Journal: Oxidative Medicine and Cellular Longevity

Article Title: Circ_0000189 Promotes the Malignancy of Glioma Cells via Regulating miR-192-5p-ZEB2 Axis

doi: 10.1155/2022/2521951

Figure Lengend Snippet: The expression characteristics of circ_0000189 in gliomas. (a) qRT-PCR was utilized to detect circ_0000189 expression in human glioma tissues and adjacent normal tissues (total = 50). (b) Circ_0000189 expression in human glioma cell lines (A172, U87, LN18, U251, LN229, and U118 cells), normal human astrocyte (NHA) cell line, and human normal brain glial cell line (HEB) was investigated by qRT-PCR. ∗∗ symbolizes P < 0.01, and ∗∗∗ symbolizes P < 0.001.

Article Snippet: Normal human astrocyte cell line (NHA), human normal brain glial cell line (HEB), and human glioma cell lines (A172, U87, LN18, U251, LN229, and U118 cells) were available from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Circ_0000189 Promotes the Malignancy of Glioma Cells via Regulating miR-192-5p-ZEB2 Axis

doi: 10.1155/2022/2521951

Figure Lengend Snippet: miR-192-5p was a target of circ_0000189 in gliomas. (a) Bioinformatics predicted the binding site between circ_0000189 and miR-192-5p. (b) qRT-PCR was utilized to explore miR-192-5p expression in the normal human astrocyte (NHA) cell line and human glioma cell lines (A172, U87, LN18, U251, and LN229, as well as U118 cells). (c) Dual luciferase report assay was performed to verify the predicted binding site between miR-192-5p and circ_0000189. (d) RIP experiment was performed to verify the direct interaction between circ_0000189 and miR-192-5p. (e) After upregulating the expression of circ_0000189 in A172 cells and knocking down the expression of circ_0000189 in U118 cells, qRT-PCR was utilized to investigate miR-192-5p expression. (f) miR-192-5p expression in normal tissues and glioma tissues was detected by qRT-PCR. (g) Analysis of the correlation between circ_0000189 expressions and miR-192-5p expression in glioma tissues. ∗ symbolizes P < 0.05, ∗∗ symbolizes P < 0.01, and ∗∗∗ symbolizes P < 0.001.

Article Snippet: Normal human astrocyte cell line (NHA), human normal brain glial cell line (HEB), and human glioma cell lines (A172, U87, LN18, U251, LN229, and U118 cells) were available from China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation

doi: 10.3390/ijms23094996

Figure Lengend Snippet: AKR1C1-4 are differentially expressed in GB cells. The expression of AKR1C1-4 was detected in a total protein extract from normal human astrocytes (HA) and different human GB cell lines. ( a ) Representative Western blots of AKR1C1-4 and α-Tubulin, which was used as a loading control. ( b ) Densitometric analysis graph. Each column represents the mean ± SEM. n = 3; * p < 0.05 U251, T98G, and LN229 vs. HA and U87 cell lines.

Article Snippet: For Western blot determination of AKR1C1-4 (37 kDa), 20 μg of normal human primary astrocytes lysate (HA; 1806, ScienCell, Carlsbad, CA, USA), and 20 μg of protein lysate of the cell lines were mixed with Laemmli 2X buffer (100 mM Tris-base pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, and bromophenol blue) were boiled for 5 min and separated in a 12% SDS-PAGE gels at 80 V. The separated proteins were then transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA) by electrophoresis in semi-dry conditions at 30 mA per membrane for 2 h. Membranes were blocked in agitation at 37 °C with a blocking solution (TBS buffer-0.1% Tween with 5% bovine serum albumin; InVitro, MEX) for 2 h; then, membranes were incubated with a mouse monoclonal AKR1C1-4 antibody (1:1000; sc-390419, Santa Cruz, CA, USA) overnight.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Neuroinflammation

