Choi, Kyung-mi Nam, Hae Yun Na, Jin Hee Kim, Seong Who Kim, Sang Yoon Kim, Kwangmeyung Kwon, Ick Chan Ahn, Hyung Jun 2024-01-20T16:31:20Z 2024-01-20T16:31:20Z 2021-09-05 2011-09 1554-8627 https://pubs.kist.re.kr/handle/201004/130033 To date, several principal methods are presently used to monitor the autophagic process, but they have some potential experimental pitfalls or limitations that make them not applicable to living cells. In order to improve on the currently developed detection methods for autophagy, we report here fluorescent peptide-conjugated polymeric nanoparticles loaded with a lysosome staining dye in their core. The fluorescent peptide is designed to be specifically cleaved by the Atg4 cysteine protease, which plays a crucial role in autophagy activation. In this study, we demonstrate that peptide-conjugated polymeric nanoparticles can be used to visualize Atg4 activity in both cell-free and cell culture systems. The fluorescence imaging of cells incubated with nanoparticles demonstrates that Atg4 activity is activated in the autophagy-induced conditions, but suppressed in the autophagy-inhibited conditions. These results indicate that Atg4 activity is correlated with autophagic flux through its own regulatory pathway. Therefore, our strategy provides an alternative detection method that can clearly distinguish between an "autophagy active" and "autophagy inactive" state in cultured cells. As our nanoparticles are highly cell-permeable and biocompatible, this detection system has general applicability to living cells and can be extended to cell-based screening to evaluate newly developed compounds. English TAYLOR & FRANCIS INC AUTOPHAGIC VACUOLES LYSOSOMAL SYSTEM IN-VITRO PROTEIN MATURATION LC3 ACIDIFICATION PATHOGENESIS DEGRADATION INDUCTION A monitoring method for Atg4 activation in living cells using peptide-conjugated polymeric nanoparticles Article 10.4161/auto.7.9.16451 1 AUTOPHAGY, v.7, no.9, pp.1052 - 1062 AUTOPHAGY 7 9 1052 1062 scie scopus 000294477100012 2-s2.0-80052388263 Cell Biology Cell Biology Article AUTOPHAGIC VACUOLES LYSOSOMAL SYSTEM IN-VITRO PROTEIN MATURATION LC3 ACIDIFICATION PATHOGENESIS DEGRADATION INDUCTION autophagy polymeric nanoparticle Atg4 fluorescence monitoring