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Samples were set up in triplicate in four independent assays (n 4) with the average values and error bars as means S.E. Green fluorescence intensities were normalized for each protein as a function of their degree of labeling. D, representative images of AC16 cells with decreasing amounts of intracellular OG I FL protein fluorescence after 12 and 24 h of OG LC-rich medium wash. Internalized AL soluble protein fluorescence decreases rapidly after the OG LC-rich medium is replaced with OG LC-free medium. C, quantification of decrease in protein-associated fluorescence emission. Also, both V L and FL AL-09 proteins internalize faster than the both I germlines. V L domains internalize faster than FL proteins. B, quantification of soluble AL protein internalization over time. Green fluorescence signal was normalized for a correct quantification and comparison. Because I FL has more OG binding sites than AL-09 V L, I FL appears more fluorescent than AL-09 V L.
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A, representative images of RFP-AC16 cells incubated with 1 M OG AL-09 V L and OG I FL proteins showing cell associated green fluorescence increasing every 4 h and localization in perinuclear regions. LC soluble proteins internalize into AC16 cells. This study emphasizes the complex LC-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real-time live-cell image analysis. This work describes our studies on potential mechanisms of AL cytotoxicity. This is another advantage for anyone who might be somewhat new to the virtual racing circuit.Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). iRacing likewise provides a number of different playing modes. There are now more than 100 different vehicles to choose from, so variety is never an issue. However, the majority of players will be impressed by the highly detailed graphics and the extremely responsive cars. A dynamic interface guarantees that no two competitions are ever alike, an amenity not often seen in similar packages. IRacing is built around a flexible platform which supports all major operating systems.
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