Journal: BioMed Research International

Article Title: lncRNA MEG3 Inhibits the Proliferation and Growth of Glioma Cells by Downregulating Bcl-xL in the PI3K/Akt/NF- κ B Signal Pathway

doi: 10.1155/2022/3729069

Figure Lengend Snippet: Expression of lncRNA MEG3 and transfection of U-251MG cells. (a) mRNA levels of lncRNA MEG3 in U-251MG cells, human astrocytes (HAs), and human tracheal epithelial cells (TECs); ∗∗ denotes p < 0.01 compared to Has and TECs. (b) Green fluorescence from U-251MG cells and U-251MG cells transfected with vector and pMEG3. (c) MEG3 mRNA levels in U-251MG cells after pMEG3 transfection. ∗∗ denotes p < 0.01 compared to U-251MG and U-251MG-vector.

Article Snippet: Human astrocytes (HAs) were obtained from iXCells (San Diego, CA, USA) and cultured in an astrocyte medium (cat no. 10HU-035, iXCells) at 37°C in a 5% CO 2 humidified atmosphere.

Techniques: Expressing, Transfection, Fluorescence, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: A New Human Blood–Retinal Barrier Model Based on Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ijms21051636

Figure Lengend Snippet: Experimental procedure followed to set up the in vitro BRB model. ( A ) Polylysine (PLL) coating; ( B ) human retinal pericytes are seeded on the bottom side of the insert; ( C ) insert is rotated of 180° and inserted into a 12-well plate containing pericytes medium; ( D ) human retinal astrocytes are seeded on a 12-well plate containing astrocytes medium; ( E ) cell incubation and adhesion; ( F ) the insert with pericytes is moved into the 12-well plate holding astrocytes; ( G ) human retinal endothelial cells are seeded on the top side of the insert; ( H ) cells are grown with a medium consisting of a mixture of the three cell lines’ media (1:1:1); ( I ) glucose was added in order to obtain high glucose conditions.

Article Snippet: Human retinal endothelial cells, human retinal pericytes, human retinal astrocytes, endothelial cell medium (ECM), fetal bovine serum (FBS), endothelial cell growth supplement (ECGS), pericyte cell medium (PM), pericyte supplement factor (PGS), astrocyte cell medium (AM), astrocytes supplement factor (AGS), PLL, and penicillin–streptomycin were purchased from INNOPROT (Derio, Bizkaia, Spain).

Techniques: In Vitro, Incubation

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Plasmid map for GDNF production

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Bone-marrow derived macrophages were transfected by electroporation using four different conditions described in Experimental section. Then, cells were washed and cultured in complete media for up to 6 days. The GDNF expression levels in cells (solid bars), and EVs collected from conditioned media (stripped bars) was assessed by ELISA on day 1 (white bars), day 4 (grey bars), and day 6 (black bars). Successful transfection was accomplished with three conditions (#2 - #4). N = 4, *p < 0.05, compared to sham-transfected macrophages (dashed line, condition #1).

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Derivative Assay, Transfection, Electroporation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Primary macrophages were transfected with GDNF-encoding p DNA by electroporation (condition #4), and EV-GDNF were collected from conditioned media on day 6. EV-GDNF were characterized for size, zeta potential, and morphology by ZetaView QUATT Nanoparticle Tracking Microscope PMX-420 ( A ), and AFM ( B ). The presence of EV-specific membrane proteins was EV-GDNF was confirmed by Wes ( C ) and quantified using Compass SW software ( D ). The bar: 1 µm.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Electroporation, Zeta Potential Analyzer, Microscopy, Membrane, Software

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: EVs samples from sham-transfected (white bars), and GDNF-transfected (black bars) macrophages were digested ( N =3) with trypsin and examined by nano-liquid chromatography tandem MS (nanoLC–MS/MS) with multiple reaction monitoring (MRM). Samples of 20 µg total protein were used, and 0.08 µg (0.4 % of the sample) was injected. No significant differences in specific proteins expression were found between sham EVs and EV-GDNF (t-tests, p < 0.05). Peptide identification is shown in . A CD81 peptide employed in other studies was not detected in these analyses. Values are means ± SD.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Liquid Chromatography, Tandem Mass Spectroscopy, Targeted Proteomics, Injection, Expressing

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Macrophages were transfected with GDNF-encoding p DNA by electroporation, and the levels of GDNF-DNA in the cells ( A ) and EVs released by these cells ( B ) were assessed. A significant amount of GDNF-DNA was detected in parent cells, as well as in the EVs. Statistical significance was assessed by One Way ANOVA corrected for multiple comparisons using the FDR. ** p < 0.01, or **** p < 0.0001.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Electroporation

