eaat2  (Novus Biologicals)

Journal: medRxiv

Article Title: Longitudinal Monitoring of Glioblastoma Small Extracellular Vesicle Evolution Using a Nanodiagnostic to Detect Emergence of Glioma Stem Cells Driving Recurrent Disease

doi: 10.1101/2024.09.23.24314250

Figure Lengend Snippet: (A) Heatmap of top 20 CNS markers’ RNA expression in TCGA and GTEx RNA-seq databases. Low expression is indicated in blue and high expression is indicated in yellow. (B) Tukey’s boxplot indicating ATP1B2 and SLC1A2 (EAAT2) genes are highly expressed in normal brain and brain tumor tissues (GBM and low-grade gliomas, LGG) in TCGA and GTEx RNA-seq databases. (C) Primary GBM tumors (n = 31) were labeled for ATP1B2 and EAAT2. Violin plots show the H-score labeling intensity across the cohort. Scale bar = 100 µm.

Article Snippet: Approximately 1 × 10 9 total sEV particles were incubated with either anti-ATP1B2 (PA526279, Thermo Fisher Scientific), anti-EAAT2 (NOVNBP120136, Novus), anti-CD9 APC (17-0098-42, Thermo Fisher Scientific), anti-CD24 (ab134375, Abcam), anti-EGFR (ab231, Abcam), anti-CD44 Alexa Fluor 488 (103016, BioLegend), or anti-CD133 Alexa Fluor 488 (FAB11331G, R&D Systems) in 50 µL of PBS, for 1 h at 37 °C.

Techniques: RNA Expression, RNA Sequencing Assay, Expressing, Labeling

Journal: medRxiv

Article Title: Longitudinal Monitoring of Glioblastoma Small Extracellular Vesicle Evolution Using a Nanodiagnostic to Detect Emergence of Glioma Stem Cells Driving Recurrent Disease

doi: 10.1101/2024.09.23.24314250

Figure Lengend Snippet: (A) Single nanoparticle analysis by nanoFCM shows the expression of CNS markers (ATP1B2 and EAAT2) in breast cancer cell line MDAMB231 (-ve) and WK1 sEVs. Average histogram measurements are the average expression of 3 GBM cell line sEVs (WK1, BAH1, FPW1). (B) Single nanoparticle analysis by nanoFCM shows the expression of GSC markers (CD24, EGFR, CD44, and CD133) as representative nanoFCM images and average histogram measurements in three GBM cell lines. (C) Representative Raman images for all of marker’s detection in GBM cell lines using GEMPAC. Scale bar = 10 µm. (D) Raman intensity measurements of sEVs from each GBM cell line with different SERS nanotags; DTNB-ATP1B2 (red), MBA-EAAT2 (blue), TFMBA-CD44 (green), MPY-CD133 (yellow), TFMBA-EGFR (purple) and MPY-CD24 (grey). MDA-MB-231 breast cancer cells and EV-free medium were used as negative controls to show biomarker specificity. Data are represented as mean ± standard error.

Article Snippet: Approximately 1 × 10 9 total sEV particles were incubated with either anti-ATP1B2 (PA526279, Thermo Fisher Scientific), anti-EAAT2 (NOVNBP120136, Novus), anti-CD9 APC (17-0098-42, Thermo Fisher Scientific), anti-CD24 (ab134375, Abcam), anti-EGFR (ab231, Abcam), anti-CD44 Alexa Fluor 488 (103016, BioLegend), or anti-CD133 Alexa Fluor 488 (FAB11331G, R&D Systems) in 50 µL of PBS, for 1 h at 37 °C.

Techniques: Expressing, Biomarker Assay

Journal: medRxiv

Article Title: Longitudinal Monitoring of Glioblastoma Small Extracellular Vesicle Evolution Using a Nanodiagnostic to Detect Emergence of Glioma Stem Cells Driving Recurrent Disease

