{"id":1132,"date":"2026-04-27T07:06:31","date_gmt":"2026-04-27T07:06:31","guid":{"rendered":"http:\/\/m-castl.org\/?p=1132"},"modified":"2026-04-27T07:06:31","modified_gmt":"2026-04-27T07:06:31","slug":"gfp-lc3-plasmid-was-kindly-provided-by-tomatsu-yoshimori","status":"publish","type":"post","link":"https:\/\/m-castl.org\/?p=1132","title":{"rendered":"\ufeffGFP-LC3 plasmid was kindly provided by Tomatsu Yoshimori"},"content":{"rendered":"<p>\ufeffGFP-LC3 plasmid was kindly provided by Tomatsu Yoshimori. == Funding Statement == This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) 1001 Grant and Sabanci University. assessments were performed to confirm that 3 UTR sequences in target genes were functional. Differential expression ofMIR376Aand the relatedMIR376Bwas compared using TaqMan quantitative PCR. == Results == Here, we exhibited that, a microRNA (miRNA) from theDLK1\/GTL2gene cluster,MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked byMIR376Aoverexpression in MCF-7 and Huh7 cells.MIR376Ashared the same seed sequence and had overlapping targets withMIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3 UTR sequences in the mRNA of these autophagy proteins were responsive toMIR376Ain luciferase assays. Antagomir assessments showed that, endogenousMIR376Awas participating to the control ofATG4CandBECN1transcript and protein levels. Moreover, blockage of endogenousMIR376Aaccelerated starvation-induced autophagic activity. Interestingly,MIR376AandMIR376Blevels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role. == Conclusions == Our findings NS-2028 underline the importance of miRNAs encoded by theDLK1\/GTL2gene cluster in stress-response control mechanisms, and introduceMIR376Aas a new regulator of autophagy. == Introduction == Two major degradation pathways, namely macroautophagy (autophagy herein) and NS-2028 the ubiquitin-proteasome system, are operational in the maintenance of cellular homeostasis. Functional at a basal level for long-lived protein degradation and organelle turnover under normal conditions, autophagy is usually rapidly upregulated in response to both extracellular (e.g. nutrient starvation, hypoxia) and intracellular (e.g. accumulation of unfolded proteins, damaged organelles, pathogens) stress factors.[1]Concerted action of several protein complexes formed by at least 32 different autophagy (ATG) proteins result in the formation of double- or multi-membrane vesicles called autophagic vesicles or autophagosomes.[2]These vesicles enwrap cargo molecules and carry them to lysosomes for degradation, resulting in the recycling of their constituents for reuse by the cell.[3],[4] Protein complexes playing a role in autophagosome formation are numerous. A key event is the accumulation of a altered lipid molecule, phosphoinositol 3-phosphate around the ER and mitochondrial membranes, marking the autophagic vesicle nucleation centers.[2]A phosphoinositol 3-kinase, VPS34, is responsible for the conversion of NS-2028 membrane associated inositol lipids into phosphoinositol 3-phosphate (PI3-P). BECN1 was discovered as a grasp regulator of the VPS34 activity and autophagosome formation.[2]Autophagic vesicle membrane elongation, growth and closure occur through the action of two ubiqituination-like protein conjugation systems. [5]The first system is rather regulatory, resulting in the covalent conjugation of a ubiquitin-like protein ATG12 to ATG5, and in the eventual formation of a larger complex including the ATG16 protein. The ATG12-5-16 complex serves as a E3 ubiquitin ligase-like enzyme for the second reaction involving <a href=\"https:\/\/www.adooq.com\/ns-2028.html\">NS-2028<\/a> covalent attachment of a lipid, phosphatidylethanolamine (PE), to a carboxy-terminal (C-ter) glycine residue of the autophagy-related MAP1LC3 (or simply LC3) protein.[6]To expose the key glycine residue for conjugation, prior C-ter cleavage of pro-LC3 by ATG4 proteins is required. Lipid conjugated LC3 is necessary for the elongation of autophagic membranes and completion of the vesicles.[7]Indeed, cells lacking one of the conjugation reaction components were shown to harbor autophagy defects.[8] Recent studies introduced microRNAs (miRNAs) as novel regulators of autophagy.[9],[10],[11]miRNAs are small non-coding RNAs serving as unfavorable regulators of gene expression.[12]By base pairing with sequences found mainly in the 3 untranslated region (3UTR) of specific mRNAs, miRNAs lead to mRNA instability and\/or translation inhibition resulting in a decrease in target gene expression.[13]A single miRNA may target tens to hundreds of mRNAs, hence may co-regulate and coordinate a number of cellular proteins and pathways at once.[14]So far, only a handful of miRNAs were shown to directly affect the autophagic activity. Among them,MIR376Bwas introduced as a new regulator of starvation and mTOR-inhibition-related autophagy.[10]MIR376Bblocked autophagy by affecting the expression of two key autophagy proteins, namely ATG4C and BECN1.MIR376Bbelongs to a miRNA gene family encoded from a gene cluster region in the human chromosome 14q32, called theDLK1\/GTL2region.[15],[16],[17]Therefore, we wondered whether other miRNAs from the same region could play a role in autophagy regulation. Here, we report that another miRNA from theDLK1\/GTL2region, namely hsa-miR-376a1 (hereafterMIR376A) made up of <a href=\"http:\/\/www.ncbi.nlm.nih.gov\/entrez\/query.fcgi?db=gene&#038;cmd=Retrieve&#038;dopt=full_report&#038;list_uids=4790\">NFKB1<\/a> a seed sequence similar to that ofMIR376B, is usually a novel NS-2028 regulator of autophagy. Overexpression ofMIR376Aattenuated starvation-induced autophagic activity and did so by modulating cellular ATG4C and BECN1 mRNA and protein levels..<\/p>\n","protected":false},"excerpt":{"rendered":"\ufeffGFP-LC3 plasmid was kindly provided by Tomatsu Yoshimori. == Funding Statement == This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) 1001 Grant and Sabanci University. assessments were performed to confirm that 3 UTR sequences in target genes were functional. Differential expression ofMIR376Aand the relatedMIR376Bwas&hellip;\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[90],"tags":[],"class_list":["post-1132","post","type-post","status-publish","format-standard","hentry","category-non-selective-at-receptors"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.5 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>\ufeffGFP-LC3 plasmid was kindly provided by Tomatsu Yoshimori - PARP Inhibitor expression in the rat pup model<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/m-castl.org\/?p=1132\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"\ufeffGFP-LC3 plasmid was kindly provided by Tomatsu Yoshimori - PARP Inhibitor expression in the rat pup model\" \/>\n<meta property=\"og:description\" content=\"\ufeffGFP-LC3 plasmid was kindly provided by Tomatsu Yoshimori. == Funding Statement == This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) 1001 Grant and Sabanci University. assessments were performed to confirm that 3 UTR sequences in target genes were functional. 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