Supplementary Materialsijms-21-00959-s001

Supplementary Materialsijms-21-00959-s001. involved with ECM redesigning is definitely suppressed by ColXV due to reduction of FGF2 translocation to FGFR1. Furthermore, ColXV induced redesigning of ECM preceding apoptosis and continued to induce apoptosis in adipocytes. Collectively, our findings establish ColXV like a basement membrane collagen with homology to ColXVIII, indicating that it is one of the positive regulators for inducing ECM redesigning and further advertising adipocyte apoptosis. = 4). (B) Gene ontology (GO) analysis of the modified genes in (A) (= 3). (C) Changes in the mRNA level of genes associated with apoptosis and ECM redesigning, which were significantly modified from RNA-seq analysis (= 4). (D) Relative mRNA expressions of Caspase-3, Caspase-9, Bax, and Bcl-2 in mice adipocytes in different organizations (= 4). (ECF) Tunnel staining of DNA fragments in adipocytes. Level pub, 100 m (= 4) (GCI) Annexin V-FITC/PI double staining and circulation cytometry analysis of adipocytes apoptosis. Level pub, 100 m (= 4). Ideals are offered as mean SEM. *< 0.05, ** = 4). (DCF) Relative mRNA and protein expressions of Caspase-3/Cleaved Caspase-3, Caspase-9/Cleaved Caspase-9, Bax, (R)-UT-155 Bcl-2, Bid, and Bad in mice adipocytes in the Control group, sh-Col15a1 group and Ad-Col15a1 group with or without PA (palmitate) treatment (= 4). (GCH) JC-1 staining of mitochondrial membrane potential in adipocytes. Scale bar, 100 m (= 4). (I) Representative images and enlarged image (bottom right) of Mito-tracker staining (Red) and Cytc (Cytochrome C) immunofluorescent staining (Green) in adipocytes in different groups. Scale bar, 100 m (= 4). Arrowheads indicate Cytc released from mitochondria to the cytoplasm. Scale bar, 20 m. Values are presented as mean SEM. *< 0.05, ** < 0.01. 2.2. The AMPK/mTORC1 Signaling Pathway was Activated and Acted as a Checkpoint during ColXV Activation of Adipocyte Apoptosis It is known that cellular energy sensor AMP-activated protein kinase (AMPK) plays a key role in maintaining adipocyte energy balance and induces energy expenditure in adiposity, with immediate significance for obesity treatment [16,17]. The mammalian target (mTOR) of rapamycin was found to be an important downstream target of AMPK. AMPK/mTOR combine as an energy checkpoint to regulate the synthesis of biomacromolecule compounds and apoptosis via multiple mechanisms, including phosphorylation of S6 kinase [18]. This reminded us to investigate whether ColXV was involved in the (R)-UT-155 regulation of (R)-UT-155 the AMPK/mTOR pathway in adipocytes. We found that ColXV enhanced AMPKA1Thr183 and AMPKA2Thr172 phosphorylation while weakening mTORC1Ser2448 and ribosome protein subunit 6 kinase 1 (S6K1Thr389) phosphorylation which showed that AMPK/mTORC1 signaling and downstream target S6K1 were disturbed by ColXV (Figure 3A, E). In addition, ColXV promoted the activation of caspase-3 and reduced the (R)-UT-155 protein expression of Bcl-2, accompanied by inactivation of mTORC1 signaling pathway (Figure 3C,G). In this study, the pathway with AMPK inhibitor Compound C (C.C) was further investigated, showing that ColXV significantly reduced AMPK phosphorylation and increased the phosphorylation level of downstream target mTORC1Ser2448. It also increased the expression of Bcl-2, in addition to weakening the activity of cleaved caspase-3 (Figure 3A-D). TUNEL staining was also verified by ColXV-induced apoptosis LATS1/2 (phospho-Thr1079/1041) antibody under C.C treatment (Figure 3I,J). Furthermore, adipocytes were treated with rapamycin, a specific phosphorylation inhibitor of mTORC1. We observed lower phosphorylation levels of mTORC1Ser2448 and S6K1Thr389, accompanied by increased activity of cleaved caspase-3 and reduced protein level of Bcl-2 (Figure 3ECH). Importantly, we found forced ColXV promoted phosphorylation (R)-UT-155 of AMPK and inhibited phosphorylation of mTORC1, while inducing apoptosis of adipocytes, either under C.C treatment or rapamycin treatment. Taken together, these findings provide strong evidence to suggest that ColXV accelerates adipocyte apoptosis via the AMPK/mTORC1/S6K1 signaling pathway. Open in a separate.