Journal: Oncology Letters

Article Title: Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma

doi: 10.3892/ol.2020.11548

Figure Lengend Snippet: CHL1 expression level is correlated with PCNA expression levels in different grades of human gliomas, and downregulation of CHL1 induces glioma cell senescence. (A) Representative immunofluorescence staining of PCNA and CHL1 in CAN and human glioma tissues (graded I–II and II). Scale bars, 50 µm. (B) Correlation between CHL1 and PCNA expression levels (n=23, 18, 85, 27 and 11 for I, I–II, II, III and IV grades, respectively). (C) Immunofluorescence staining of CHL1 and PCNA in U-87 MG cells treated with either a control siRNA or an siRNA targeting CHL1 for 48 h. Downregulation of CHL1 resulted in the reduction of PCNA expression levels. Scale bars, 25 µm. (D) Enhanced staining of senescence-associated β-galactosidase was observed in cells with CHL1 knockdown, suggesting that CHL1 may function by delaying the senescence of glioblastoma cells. CHL1, cell adhesion molecule L1 like; PCNA, proliferation cell nuclear antigen; si, small interfering; con, control.

Article Snippet: The human glioblastoma U-87 MG cell line (cat. no. CL-0238; unknown origin) and the human glioma U251 cell line (cat. no. CL-0237) were purchased from Procell Life Science and Technology Co., Ltd.

Techniques: Expressing, Immunofluorescence, Staining, Control, Knockdown

Journal: Oncology Letters

Article Title: Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma

doi: 10.3892/ol.2020.11548

Figure Lengend Snippet: Western blot analysis of CHL1 expression levels in cultured SHG-44 and U251 human glioma and U-87 MG human glioblastoma cells in response to 48 h of treatment with (A) NRG1α or (Β) NRG1β at doses ranging from 0 to 5.0 nM. (C) A total of 3 siRNAs targeting NRG1 reduced NRG1 and CHL1 expression levels. *P<0.05. CHL1, cell adhesion molecule L1 like; NRG1, neuregulin 1; si, small interfering; Mr, molecular mass.

Article Snippet: The human glioblastoma U-87 MG cell line (cat. no. CL-0238; unknown origin) and the human glioma U251 cell line (cat. no. CL-0237) were purchased from Procell Life Science and Technology Co., Ltd.

Techniques: Western Blot, Expressing, Cell Culture

Journal: Oncology Letters

Article Title: Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma

doi: 10.3892/ol.2020.11548

Figure Lengend Snippet: Expression levels and localization of CHL1 in U251 human glioma cells and U-87 MG human glioblastoma cells in response to treatment with 2.5 nM NRG1s. U251 and U-87 MG cells were treated with 2.5 nM NRG1α and NRG1β for 48 h. The cells were then fixed and immunostained for CHL1 (green) and p-Neu (red), and then counterstained with DAPI (blue). Representative immunofluorescence staining showing the effects of NRG1α and NRG1β on CHL1 expression levels and localization in (A) U251 and (B) U-87 MG cells., Scale bars 30 and 20 µm, respectively. CHL1, cell adhesion molecule L1 like; NRG1, neuregulin 1; NEU, ErbB2 receptor; p, phosphorylated.

Article Snippet: The human glioblastoma U-87 MG cell line (cat. no. CL-0238; unknown origin) and the human glioma U251 cell line (cat. no. CL-0237) were purchased from Procell Life Science and Technology Co., Ltd.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Oncology Letters

Article Title: Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma

doi: 10.3892/ol.2020.11548

Figure Lengend Snippet: Effects of NRG1s on the expression levels of p-ERK1/2 in U-87 MG cells. Representative images showing western blot analysis for p-ERK1/2 in SHG-44, U251 and U-87 MG human glioma/glioblastoma cells in response to (A) NRG1α or (B) NRG1β at doses ranging from 0 to 5.0 nM. NRG1, neuregulin 1; p, phosphorylated.

