Autophagy as a promising target for drug therapy

Autophagy and its dysregulation have been found in neurodegenerative diseases and cancerplay, give play to, bring into playIt plays an important role, therefore, discovering new therapeutic targets in this process has become a promising drug treatment method. Autophagy is a regulatory process that degrades and recovers damaged proteins and organelles under cellular stress1，2.byA sac called an autophagosomeBubble throughForm a double membrane around the cell components marked as damagedconductassembly1Autophagosome vesicles then fuse with lysosomes, delivering their contents for degradation by lysosomal hydrolases.

Autophagy has a variety of complex physiological and pathophysiological functions, such as adaptation to nutritional starvation, elimination of damaged intracellular proteins and organelles, cell development, anti-aging, elimination of microorganisms, cell death, tumor suppression and antigen presentation2Mitochondrial fission is the selective degradation of mitochondria by autophagy3This process usually involveswithEliminate defective mitochondria after cellular damage or stress.

Materials

• PC12Human neuroblastoma cell line（ATCC）

• 384 Orifice plate（Greiner）

• Cyto-id ®Autophagy detection kit（ENZO Life Sciences）

• Orange mitochondrial dye

• Hoechst 33342

• ImageXpress® PicoAutomated Cell Imaging System（Molecular Devices）

• CellReporterXpress Data collection and analysis software（Molecular Devices）

advantage

The effect of quantitative compounds on autophagy process

Using nuclear markers to segment cells, detect and characterize small cellsThe target object

Using automatic imaging to ensure reliable statistical results

Using automated cell imaging to detect and quantify autophagosome particles

Evaluate the impact on autophagy

In this application manualWe evaluated itImageXpressPicoThe efficiency of automated cell imaging system for detecting and quantifying autophagosome particles.PC12The human neuroblastoma cell line is used as a detection and development model. Transforming cells into 6，000 acell/Inoculate into the hole384Incubate in the orifice plate48Hour. To evaluate the impact on autophagy,makeTreat cells with different concentrations of chloroquine or verapamil48Hour. VerapamilcanInducing autophagy, while chloroquinecanInhibition of autophagosome degradation leading to particle accumulation.compoundAfter processing, useCyto-id ®Autophagy detectionReagent kit for activityStaining of cells toshowTraceAutophagosome. In addition, we used fluorescent mitochondrial dyes to detect mitochondria and employedHoechstIdentify cell nucleus（concentrationrespectively 0.2 MMand 1 MM）. usecollocation20Xor40Xobjective lensofImageXpressPicoSystem and three detection channels（FITCTheTRITCandDAPI）Regarding cellsPerform imaging. In20Xobjective lensCollect one image for each hole below40XUnder the objective lensEach holecollect2-4Create images to ensure reliable statistical results. useCellReporterXpressAutophagy or mitochondrial application schemes in image acquisition and analysis software are used to analyze images.

image 1：Autophagosome detection.The above figure shows thepassAutophagy inducer chloroquine（30 pM）Handle andmakeuseCyto-idDyeing with dyesPC12Representative autophagic cell images and analysis masks of cells. InCellReporterXpressIn the software, point segmentation is used to identify autophagic granules. nucleusdisplay asBlue, mitochondriadisplay asOrange, autophagydisplay asGreen. downimagedisplayThe use ofStain untreated cells with orange mitochondrial integrity dye.Image and analysis mask for mitochondrial detection..

These algorithms canwithSmall detection and characterizationThe target objectLike autophagosomes or mitochondria in the cytoplasm, while using nuclear markers to segment cells. Quantitative detection of autophagydetectionIncluding the total or average number of particles, the total or average area of particles (subcellular objects), and their intensity. Using chloroquine（30 MM）ProcessingafterRepresentative images and analysis masks of cells are shown in the figure1As shown. inspectmeasureChloroquine and VerapamilProcessed cellsConcentration reaction and evaluationEC50Value. Total autophagosome countThe data shows that,Chloroquine and Verapamil treatmentcellAutophagy levels increase separately4.6Beihe and3.3times（EC50The values are respectively4.0and3.6 MM）.

Conclusion

This method andCyto-iddyecombineCan be used to detect autophagic granules and can be used to detect and quantify the effects of compounds on autophagy processes.

referenceliterature

1. 1. Mizushima, N; Komatsu, M (2011). Autophagy: renovation of cells and tissues. Cell. 147: 728–41

2. 2. Mizushima, N; (2007). Autophagy: process and function, Genes & Dev. 21: 2861-2873