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: The effect of EV-GDNF on motor functions and activity was assessed in Wire hanging test, and Rotarod test ( A, B ), as well as in OFA tests ( C, D ). ( A, B ) Transgenic mice were i.n . injected with EV-GDNF (triangles, 3×10 9 particles/10 µL/mouse), or sham EVs (empty circles, 3×10 9 particles/10 µL/mouse), or saline (filled circles, 10 µL/mouse). Wild type mice were i.n . injected with saline (filled squares, 10 µL/mouse) were used as controls. Wire hanging test ( A ), and Rotarod test ( B ) demonstrated significant improvements in motor functions upon treatment with EV-GDNF. ( C, D ) OFA tests at 12 mo. demonstrated improved behavior in EV-GDNF treated PD mice (striped bars) compared to PD mice treated with saline (white bars) that was similar as in healthy WT mice (black bars) including decreases in the hyperactivity and anxiety-like behavior. The differences between sham EVs and saline in PD mice were inconclusive. Values are means ± SEM ( N = 10), * p < 0.05, ** p < 0.005, and # p < 0.05, as compared to WT control.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Activity Assay, Transgenic Assay, Injection, Saline, Control

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old, N = 10) were i.n . injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, and brain slides were stained with TH, a marker for dopaminergic neurons ( A ); or Ab to CD11b for activated microglia ( B ). The images indicate significant preservation of TH-positive neurons and decrease in microglial activation in Parkin Q311(X)A mice upon EV-GDNF treatment compared to PD mice treated with saline. The administration of sham EVs did not cause significant therapeutic effects.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining, Marker, Preserving, Activation Assay

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brains were removed post-mortem, and homogenized in cell lysis buffer. Elevated cytokine levels in the brain, were recorded in PD mice treated with saline and Sham EVs. Administration of EV-GDNF significantly decreased pro-inflammatory molecules in the brain compared with PD mice treated with saline. N = 4, # p < 0.05 compared to healthy WT animals; * p < 0.05 compared to PD mice treated with saline, $ p <0.05 compared to PD mice treated with saline and sham EVs.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Lysis

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or ( 3 ) EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse). Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining ( A – D ) and H&E staining ( E – H ). The obtained bright light images show lower number of Nissl bodies with neuronal shrinkage ( B ) and damages tissues with degeneration in the neurons ( F ) in PD mice treated with saline when compared to WT mice ( A, E ). Histological analysis indicate neuroprotective effects in the brain of PD mice treated with EV-GDNF with healthy morphology in tissue structure and high integrity of neurons ( C, G ) when comparted to PD mice treated with saline ( B, F ). The administration of sham EVs did not have significant therapeutic effect in PD mice ( D, H ). Black arrows, degenerated neurons; blue arrows, elongated irregular nuclear morphology.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse) weekly three times. Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining. The obtained bright light images show lower number of Nissl bodies with neuronal shrinkage and damages tissues with degeneration in the neurons in PD mice treated with saline when compared to WT mice. Histological analysis indicates neuroprotective effects in the brain of PD mice treated with GDNF-EVs with healthy morphology in tissue structure and high integrity of neurons when comparted to PD mice treated with saline. The administration of sham EVs did not have significant therapeutic effect in PD mice.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. old) were intranasally injected with: saline (10 µL/mouse), or EV-GDNF (3×10 9 particles/10 µL/mouse), or sham EVs (3×10 9 particles/10 µL/mouse) weekly three times. Wild type control mice were intranasally injected with saline (10 µL/mouse). Animals were sacrificed at mo. 16, brain slides were stained with Nissl staining. The obtained bright light images show damaged tissues with degeneration in the neurons in PD mice treated with saline when compared to WT mice. Histological analysis indicates neuroprotective effects in the brain of PD mice treated with GDNF-EVs with healthy morphology in tissue structure when comparted to PD mice treated with saline. The administration of sham EVs did not have significant therapeutic effect in PD mice.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline, Control, Staining

Journal: bioRxiv

Article Title: Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease

doi: 10.1101/2022.05.25.493424

Figure Lengend Snippet: Transgenic mice (4 mo. of age) were i.n . injected with saline, or EV-GDNF, or sham EVs (3×10 9 particles/10 µL/mouse, once a week, 3x weeks). At 16 mo. of age total weigh of the animals was recorded. No gross toxicity manifested in the losing weight was detected in mice injected with EV-GDNF and well as sham EVs.