Article Title: Astrocytic DLL4-NOTCH1 signaling pathway promotes neuroinflammation via the IL-6-STAT3 axis

doi: 10.1186/s12974-024-03246-w

Figure Lengend Snippet: DLL4 is expressed by CNS astrocytes and upregulated during chronic neuroinflammation. ( A-D ) Human astrocytes (NA) were cultured until confluence, starved for 24 h and treated for 24 h with PBS 1X (control) versus 10 ng/mL of recombinant IL-1β. ( A ) DLL4 and ( B ) VIM expression were quantified by qRT-PCR. β-ACTIN was used as a reference ( n = 6). ( C ) Representative western blot (WB) for DLL4 and β-ACTIN (ACTB) protein expression level are shown. ( D ) DLL4 protein expression level was quantified by western blot. ACTB was used as a reference. ( n = 6). ( E-G ) C57Bl/6 females (4 animals/group) were induced with EAE versus Freund adjuvant alone (Ctrl). At day 13 post induction, mice were sacrificed and spinal cord neurovascular units were isolated. ( E ) Dll4 , ( F ) Hey1 , and ( G ) Jag1 expression were measured via qRT-PCR in both groups (MOG 35 − 55 versus Ctrl) ( n = 4). ( H ) Spinal cord sections were harvested from MOG 35 − 55 EAE-induced versus Ctrl C57Bl/6 females and immuno-stained with anti-DLL4 (in red) and anti-GFAP (in green) antibodies. Representative DLL4/GFAP staining is shown. ( I ) DLL4 positive areas were quantified (Ctrl mice n = 6 and MOG 35 − 55 EAE-induced mice n = 6). ( J ) Human cortical sections from non-pathological donors and patients with MS lesions were immunostained with anti-DLL4 (in green) and anti-GFAP (in red) antibodies. Nuclei were stained with DAPI (in blue). Representative DLL4/GFAP staining is shown. DLL4 quantification in the human cortical sections cannot be provided as the number of samples available (3 /group) limits us. ( K ) 12-week-old C57Bl/6 females were induced with EAE. At day 5, 11, 18 and 24 post induction, mice were sacrificed (4–5 animals per group) and spinal cord cervical, thoracic and lumbar sections were harvested and immuno-stained with anti-DLL4 and anti-GFAP antibodies. DLL4 positive areas in the 3 sections were quantified at each time point. Statistical significance was determined by using a Mann-Whitney U test or 2 ways Anova followed by Holm-Sidak’s multiple comparisons test

Article Snippet: First, we showed that DLL4 is strongly upregulated in reactive astrocytes under inflammatory conditions in vitro using CNS normal human astrocytes (NA) from ScienCell treated with IL-1β (interleukin-1β), a central pro-inflammatory cytokine driving multiple sclerosis pathophysiology (Fig. , A, C-D) and in vivo (Fig. , E), in the neurovascular unit, using experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis using MOG 35 − 55 to induce chronic neuroinflammation in C57BL/6 mice.

Techniques: Cell Culture, Control, Recombinant, Expressing, Quantitative RT-PCR, Western Blot, Adjuvant, Isolation, Staining, MANN-WHITNEY

Journal: International Journal of Oncology

Article Title: Glioma-amplified sequence KUB3 influences double-strand break repair after ionizing radiation

doi: 10.3892/ijo.2013.1937

Figure Lengend Snippet: (A) Correlation between KUB3 amplification and DSB repair efficiency in glioma derived cell cultures and normal human astrocytes. Following radiation with 1 Gy, γ-H2AX foci were counted in glioblastoma cell lines TX3095, TX3868, H346 and H385, in astrocytoma (WHO II) T6468 and in normal human astrocytes (NHA). Percentage of remaining γ-H2AX foci (indirect readout for DSBs) is indicated on the y-axis. The repair time is plotted on the x-axis. (B) Expression and amplification status of glioma derived cell lines, normal brain and normal human astrocytes. Total protein extract (30 μ g) was separated by SDS-PAGE. The membrane was immunostained with anti-KUB3. β-actin staining confirmed equal loading. Arrows indicate KUB3 at 32 kDa.

Article Snippet: Normal human astrocytes were obtained from CellSystems and maintained in ABM (astrocyte basal medium) without L-glutamine (Clonetics, Cambrex Bio Science, Walkersville, MD, USA).

Techniques: Amplification, Derivative Assay, Expressing, SDS Page, Membrane, Staining