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-...心脏中受损线粒体的蓄积与心力衰竭密切相关。线粒体自噬（mitophagy）是一种特异性靶向受损线粒体的自噬降解系统。我们此前的研究表明，Bcl2样蛋白13（Bcl2-like protein 13，Bcl2-L-13）可介导哺乳动物细胞中的线粒体自噬与线粒体分裂。然而，Bcl2-L-13在体内的功能仍不明确。本研究证实，Bcl2-L-13基因敲除小鼠以及将Bcl2-L-13上的磷酸化位点（Ser272）突变为丙氨酸的敲入小鼠，在受到压力超负荷刺激时会出现左心室功能障碍。线粒体分裂与线粒体自噬的减弱会导致这些小鼠心脏的ATP生成受损。此外，我们鉴定出AMP活化蛋白激酶α2（AMPKα2）是负责Bcl2-L-13 Ser272位点磷酸化的激酶。上述结果表明，Bcl2-L-13及其磷酸化在维持心脏功能中发挥重要作用。此外，应激刺激下的线粒体自噬活性幅度可被modu…… 在初筛实验中，我们使用了Silencer™人激酶小干扰RNA（siRNA）文库（每个基因对应3条siRNA），共靶向708个基因（赛默飞世尔科技，货号A30079）。96孔细胞培养板的每个孔预先铺有3.6 pmol的siRNA与0.24 mL的Lipofectamine RNAiMAX（Invitrogen）。我们以终浓度30 nM的siRNA对3000株稳定表达HA-Bcl2-L-13的HEK293A细胞进行反向转染。以二甲基亚砜（DMSO）处理的样本作为阳性对照。 转染72小时后，添加15 mM CCCP与100 nM巴弗洛霉素A1（bafilomycin A1）共同孵育4小时以诱导线粒体自噬。随后用PBS配制的4%多聚甲醛固定细胞，并用PBS配制的0.1% Triton X-100进行透化处理。将细胞与抗磷酸化Bcl2-L-13（Ser272）抗体在4℃下孵育过夜，之后在室温下与山羊抗Alexa 568二抗孵育1小时。洗涤完成后，通过荧光显微镜（基恩士BZ-X700；20倍物镜，2倍数字放大……）自动扫描获取图像。 # 首轮及第二轮筛选中获取的显微图像 https://doi.org/10.5061/dryad.9cnp5hqsx ## 数据与文件结构说明 本次初筛使用靶向708个基因的siRNA文库（每个基因对应3条siRNA）。通过添加CCCP诱导线粒体自噬，筛选可减少CCCP处理诱导的磷酸化Bcl2-L-13（Ser272）阳性斑点数量的候选靶点。为缩小候选范围，我们开展了第二轮筛选：在敲低候选靶点后，采用抗ATP合酶与抗LC3B抗体的荧光免疫细胞化学法，评估CCCP诱导的线粒体自噬水平。由于靶向效应激酶的敲低会削弱Bcl2-L-13介导的线粒体自噬，因此我们筛选出可减少CCCP处理诱导的线粒体自噬数量的候选靶点。 ### 文件与变量 每块板的靶点基因已在Contents.xlsx中列明。 #### 文件：2nd_scr_plate_1_1-...