Article Snippet: The human glioblastoma U-87 MG cell line (cat. no. CL-0238; unknown origin) and the human glioma U251 cell line (cat. no. CL-0237) were purchased from Procell Life Science and Technology Co., Ltd.

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Anticancer effect of the oncolytic Newcastle disease virus harboring the PTEN gene on glioblastoma

doi: 10.3892/ol.2024.14752

Figure Lengend Snippet: PTEN restoration suppresses tumorigenesis in orthotopic GBM mouse through infection of rNDV. (A) Mice were orthotopically injected with U87-MG-Luc2 cells. A total of 40 days after tumor cell injection, the mice were infected with rNDV-PTEN or rNDV via i.v. injection. (B) Body weight of in vivo mouse GBM models. (C) Survival rate of orthotopic GBM mouse models following rNDV-PTEN, rNDV or PBS injections (n=5). (D) Bioluminescent images of luciferase activity. Bioluminescent images were taken with IVIS Lumina XR and analyzed using Living Image Software (n=5). (E) MRI in orthotopic GBM mouse models. Representative MRI image of orthotopic GBM mouse models, taken 50 days after tumor injection (rNDV-PTEN, rNDV or PBS was injected 5 times). These data are expressed as the fold change in expression compared with PBS injected mice. *P<0.05 vs. PBS-injected mice. PTEN, phosphatase and tensin homolog; GBM, glioblastoma; rNDV, recombinant Newcastle disease virus; MRI, magnetic resonance imaging; i.v., intravenous.

Article Snippet: Human GBM cells, U87-MG (cat no. HTB-14; GBM of unknown origin), U87-MG-luc2 (cat. no. HTB-14-LUC2; GBM of unknown origin), T98G (cat. no. CRL-1690) and CCF-STTG1 (cat. no. CRL-1718), were purchased from American Type Culture Collection (ATCC).

Techniques: Infection, Injection, In Vivo, Luciferase, Activity Assay, Software, Expressing, Recombinant, Virus, Magnetic Resonance Imaging

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: Characterization of TNTs formed upon treatment with α-SYN protofibrils in astroglia cells (A) The red arrow in the DIC image (a’) indicates TNT formed between two astrocytes on α-SYN protofibril treatment. The bottom panel shows a 3D volume view of Phalloidin and GFAP-positive astrocytes connected by both long (a’’) and short TNTs (b). (B and C) DIC images of U-87 MG cells show TNTs (B) and TMs (C). The cells were stained with phalloidin and β-tubulin; both bottom panels show 3D volume views of TNT as an actin-positive hovering structure (B) and tubulin-positive TM at the surface (C). Red arrows indicate actin-positive TNTs and blue arrows indicate β-tubulin-positive tumor microtubes (TM), respectively. (D and E) Characterization of GJ-negative (D) and -positive (E) TNTs based on Phalloidin and connexin43 staining at the tip of the TNT in U-87 MG cells. Pink arrows indicate close-ended TNT. The bottom panel shows 3D volume views of the same. (F) Closed-ended TM characterized by the presence of connexin43 staining, indicated by pink arrows. The bottom panel shows 3D volume views of the TM at the surface. (G) TNT-like structures (red arrows) are detectable in DIC images as focused structures at z = 4. At z = 0, filopodia-like extensions (magenta arrows) on the substratum are at focus. (H and I) U87MG cells treated with 1μM α-SYN protofibrils for 3h and 6h, stained with lysotracker and MitoTracker, respectively. Red arrows indicate lysotracker and MitoTracker through TNTs at 3h and 6h. Movement of lysotracker (H) and MitoTracker (I) positive vesicles through TNTs were tracked using the TrackMate plug-in of Fiji. (J) Quantification of the percentage of TNTs and (K) percentage of GJ-negative TNTs, GJ-positive TNTs, and TMs from the confocal z-stack images of U-87 MG cells. Quantification of length (L) and diameter (M) of TNTs formed by U-87 MG cells, U251 cells, and astrocytes. Quantifications are done from 10 to 15 image frames of a set and each image frame has 10–20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA (2J and 2K) and one-way ANOVA (2L and 2M). n = 3.

