Stained with BODIPY and DAPI and analyzed by confocal microscopy. The perinuclear zone is shown; arrowheads indicate LDs in the vicinity in the nucleus. (C) Fixed cells stained with BODIPY, DAPI, and anti-CLNX antibody and analyzed by confocal microscopy. The 3D perinuclear zone is shown. The arrowhead indicates LD related closely using a CLNX-positive perinuclear endoplasmic reticulum structure. (D) Cells processed as in B. An intranuclear LD. (E) Fixed cells stained with BODIPY and DAPI and analyzed by confocal microscopy. The basal zone is shown. Scale bars, 4 m (B ). (F) Fixed cells stained with BODIPY, DAPI, anti-CLNX antibody, and fluorescent-phalloidin (F-actin). The XZ-projection of XY-stack acquisitions in two different fields, illustrating the presence of LDs in the basal pole in the cells, inside the perinuclear vicinity, and inside nucleus.Tazemetostat Scale bar, 12 m. (G) Polarized and differentiated Caco-2/TC7 enterocytes treated with lipid micelles for the indicated instances and fixed and stained with BODIPY, as within a .Retifanlimab Confocal acquisitions have been analyzed using the BODIPY signal. The imply volume of LD (in m3) was quantified in every situation for intranuclear (nuclear LD; gray), perinuclear (PN LD; light blue), and basal LDs (dark blue) and represented graphically (n = 5 independent experiments). (H) Polarized and differentiated Caco-2/TC7 enterocytes treated with lipid micelles for 24 h in presence (NOC) or absence (CTRL) of nocodazole, fixed, and stained with DAPI and BODIPY. The XZ-projection of XY-stack acquisitions is shown in both conditions. 120 | S. A. Khaldoun et al.Molecular Biology on the CellAlimentary lipid supply triggers autophagic response in enterocytes in vivo and in vitroAutophagy is involved in cytosolic LD clearance in hepatocytes, a phenomenon described as macrolipophagy (Singh et al., 2009; Singh and Cuervo, 2012; Weidberg et al., 2009). Hepatocytes and enterocytes share the ability to take care of huge amounts of neutral lipids, and they both have triglyceride-rich lipoprotein synthesis machinery positioned at the ER membrane (Mansbach and Siddiqi, 2010).PMID:28739548 However, lipid delivery to each and every cell form is different. We hence wondered whether acute delivery of dietary lipids to enterocytes could induce an autophagic response. We initially addressed this query in vivo using mice fed a normal meal or possibly a meal supplemented with 150 l of olive oil, which induces massive cytosolic LD look in mouse enterocytes (Bouchoux et al., 2011). Mouse intestinal epithelium was recovered, and lipidated LC3 (LC3II) was analyzed by SDS AGE/Western blot. LC3-II will be the posttranslationally modified type of LC3, that is associated with autophagosome membranes and can be distinguished from its precursor, LC3-I, by SDS AGE due to its improved electrophoretic mobility (apparent molecular weight of 16 kDa for LC3-I vs. 14 kDa for LC3-II; Kabeya et al., 2000). We discovered that the volume of LC3II was increased 60 min (unpublished data) and 180 min (Figure two, A and B) soon after meals intake in mice fed the oily meal as compared with mice fed typical meal, suggesting that oil delivery was adequate to induce an autophagic response in enterocytes in vivo. For additional analysis, we made use of the differentiated human Caco-2/TC7 enterocytes supplied or not with complex lipid micelles inside the apical medium. We first observed by light microscopy that a 24-h lipid micelle remedy induced a threefold increase within the number of LC3 autophagosome-related dotted structure.