The peroxisome is a dynamic organelle with variety in number, size, shape, and activity in different cell types and physiological states. Recent studies have implicated peroxisomal homeostasis in ferroptosis susceptibility. Here, we developed a U-2OS cell line with a fluorescent peroxisomal tag and screened a target-selective chemical library through high-content imaging analysis. U-2OS cells stably expressing the mOrange2-Peroxisomes2 tag were generated to screen a target-selective inhibitor library. The nuclear DNA was counterstained with Hoechst 33342 for cell cycle analysis. Cellular images were recorded and quantitatively analyzed through a high-content imaging platform. The effect of selected compounds on ferroptosis induction was analyzed in combination with ferroptosis inducers (RSL3 and erastin). Flow cytometry analysis was conducted to assess the level of reactive oxygen species (ROS) and cell death events. Through the quantification of DNA content and peroxisomal signals in single cells, we demonstrated that peroxisomal abundance was closely linked with cell cycle progression and that peroxisomal biogenesis mainly occurred in the G1/S phase. We further identified compounds that positively and negatively regulated peroxisomal abundance without significantly affecting the cell cycle distribution. Some compounds promoted peroxisomal signals by inducing oxidative stress, while others regulated peroxisomal abundance independent of redox status. Importantly, compounds with peroxisome-enhancing activity potentiated ferroptosis induction. Our findings pinpoint novel cellular targets that might be involved in peroxisome homeostasis and indicate that compounds promoting peroxisomal abundance could be jointly applied with ferroptosis inducers to potentiate anticancer effect.

中文翻译：

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高内涵图像筛选以确定过氧化物酶体和铁死亡的化学调节剂

过氧化物酶体是一种动态细胞器，在不同细胞类型和生理状态下其数量、大小、形状和活性各不相同。最近的研究表明过氧化物酶体稳态与铁死亡易感性有关。在这里，我们开发了带有荧光过氧化物酶体标签的U-2OS细胞系，并通过高内涵成像分析筛选了目标选择性化学库。生成稳定表达 mOrange2-Peroxisomes2 标签的 U-2OS 细胞来筛选靶点选择性抑制剂库。用 Hoechst 33342 复染核 DNA 以进行细胞周期分析。通过高内涵成像平台记录并定量分析细胞图像。结合铁死亡诱导剂（RSL3 和erastin）分析所选化合物对铁死亡诱导的影响。进行流式细胞术分析以评估活性氧（ROS）水平和细胞死亡事件。通过对单细胞中DNA含量和过氧化物酶体信号的定量，我们证明过氧化物酶体丰度与细胞周期进程密切相关，并且过氧化物酶体生物发生主要发生在G1/S期。我们进一步鉴定了能够正向和负向调节过氧化物酶体丰度而不显着影响细胞周期分布的化合物。一些化合物通过诱导氧化应激促进过氧化物酶体信号，而其他化合物则独立于氧化还原状态调节过氧化物酶体丰度。重要的是，具有过氧化物酶体增强活性的化合物可增强铁死亡的诱导。我们的研究结果查明了可能参与过氧化物酶体稳态的新细胞靶标，并表明促进过氧化物酶体丰度的化合物可以与铁死亡诱导剂联合应用以增强抗癌作用。