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: Donor PC therapy restores CMA in host PCs in damaged tissue. (A) Representative images showing co-localization of puncta pattern expression of LAMP-2A protein (green) with a PC marker, α-SMA (red), analyzed in host PCs of microvessels (v) of control mice, in mice brain with lesioned areas (injured) and treated intravenously with control adMSCs and with donor brain WT or KO PCs. Nuclei were stained with DAPI (blue). Scale bar: 100 µm. Right panels show bigger magnification of double positive cells. ( B ) Representative images showing co-localization of puncta pattern expression of LAMP-2A protein (in green) with a PC marker, PDGFRβ (in red), analyzed in host PCs of microvessels (v) of control mice, in mice brain with lesioned areas and treated intravenously with control adMSCs and with donor brain WT or KO PCs. Right panels show bigger magnification of double positive cells. Nuclei were stained with DAPI (blue). Scale bars: 100 µm. (C) Representative images showing co-localization of puncta pattern expression of LAMP-2A protein (in green) with a PC marker, CD146 (in red), analyzed in host PCs of microvessels (v) of control mice, in mice brain with lesioned areas and treated intravenously with donor brain PC. Right panels show bigger magnification of double positive cells (Scale bars: 20 µm). Nuclei were stained with DAPI (blue). Scale bars: 100 µm. ( D ) Relative quantification of percentage of PCs in brain microvessels expressing the PC marker α -SMA (n = 40 α -SMA + PCs per condition), PDGFRβ (n = 20 PDGFRβ + PCs per condition), or CD146 (n = 35 CD146 + PCs per condition) and the CMA receptor LAMP-2A (PC/LAMP-2A +/+ ) over total PCs. All data represent mean ± SD obtained from at least, three experiments represented as dots, independently; * p < 0.05; ** p < 0.01; *** p < 0.001; ns indicates no statistical significance (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: For fluorescent double labelling, mouse anti-alpha-smooth muscle actin (α-SMA; Abcam, ab7817), goat anti-platelet derived growth factor receptor beta (PDGFRβ; R&D Systems, BAF1042), or rat CD146-APC (Miltenyi Biotec, 130–118-408) antibodies were used in combination with rabbit anti-LAMP-2A (Invitrogen, 51–2200) antibody.

Techniques: Expressing, Marker, Control, Staining, Quantitative Proteomics

Journal: Journal of Extracellular Biology

Article Title: Development of a Novel Extracellular Vesicle‐Based Biomarker Approach for Pediatric High‐Grade Glioma

doi: 10.1002/jex2.70117

Figure Lengend Snippet: PTPRZ1 and GLAST are reliable cell surface markers for radial glia (RG)‐like identity . (A) Single cell RNA‐seq analysis revealed 3 candidate genes highly specific to RG: GLAST, PTPRZ1 and ADGRV1 . (B) Multiple linear regression analysis showed the highest fitness with the combination of PTPRZ1 and GLAST (C) Double positive (DP) cells mainly fell into the RG and dividing cell cluster. (D) Elevated expression of other RG marker ( VIM, HES1, PAX6 and FABP7) in DP cells. Statistical analysis was performed using the Wilcoxon rank sum test (****: p < 0.0001).

Article Snippet: For nano‐flow cytometry analysis, 3 μL of SJ‐HGGX42‐derived EVs were incubated overnight at 4 degrees with gentle rotation with 1 μL of the antibody solution containing GLAST antibody conjugated to APC (1:800; Miltenyi Biotec, ACSA‐1, cat. no. 130‐123‐555, mouse monoclonal IgG2a) or PTP zeta antibody conjugated with FITC (1:1600; Bioss, cat. no. bs‐11327R‐FITC, rabbit polyclonal IgG).

