Journal: Journal of Advanced Research
Article Title: Chaperone-mediated autophagy sustains pericyte stemness necessary for brain tissue homeostasis
doi: 10.1016/j.jare.2025.04.015
Figure Lengend Snippet: CMA maintains a functional MSCs-like secretome in PCs. ( A ) Representative pictures show periodontal ligament stem cells (PDLSCs) that can spontaneously differentiate into an osteogenic lineage (T0). ALP, Alcian Blue, and Oil Red positive PDLSCs are shown after being incubated for 14 days (T14) with the control vehicle (veh), with supernatants from WT PCs (WT sup) or with supernatants from KO PCs (KO sup). Scale bar: 100 µm. ( B-D ) Graph represents the quantification of levels of positive cells for ALP activity (osteogenic lineage), alcian blue (chondrocytes), and Oil Red O (adipocytes) staining, respectively. All data represent mean ± SD obtained from at least five experiments represented as dots, independently; *** p < 0.001; **** p < 0.0001 (ANOVA with Tukey’s post-test). ( E ) Quantification of the mRNA expression by qPCR of some genes involved in osteogenesis differentiation. Data show specifically the gene expression levels in PDLSCs (expressed as folds over control vehicle and normalized to Actb as the housekeeping reference gene expression) incubated with WT PCs sup or KO PCs sup. All data represent mean ± SD obtained from at least four experiments represented as dots, independently; ** p < 0.001; *** p < 0.0001 (ANOVA with Tukey’s post-test). ( F ) Venn diagram of the secretome analysis of identified proteins in WT PCs and KO PCs. WT PCs secrete 10 exclusive proteins, sharing 15 proteins in common with KO PCs, which have 3 specific secreted proteins. ( G ) Chart shows the analysis of functional groups associated with pro-regenerative (left) and pro-inflammatory (right) mechanisms. These groups represent the percentage of proteins identified in both the secretome of WT PCs (right) and KO PCs (right) cultures from the total amount of identified proteins by mass spectrometry. The ratio between KO and WT PCs represents the most secreted proteins by each cell type. Twelve (34 %) of the proteins secreted by WT PCs were related to tissue development and cell differentiation functions (such as UHRF1 (ICBP90)-binding protein 1, follistatin-related protein 1, and SPARC), nine (26 %) were related to wound healing (such as plasminogen activator inhibitor 1 and perlecan), nine (26 %) to extracellular matrix organization (such as beta-actin-like protein 2) and five (17 %) to angiogenesis functions (such as antithrombin III). On the other hand, four (45 %) of the proteins secreted by KO PCs were related to extracellular matrix assembly (such as dipeptidyl aminopeptidase-like protein 6 and collagen alpha-1 chain), three (33 %) were related to inflammatory response (such as complement C3), and two (22 %) were related to lipid metabolism (such as long-chain-fatty-acid-CoA ligase 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Article Snippet: Perlecan (5 μg/mL; Origene #TP762044) and follistatin (500 ng/mL; Thermofisher #120–13), alone or combined, were added every 3 days for 7 days.
Techniques: Functional Assay, Incubation, Control, Activity Assay, Staining, Expressing, Gene Expression, Mass Spectrometry, Cell Differentiation, Binding Assay