlsx and positive and negative controls are included. #### File: plate\_No\_01.zip **Description:**  Images from plate 1 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for c-abl oncogene 1, non-receptor tyrosine kinase.)  #### File: plate\_No\_02.zip **Description:** Images from plate 2 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for ataxia telangiectasia mutated.) #### File: plate\_No\_03.zip **Description:** Images from plate 3 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for calcium/calmodulin-dependent protein kinase (CaM kinase) II beta.) #### File: plate\_No\_04.zip **Description:** Images from plate 4 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for creatine kinase, mitochondrial 1B.) #### File: plate\_No\_05.zip **Description:** Images from plate 5 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for diacylglycerol kinase, beta 90kDa.) #### File: plate\_No\_06.zip **Description:** Images from plate 6 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for EPH receptor A7.) #### File: plate\_No\_07.zip **Description:** Images from plate 7 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for fms-related tyrosine kinase 1.) #### File: plate\_No\_08.zip **Description:** Images from plate 8 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for glycogen synthase kinase 3 beta.) #### File: plate\_No\_09.zip **Description:** Images from plate 9 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for Janus kinase 2.) #### File: plate\_No\_10.zip **Description:** Images from plate 10 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for secretoglobin, family 2A, member 1.) #### File: plate\_No\_11.zip **Description:** Images from plate 11 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for NME/NM23 nucleoside diphosphate kinase 4.) #### File: plate\_No\_12.zip **Description:** Images from plate 12 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for pyruvate dehydrogenase kinase, isozyme 1.) #### File: plate\_No\_13.zip **Description:** Images from plate 13 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for pim-1 oncogene.) #### File: plate\_No\_14.zip **Description:** Images from plate 14 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for protein kinase C, delta.) #### File: plate\_No\_15.zip **Description:** Images from plate 15 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for mitogen-activated protein kinase 7.) #### File: plate\_No\_16.zip **Description:** Images from plate 16 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for phosphoribosyl pyrophosphate synthetase-associated protein 2.) #### File: plate\_No\_17.zip **Description:** Images from plate 17 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for ribosomal protein S6 kinase, 70kDa, polypeptide 1.) #### File: plate\_No\_18.zip **Description:** Images from plate 18 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for aurora kinase C.) #### File: plate\_No\_19.zip **Description:** Images from plate 19 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for vaccinia related kinase 1.) #### File: plate\_No\_20.zip **Description:** Images from plate 20 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for phosphoinositide-3-kinase, regulatory subunit 3 (gamma).) #### File: plate\_No\_21.zip **Description:** Images from plate 21 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for receptor-interacting serine-threonine kinase 2.) #### File: plate\_No\_22.zip **Description:** Images from plate 22 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for protein kinase, membrane associated tyrosine/threonine 1.) #### File: plate\_No\_23.zip **Description:** Images from plate 23 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for A kinase (PRKA) anchor protein 7.) #### File: plate\_No\_24.zip **Description:** Images from plate 24 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for v-akt murine thymoma viral oncogene homolog 3 (protein kinase B, gamma).) #### File: plate\_No\_25.zip **Description:** Images from plate 25 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for calcium/calmodulin-dependent protein kinase kinase 2, beta.) #### File: plate\_No\_26.zip **Description:** Images from plate 26 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for twinfilin actin-binding protein 2.) #### File: plate\_No\_27.zip **Description:** Images from plate 27 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for SIK family kinase 3.) #### File: plate\_No\_28.zip **Description:** Images from plate 28 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for SFRS protein kinase 3.) #### File: plate\_No\_29.zip **Description:** Images from plate 29 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for hormonally up-regulated Neu-associated kinase.) #### File: plate\_No\_30.zip **Description:** Images from plate 30 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for sterile alpha motif and leucine zipper containing kinase AZK.) #### File: plate\_No\_31.zip **Description:** Images from plate 31 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for phosphatidylinositol 4-kinase type 2 alpha.) #### File: plate\_No\_32.zip **Description:** Images from plate 32 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for calcium/calmodulin-dependent protein kinase ID.) #### File: plate\_No\_33.zip **Description:** Images from plate 33 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for ceramide kinase.) #### File: plate\_No\_34.zip **Description:** Images from plate 34 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for MORN repeat containing 1.) #### File: plate\_No\_35.zip **Description:** Images from plate 35 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for serine/threonine kinase 40.) #### File: plate\_No\_36.zip **Description:** Images from plate 36 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for STE20-related kinase adaptor alpha.) #### File: plate\_No\_37.zip **Description:** Images from plate 37 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 210kDa-like.) #### File: plate\_No\_38.zip **Description:** Images from plate 38 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for chromosome 9 open reading frame 96.) #### File: plate\_No\_39.zip **Description:** Images from plate 39 in the primary screening. Each folder contains images for a target gene. The target gene names are shown in Contents.xlsx. (e.g., The forder XY01 contains images for EPHA10.) #### File: Contents.xlsx **Description:** The list of target genes of the each plate.