Techniques: RNA Sequencing, Expressing, Marker

Journal: Journal of Extracellular Biology

Article Title: Development of a Novel Extracellular Vesicle‐Based Biomarker Approach for Pediatric High‐Grade Glioma

doi: 10.1002/jex2.70117

Figure Lengend Snippet: Expression of RG markers in pHGG patient‐derived cell lines . (A) RT‐qPCR of various HGG patient‐derived, control cells, and astrocytes showed the expression of RG‐like markers ( n = 3). (B) Representative flow cytometry density plots of HGG patient‐derived cell lines and astrocytes showing the percentage of double‐positive (PTPRZ1 + /GLAST + ) cells. The DP gate was set based on GLAST/PTPRZ1 expression on SH‐SY5Y (negative control) (C) Quantification of flow cytometry data, where each dot represents a biological replicate, and error bars indicate standard deviation ( n = 3–4). Statistical analysis was performed using the Kruskal‐Wallis test with multiple comparisons. *: p < 0.05, **: p < 0.01. (D) Live time‐lapse imaging from patient‐derived cell line SJ‐HGGX42 showing mitotic somal translocation (MST). Images acquired using 10X bright‐field microscopy over a 48‐h period with 2‐min intervals, under standard incubator conditions (37°C, 5% CO2).

Article Snippet: For nano‐flow cytometry analysis, 3 μL of SJ‐HGGX42‐derived EVs were incubated overnight at 4 degrees with gentle rotation with 1 μL of the antibody solution containing GLAST antibody conjugated to APC (1:800; Miltenyi Biotec, ACSA‐1, cat. no. 130‐123‐555, mouse monoclonal IgG2a) or PTP zeta antibody conjugated with FITC (1:1600; Bioss, cat. no. bs‐11327R‐FITC, rabbit polyclonal IgG).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Control, Flow Cytometry, Negative Control, Standard Deviation, Imaging, Translocation Assay, Microscopy

Journal: Journal of Extracellular Biology

Article Title: Development of a Novel Extracellular Vesicle‐Based Biomarker Approach for Pediatric High‐Grade Glioma

doi: 10.1002/jex2.70117

Figure Lengend Snippet: EVs derived from RG‐like glioma cells exhibited detectable levels of PTPRZ1 and GLAST markers . (A) Illustration of workflow for EV collection and characterization. (B) Nanoparticle tracking analysis (NTA) showing the size distribution of SJ‐HGGX42 EVs obtained by Nanosight NS300 (C) Transmission Electron Microscopy (TEM) image of EVs isolated from SJ‐HGGX42 cells by ExoQuick‐TC. (D) Western blot analysis of EV surface markers (CD63 and CD81) and negative control Calnexin. (E) Representative flow cytometry histograms of HGG patient‐derived EVs and astrocyte‐derived EVs, showing the APC signal (PTPRZ1 + /GLAST + EVs). The gate was set based on the control (secondary antibody only). (F) Quantification of flow cytometry data. Each dot represents a biological replicate and error bars indicate standard deviation ( n = 3–6). (G) Quantification of flow cytometry data of HGG patient‐derived EVs (SJ‐HGGX42) and astrocyte‐derived EVs isolated by ultracentrifugation, showing the APC signal (PTPRZ1 + /GLAST + EVs). Each dot represents a biological replicate and error bars indicate standard deviation ( n = 3). Statistical analysis was performed using one‐way ANOVA with multiple comparisons. **: p < 0.01, ****: p < 0.0001.

Article Snippet: For nano‐flow cytometry analysis, 3 μL of SJ‐HGGX42‐derived EVs were incubated overnight at 4 degrees with gentle rotation with 1 μL of the antibody solution containing GLAST antibody conjugated to APC (1:800; Miltenyi Biotec, ACSA‐1, cat. no. 130‐123‐555, mouse monoclonal IgG2a) or PTP zeta antibody conjugated with FITC (1:1600; Bioss, cat. no. bs‐11327R‐FITC, rabbit polyclonal IgG).

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Western Blot, Negative Control, Flow Cytometry, Control, Standard Deviation