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: Staining

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: TNT biogenesis pathways in cell proliferation (A) Absorbance measured at 570 nm after MTT assay performed in astrocytes treated with varying concentrations (0.5μM, 1μM, 2μM, and 3μM) of α-SYN protofibrils at 3h, 6h, 12h and 24h. (B) Cell number quantification of U-87 MG cells treated with 1μM α-SYN protofibrils after 3h, 6h, 12h, 18h and 24h. (C) MTT absorbance estimated at 24h on treatment with varying concentrations (0.5μM, 1μM, 2μM, and 3μM) of α-SYN protofibrils in U-87 MG cells. (D) Estimation of MTT absorbance at 12h and 24h after treatment with 1μM α-SYN protofibrils in U251 cells. (E) Fluorescence images of astrocytes and U87MG cells treated with 1μM α-SYN protofibrils for 3h–24h were stained with proliferation marker Ki67 along with nuclear stain. (F and G) Quantification of the intensity of Ki67 per cell in astrocytes and U-87 MG respectively. Quantifications are done from 15 image frames of a set and each image frame has 20–25 cells. (H) Flow chart depicting the mode of action of actin inhibitors. (I) DiD (membrane dye) stained, control and 1μM α-SYN protofibrils treated U-87 MG cells pre-treated (before 30min) with 3 μM IPA3, 0.5 μM cytochalasin D, 50 μM CK-666 (Arp2/3 inhibitor), 75 μM blebbistatin and 5 μM Y-27632 (ROCK inhibitor). Red arrows indicate the formation of TNT-like structures observed at 3 h and (K) shows the quantification of TNT numbers. (J) representative images showing cell numbers with the above-mentioned treatment with inhibitors and α-SYN protofibrils at 24 h. (L and M) quantification of MTT absorbance and cell numbers of the same, respectively. Quantifications are done from 5 image frames of a set and each image frame has 15–20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA, and only graph 6B was analyzed using one-way ANOVA. n = 3.

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: MTT Assay, Fluorescence, Staining, Marker, Membrane, Control

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: FAK translocation, ROCK remodulation, and activation of proliferation pathway (A) Estimation of uptake of α-SYN-TMR protofibrils by U251 cells treated with 1μM α-SYN protofibrils and pre-treated (before 30min) with 0.5 μM cytochalasin D and 5 μM Y-27632 (ROCK inhibitor) by flow cytometry. (B) Quantification of percentage gated fluorescence intensity of α-SYN-TMR protofibrils in the above experiment. (C) Fluorescence images of maximum intensity projected confocal z-stacks in astrocytes stained with Phalloidin (green) and FAK (red). The upper panel has nucleus stained with DAPI and the lower panel is without DAPI. White arrows indicate the nuclear colocalization of FAK, red arrows indicate TNTs and pink arrows indicate FAK at focal adhesion. (D) Quantification of FAK intensity in the nucleus of the cells with and without TNTs. (E and G) Fluorescence images of maximum intensity projected confocal z-stacks in U-87 MG cells stained with FAK (red) and pFAK (red) with DAPI, respectively. (F and H) Quantification of FAK and pFAK fluorescence intensity per cell in the nucleus. Quantifications are done from 15 image frames in a set, and each image frame has 15–20 cells. (I) Western blot images showing the change in ROCK1 and ROCK2 expressions with time on 1μM α-SYN protofibril treatment in U-87 MG cells. (J) Quantification of ROCK1 and ROCK2 western blots. Full-length western blots of four repeats are represented in Figure S9 . (K) Western blot images showing increased intensity of pERK1/pERK2, NF-κB, and Cdk1 with time on 1μM α-SYN protofibril treatment in U-87 MG cells. Full-length western blots of three repeats for pERK1/pERK2 and NF-κB and five repeats for Cdk1 were represented in Figure S10 . (L) Quantification of pERK1/pERK2, NF-κB, and Cdk1 western blots. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. n = 3.

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: Translocation Assay, Activation Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet:

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: Recombinant, Staining, Software