characteristic of oxidative stress-induced senescence Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence Autophagy (PMID: 27791464 ; IF= 8.593) 报告人：董浩 IMI CONFIDENTIAL

Background 细胞衰老 1. 线粒体功能障碍 与ROS 线粒体受损，ROS过量产生，引发细胞衰老。 2. 自噬与细胞衰老 bla Background 衰老（Senescence）机体对环境的生理和心理适应能力进行性降低、逐渐趋向死亡的现象。衰老可分为两类：生理性衰老和病理性衰老。 细胞衰老 1. 线粒体功能障碍 与ROS 线粒体受损，ROS过量产生，引发细胞衰老。 2. 自噬与细胞衰老 二者关系不明确，不同条件下存在正负向不同关系。 3. 线粒体、溶酶体、自噬、衰老间关系 线粒体与衰老、溶酶体与自噬有关，四者之间是否有联系。 IMI CONFIDENTIAL bla

Background 1. 自噬体 自噬体、自噬溶酶体数目、LC3蛋白聚集程度。 2. 自噬流 自噬体形成，到溶酶体降解清除的整个动态过程。 bla Background 自噬体与自噬流 1. 自噬体 自噬体、自噬溶酶体数目、LC3蛋白聚集程度。 检测方法：电镜、GFP-LC3 2. 自噬流 自噬体形成，到溶酶体降解清除的整个动态过程。 检测方法： Western blot、流式细胞术、免疫荧光等 检测 LC3蛋白的动态降解过程 LC3B-I/II 的转化以及细胞定位、 自噬蛋白 SQSTM1/p62 长寿命蛋白自噬性降解（放射同位素标记） IMI CONFIDENTIAL bla

Background 研究目的：自噬与细胞衰老的关系 1. 明确氧化应激诱导的衰老细胞中自噬流的变化。 1. 明确氧化应激诱导的衰老细胞中自噬流的变化。 2. 探明线粒体、溶酶体功能障碍对自噬的影响。 3. 研究自噬调节剂对自噬功能与细胞衰老进程的影响。 IMI CONFIDENTIAL

Materials and Methods 复制性衰老：成纤维细胞传代一定次数 bla 1. 细胞模型 复制性衰老：成纤维细胞传代一定次数 早衰(Stress-induced premature senescence， SIPS)：过氧化氢处理 H2O2- SIPS 细胞系：murine fibroblast NIH3T3 human lung fibroblast MRC-5 2. 细胞衰老检测： a. SA-GLB1/SA-b-gal staining试剂盒（蓝绿色荧光） b. 衰老相关的异染色质簇集(SAHF): DAPI染色 IMI CONFIDENTIAL bla

Materials and Methods 3. 自噬检测： a. LC3：转染 mRFP-LC3、 mRFP-GFP-LC3 b. 电镜、IL-6 、SQSTM1/p62 4. 溶酶体检测： a. Lysosome labeling with LysoTracker Red DND-99（红色荧光） b. Lysosomal acid phosphatase assay 试剂盒（酸性磷酸酶 ACP2 ） c. 组织蛋白酶CTSB: WB、活性检测（试剂盒） 5. 线粒体检测： 线粒体膜电位（荧光法）、线粒体DNA（qPCR）、cytochrome c oxidase（试剂盒）、ATP（试剂盒） IMI CONFIDENTIAL

Materials and Methods 6. 胞内ROS检测： DCFH-DA combined with flow cytometry ， DCFH-DA turns into a green fluorescent molecule called DCF when oxidized by ROS 其他方法: Western Blot、Real-time PCR、Knockdown of Atg5 by shRNA IMI CONFIDENTIAL

Results 1. H2O2 treatment induced cellular senescence with intracellular ROS elevation SAHF IMI CONFIDENTIAL

Results 1. H2O2 treatment induced cellular senescence with intracellular ROS elevation p53 抑癌因子 IMI CONFIDENTIAL

Autophagic structure increase accompanied with impaired flux of autophagy in senescent cells 溶酶体 IMI CONFIDENTIAL

Autophagic structure increase accompanied with impaired flux of autophagy in senescent cells LC3 溶酶体活性抑制剂 IMI CONFIDENTIAL

Autophagic structure increase accompanied with impaired flux of autophagy in senescent cells 蛋白合成抑制剂 平衡盐溶液：自噬诱导剂 IMI CONFIDENTIAL

3. Chronic blockage of autophagicflux was sufficient to induce cellular senescence 溶酶体活性抑制剂 IMI CONFIDENTIAL

3. Chronic blockage of autophagicflux was sufficient to induce cellular senescence IMI CONFIDENTIAL

3. Chronic blockage of autophagic flux was sufficient to induce cellular senescence 15 d IMI CONFIDENTIAL

4. MTOR-dependent autophagy activators attenuated senescence through the restoration of autophagy flux 4种自噬激活剂 IMI CONFIDENTIAL

4. MTOR-dependent autophagy activators attenuated senescence through the restoration of autophagy flux 3种自噬抑制剂 IMI CONFIDENTIAL

4. MTOR-dependent autophagy activators attenuated senescence through the restoration of autophagy flux IMI CONFIDENTIAL

4. MTOR-dependent autophagy activators attenuated senescence through the restoration of autophagy flux IMI CONFIDENTIAL

Lysosomal and mitochondrial dysfunction occurs during SIPS development 组织蛋白酶 酸性磷酸酶2 IMI CONFIDENTIAL

Lysosomal and mitochondrial dysfunction occurs during SIPS development CTSB 活性 IMI CONFIDENTIAL

Lysosomal and mitochondrial dysfunction occurs during SIPS development 线粒体去极化 膜电位降低 IMI CONFIDENTIAL

Lysosomal and mitochondrial dysfunction occurs during SIPS development ROS 水平 线粒体氧化磷酸化解偶联剂：抑制磷酸化过程，降低ROS产生 IMI CONFIDENTIAL

Lysosomal and mitochondrial dysfunction occurs during SIPS development IMI CONFIDENTIAL

6. Temporally sequential dysfunction of mitochondria and lysosomes during SIPS development IMI CONFIDENTIAL

6. Temporally sequential dysfunction of mitochondria and lysosomes during SIPS development 溶酶体成熟诱导剂 ROS 水平 CTSB 活性 IMI CONFIDENTIAL

6. Temporally sequential dysfunction of mitochondria and lysosomes during SIPS development N-乙酰半胱氨酸：抗氧化剂 IMI CONFIDENTIAL

6. Temporally sequential dysfunction of mitochondria and lysosomes during SIPS development IMI CONFIDENTIAL

6. Temporally sequential dysfunction of mitochondria and lysosomes during SIPS development IMI CONFIDENTIAL

7. Autophagy modulators regulated the function of mitochondria and lysosomes in senescent cells 线粒体膜电位 IMI CONFIDENTIAL

7. Autophagy modulators regulated the function of mitochondria and lysosomes in senescent cells ROS 水平 IMI CONFIDENTIAL

7. Autophagy modulators regulated the function of mitochondria and lysosomes in senescent cells IMI CONFIDENTIAL

7. Autophagy modulators regulated the function of mitochondria and lysosomes in senescent cells CTSB 活性 IMI CONFIDENTIAL

bla Thanks! IMI CONFIDENTIAL bla