Journal: International journal of biological sciences

Article Title: Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells.

doi: 10.7150/ijbs.11943

Figure Lengend Snippet: Figure 5. Exosomal VASN was transferred into HUVECs through HSPGs pathways. (A) HUVECs were co-cultured with HepG2-derived exosomes in presence of different dose of heparin (0, 1 μg/ml, 5 μg /ml, 10 μg /ml) for 24hr. The whole cell lysates of HUVECs were subjected to western blotting using the anti-VASN antibody. GAPDH was used as a loading control. (B) Western blot analysis of VASN express level in HepG2, L02, HUVECs, HeLa, U937 cells; (C) Exosomal VASN cannot be transferred to HeLa, U937 and L02; (D) Western blot analysis of HSPG2 express level in HepG2, L02, HUVECs, HeLa, U937 cells.

Article Snippet: Rabbit anti-HSPG2 antibody was purchased from ProteinTech, China.

Techniques: Cell Culture, Derivative Assay, Western Blot, Control

Journal: Physiological Reports

Article Title: Perlecan deficiency causes endothelial dysfunction by reducing the expression of endothelial nitric oxide synthase

doi: 10.14814/phy2.12272

Figure Lengend Snippet: RNA expression of eNOS in human aortic endothelial cells (HAECs) treated with HSPG2 siRNA. Analyses of perlecan and eNOS RNA expression levels in the HAECs treated with or without control siRNA and 20 or 40 nmol of Perlecan siRNA using qPCR. RNA expressions were normalized to that of GAPDH and are indicated as relative to GAPDH. (A) Perlecan expression in HAECs was significantly decreased by approximately 90% following Perlecan siRNA treatment. (B) eNOS expression showed a significant decrease of approximately 50% following treatment with Perlecan siRNA. The bars indicate the mean ± SEM ( n = 3), *** P < 0.001 versus control cells.

Article Snippet: Perlecan siRNA and control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: RNA Expression, Control, Expressing

Journal: Physiological Reports

Article Title: Perlecan deficiency causes endothelial dysfunction by reducing the expression of endothelial nitric oxide synthase

doi: 10.14814/phy2.12272

Figure Lengend Snippet: RNA expression levels of eNOS in HAECs treated with heparinase III. (A) A time course of experimental protcol using heparitinase III. (B) The heparan sulfate chains were removed from HAECs using heparinase III. After a 1 h treatment with or without heparinase III, the cells were fixed immediately (time point 0) or 24 h later (time point 24 h), and immunostaining was performed with 10E4 or 3G10 (Green), and perlecan (Red) antibodies. Successful heparinase III digestion is indicated by negative staining of 10E4 (c and g) and positive staining of 3G10 (d and h). At 24 h culture after heparinase III treatment, heparan sulfate chains were not detected (g and h). (C) Analysis of eNOS RNA expression levels in HAECs treated with or without heparinase III using qPCR. eNOS RNA expressions was normalized to that of GAPDH and it is indicated as relative to GAPDH. eNOS expression levels in the heparinase III treated HAECs was not significantly different compared with that of the non‐treated HAECs at 1, 12, and 24 h later. The bars indicate the mean ± SEM ( n = 3)

Article Snippet: Perlecan siRNA and control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: RNA Expression, Immunostaining, Negative Staining, Staining, Expressing

Journal: Physiological Reports

Article Title: Perlecan deficiency causes endothelial dysfunction by reducing the expression of endothelial nitric oxide synthase

doi: 10.14814/phy2.12272

Figure Lengend Snippet: RNA expression level of eNOS in Perlecan knockdown HAECs on perlecan recombinant protein. qPCR analysis of eNOS RNA expression level in HAECs treated with Perlecan or Control siRNA and seeded on a plate coated with or without perlecan recombinant protein (rPerlecan). The level of the eNOS RNA expression was normalized to that of GAPDH and it is indicated as relative to GAPDH. The eNOS expression level in HAECs treated with Perlecan siRNA was significantly decreased compared to that of Control siRNA treated HAECs. HAECs treated with Perlecan siRNA and plated on rPerlecan was no significantly different compared with that of Control siRNA treated HAECs. The bars indicate the mean ± SEM ( n = 4) *** P < 0.001 versus Control siRNA treated HAECs without rPerlecan, ### P < 0.001 versus Perlecan siRNA treated HAECs without rPerlecan.

Article Snippet: Perlecan siRNA and control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: RNA Expression, Knockdown, Recombinant, Control, Expressing

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

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: Stemness and anti-inflammatory properties of the CMA-dependent secretome of PCs. ( A ) Representative images of PDLSCs differentiation after incubation with proteins from WT PC supernatant (sup): perlecan (PLC, 5 μg/mL) and follistatin (FLT, 500 ng/mL), alone or combined, and vehicle (veh) for 14 days (T14). Scale bar: 100 µm. ( B-D ) Graphs represents the quantification of levels of positive cells for ALP activity, alcian blue, and Oil Red O staining, respectively. All data represent mean ± SD obtained from at least three experiments represented as dots, independently; * p < 0.05; *** p < 0.001; **** p < 0.0001; ns indicates no statistical significance (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 (expressed as folds over control vehicle and normalized to Actb as the housekeeping reference gene expression) in PDLSCs incubated with PLC and FLT, alone or combined. All data represent mean ± SD obtained from at least three experiments represented as dots, independently; *** p < 0.001; **** p < 0.0001 (ANOVA with Tukey’s post-test). ( F ) Quantification of the mRNA expression by qPCR of some genes involved in inflammatory responses in the microglial cell line BV2 (CT: control) activated with LPS (1 μg/mL) for 24 h (veh) and then incubated with WT PCs sup or KO PCs sup for 24 h after activation (expressed as folds over control non-activated and normalized to Actb and Hrtp1 as housekeeping reference gene expression). Concentrated cultured media (veh) was used as control in activated cells. All data represent mean ± SD obtained from at least four experiments represented as dots, independently; * p < 0.05; ** p < 0.01 (ANOVA with Tukey’s post-test). ( G ) Levels of inflammatory cytokines TNF-α and ( H ) IL-1β secreted by unstimulated microglial cells (CT) or activated with LPS (1 μg/mL) for 24 h (veh) and incubated with WT PCs sup or KO PCs sup for 48 h after stimulation. Concentrated cultured media (veh) was used as control in activated cells. All data represent mean ± SD obtained from at least four experiments represented as dots, independently; * p < 0.05; ** p < 0.01 (ANOVA with Tukey’s post-test). (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: Incubation, Activity Assay, Staining, Expressing, Gene Expression, Control, Activation Assay, Cell Culture