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Macroautophagy (hereafter autophagy) is one of the adaptive pathways that contribute to cancer cell chemoresistance. Despite the fact that autophagy can both promote and inhibit cell death, there is mounting evidence that in the context of anticancer treatment, it predominantly functions as a cell survival mechanism. Therefore, silencing of key autophagy genes emerges as a potent strategy to reduce...
Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo “microenvironment” compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and...
We applied fluorescence microscopy‐based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)‐to‐Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi‐to‐ER relocalization of GalT‐CFP (β1,4‐galactosyltransferase I‐cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash‐out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER‐to‐Golgi trafficking. Nine of them interfered with the rate of BFA‐induced redistribution of GalT‐CFP from the Golgi complex to the ER, six of them interfered with GalT‐CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash‐out and six of them were positive effectors in both assays. Notably, our live‐cell approach captures regulator function in ER‐to‐Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.
A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or...
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