doi: 10.1101/2024.09.23.24314250

Figure Lengend Snippet: (A) GBM patient samples (n = 36) sEV size distribution. (B) GBM sEV concentration in pre-op and post-op. (C) Raman intensity measurements of each marker with SERS images in pre-op and post-op in the representative GBM patient, DTNB-ATP1B2 (red), MBA-EAAT2 (blue), TFMBA-CD44 (green), MPY-CD133 (yellow), TFMBA-EGFR (purple), and MPY-CD24 (grey). Data are represented as mean ± standard error (n = 4). (D) All the GBM stem cell markers were normalized by CNS markers and sum as GEMPAC score. Comparison of GEMPAC score between pre-op and post-op. The proportion of each normalized GSC marker in (E) pre-op and (F) post-op. Kaplan-Meier plots of overall survival by GEMPAC score. Survival outcome differences in pre-op (G) and post-op (H). (I) ROC curve analysis for GEMPAC score between short and long overall survival groups. Next, we investigated if the GEMPAC score was able to predict overall survival in a cohort of 24 patients who received only standard care temozolomide (TMZ) and radiotherapy (RT). Patients were categorized into either high (> median GEMPAC score) or low (< median GEMPAC score) groups. Before surgical resection there was no correlation in overall survival (p = 0.4936) , however, after surgical resection, patients with a high GEMPAC score had significantly shorter survival compared to patients with a low GEMPAC score (p = 0.0055) . To investigate the sensitivity and specificity of the GEMPAC score for predicting overall survival, a 1-year overall survival was assessed by receiver operating characteristic (ROC) curves. In this context, the area under the curve (AUC) for predicting overall survival after surgical resection (post-op) was 0.79 (95% CI 0.6-0.98) . These results indicate the GEMPAC score from circulating GBM sEVs could predict patients’ survival after surgery, likely reflecting the remaining tumor burden after surgical resection which is a predictor of survival after resection.

Article Snippet: Approximately 1 × 10 9 total sEV particles were incubated with either anti-ATP1B2 (PA526279, Thermo Fisher Scientific), anti-EAAT2 (NOVNBP120136, Novus), anti-CD9 APC (17-0098-42, Thermo Fisher Scientific), anti-CD24 (ab134375, Abcam), anti-EGFR (ab231, Abcam), anti-CD44 Alexa Fluor 488 (103016, BioLegend), or anti-CD133 Alexa Fluor 488 (FAB11331G, R&D Systems) in 50 µL of PBS, for 1 h at 37 °C.

Techniques: Concentration Assay, Marker, Comparison

Journal: Heliyon

Article Title: Morphological changes in perisynaptic astrocytes induced by dopamine neuronal degeneration in the striatum of rats.

doi: 10.1016/j.heliyon.2024.e27637

Figure Lengend Snippet: Fig. 2. Western Blotting for GFAP, GLAST, GLT1, GS expressed in astrocytes. Image A (For original uncropped images, see Appendix. Fig. 2A-GFAP and Fig. 2A-GAPDH in the supplementary materials.), western blotting reveals the distinct GFAP protein bands (52KD) across both the control and 6OHDA groups, and we also chart the semi-quantitative outcomes delineating the protein expression levels of GFAP in the two groups using T test. In image B (For original uncropped images, see Appendix. Fig. 2B-GLAST and Fig. 2B-GAPDH in the supplementary materials.), western blotting shows the distinct GLAST protein bands (59KD) across both the control and 6OHDA groups, and we also chart the semi-quantitative outcomes delineating the protein expression levels of GLAST in the two groups using T test. As for image C (For original uncropped images, see Appendix. Fig. 2C-GLT1 and Fig. 2C-GAPDH in the supplementary materials.), western blotting demonstrates the distinct GLT1 protein bands (62KD) across both the control and 6OHDA groups, and we also chart the semi-quantitative outcomes delineating the protein expression levels of GLT1 in the two groups using T test. Finally, in image D (For original uncropped images, see Appendix. Fig. 2D-GS and Fig. 2D-GAPDH in the supplementary materials.), western blotting reveals the distinct GS protein bands (42KD) across both the control and 6OHDA groups, and we also chart the semi-quantitative outcomes delineating the protein expression levels of GS in the two groups using T test, explicitly demonstrating the heightened expression in the 6OHDA group consequent to DA depletion in contrast to the control group. The asterisk (*) emphasizes statistical significance (*P < 0.05) between the control and 6OHDA groups.

Article Snippet: After blocking with 5% nonfat dry milk for 2 h, the membranes were incubated overnight at 4 ◦C with the following primary antibodies: mouse anti-GFAP (1:5000, catalog no. MAB3402, Millipore), GLAST (1:1000, catalog no. #4166S, CST), GLT1 (1:1000, catalog no. #3838S, CST), GS (1:1000, catalog no. #9832S, CST) and rabbit anti-GAPDH (1:1000, catalog no. mAB5174, CST).

Techniques: Western Blot, Control, Expressing