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

Murakawa, Tomokazu 1 ; Ito, Jumpei2; Rusu, Mara-Camelia3; Taneike, Manabu1; Omiya, Shigemiki2; Moncayo-Arlandi, Javier3; Nakanishi, Chiaki2; Sugihara, Ryuta1; Nishida, Hiroki1; Mine, Kentaro1; Fleck, Roland1; Zhang, Min3; Nishida, Kazuhiko3; Shah, Ajay M.3; Yamaguchi, Osamu4; Sakata, Yasushi1; Otsu, Kinya2

Published Nov 21, 2024 on Dryad. https://doi.org/10.5061/dryad.9cnp5hqsx

Abstract

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.

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.

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.