Article Snippet: Human GDNF cDNA (NM_199234) was provided by OriGene (Rockville, MD, USA) that was propagated in DH5α E.coli, followed by purification Giga-prep kits (Qiagen, Valencia, CA, USA).

Techniques: Transgenic Assay, Injection, Saline

Journal: Nature Communications

Article Title: Targeting leucine-rich repeat kinase 2 overcomes resistance to oncolytic herpes simplex virus-based therapies in glioblastoma

doi: 10.1038/s41467-026-70132-9

Figure Lengend Snippet: a Western blot analysis of LRRK2 and phosphorylated Rab10 (p-Rab10) protein expression in different GBM cell lines. The samples derive from the same experiment, but different gels for LRRK2, another for p-Rab10 and another for GAPDH were processed in parallel. b EC 50 values were determined in GBM cell lines treated with escalating titers of virus, with or without LRRK2-IN-1 (2.5 μM), for 72 h. c Pearson correlation analysis between the EC 50 values for oHSV-GFP and relative expression levels of LRRK2 or p-Rab10 in different GBM cell lines. Quantified LRRK2 expression was derived from band densities shown in Fig. 8a. d–g Effects of LRRK2-IN-1 on oHSV-GFP-mediated cytotoxicity in various normal cell types. EC 50 shifts were determined in primary astrocytes ( d ) human PBMCs-derived macrophages ( e ) iPSCs-derived neurons ( f ) or human microglia cells ( g ) treated with escalating titers of virus with or without LRRK2-IN-1 (2.5 μM), for 48 h. h Western blot analysis of LRRK2 protein expression in two GBM PDX tumors. The samples derive from the same experiment, but different gels for LRRK2 and another for actin were processed in parallel. i Experimental timeline for the PDX mouse model. NOD/SCID mice were subcutaneously inoculated with GBM PDX tumors. Once the tumor volume reached approximately 100–150 mm 3 , mice were treated with vehicle, oHSV-GFP virus (1 × 10 7 PFU, intratumorally every 2 days for a total of three injections), LRRK2-IN-1 (25 mg/kg, intraperitoneally every 2 days for a total of three injections), or a combination (Combo). j , k Tumor growth was monitored over time in GBM-PDX-1 ( n = 7) and GBM-PDX-2 ( n = 5). Western blot analyses were independently repeated three times for panel a, and twice for panel h, with similar results. The data are presented as the mean ± SD from three independent experiments ( b , d – g ), and P values were calculated using Spearman’s correlation test ( c ) and repeated measures ANOVA ( j , k ).

Article Snippet: Human astrocytes were purchased from iXCell Biotechnologies and cultured in astrocyte medium according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Virus, Derivative Assay

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: CYBC1 Drives Glioblastoma Progression via Reactive Oxygen Species and NF-κB Pathways

doi: 10.4143/crt.2024.827

Figure Lengend Snippet: Analysis of cytochrome b-245 chaperone 1 (CYBC1) expression. (A) CYBC1 mRNA expression in different glioma types. Non-tumor, n=4; oligodendroglioma, n=191; oligoastrocytoma, n=130; astrocytoma, n=194; glioblastoma (GBM), n=152. One-way ANOVA with Tukey’s multiple comparison test was conducted for statistical analysis. (B) CYBC1 expression in non-tumors and gliomas. Non-tumor, n=4; GBM, n=156. This data was derived from the GlioVis database ( http://gliovis.bioinfo.cnio.es/ ). TCGA, The Cancer Genome Atlas.(C) Differential mRNA expression of the CYBC1 gene in Gene Expression Omnibus (GEO) datasets (accession number: GSE15824 ). Non-tumor, n=5; GBM, n=12. (D) Kaplan-Meier survival curves derived from GEPIA ( http://gepia.cancer-pku.cn/index.html ). (E) CYBC1 immunostaining in normal cortex, low-grade glioma (LGG), and high-grade glioma (HGG). Data are derived from the Human Protein Atlas. (F) CYBC1 immunostaining in normal (n=10), astrocytoma (n=22), and GBM (n=23) tissue microarrays (GL807a). Scale bars=500 μm. (G) Western blot analysis of CYBC1 expression in normal human astrocytes (NHA) and GBM cell lines. Immunofluorescence (H) and immunoblot (I) assay demonstrating CYBC1 localization in GBM cells. Scale bars=200 μm. n.s, not significant.

Article Snippet: U373 and U87 cells were purchased from the Korean Cell line bank, and normal human astrocytes (NHA) were purchased from iXCells Biotechnologies.

Techniques: Expressing, Comparison, Derivative Assay, Gene Expression, Immunostaining, Western Blot, Immunofluorescence