Journal: Cancers

Article Title: Synthetic Cannabinoids Influence the Invasion of Glioblastoma Cell Lines in a Cell- and Receptor-Dependent Manner

doi: 10.3390/cancers11020161

Figure Lengend Snippet: Analysis of the microRNA (miR) profile in glioblastoma cell lines after treatment with agonists (ACEA, 10 µM; JWH133, 10 µM) and antagonists (AM281, 1 µM; AM630, 1 µM) for cannabinoid receptors (CB) 1 and CB 2 . Each sample was run in duplicates and quantified by 2 −ΔΔC T (DCT) method. Small RNAs, SNORD44 and let-7a, were chosen as reference genes and serve as a reference according to a NormFinder analysis. ( a ) Expression of miR-21 in U-138 MG ( n = 6–8), LN229 ( n = 7–8) and U-87 MG ( n = 9–10). ( b ) Expression of miR-27a in U-138 MG ( n = 6–7), LN229 ( n = 6–8) and U-87 MG ( n = 9–10). ( c ) Expression of miR-34a in U-138 MG ( n = 6–7), LN229 ( n = 6–8) and U-87 MG ( n = 9–10). ( d ) Expression of miR-210 in U-138 MG ( n = 6–7), LN229 ( n = 6–8) and U-87 MG ( n = 8–10). ( e ) Expression of miR-423-5p in U-138 MG ( n = 5–7), LN229 ( n = 5–7) and U-87 MG ( n = 9–10). ( f ) No significant differences could be observed in the expression of miRs 21, 27a, 34a, 210, and 423-5p between the control groups.

Article Snippet: U-87 MG and LN229 cells were purchased from the American Type Culture Collection (U-87 MG: ATCC HTB-14; LN229: ATCC CRL-261, Manassas, VA, USA) and U-138 MG cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Expressing, Control

Journal: Cancers

Article Title: Synthetic Cannabinoids Influence the Invasion of Glioblastoma Cell Lines in a Cell- and Receptor-Dependent Manner

doi: 10.3390/cancers11020161

Figure Lengend Snippet: No changes in the proliferation index could be observed in U-138 MG, LN229, and U-87 MG cell lines after treatment with agonists (ACEA, 10 µM; JWH133, 10 µM) and antagonists (AM281, 1 µM; AM630, 1 µM) for CB 1 and CB 2 for 24 h. Differences occurred in the basal level of proliferation between the cell lines. Control groups of U-138 MG, LN229, and U-87 MG cell lines were compared in the ratio of positive cells for ( a ) Ki67 ( n U138 = 4, n LN229 = 3, n U87 = 4) and ( b ) bromodeoxyuridine (BrdU) ( n U138 = 3, n LN229 = 4, n U87 = 3). All cell lines had a significantly different expression of S-phase marker Brdu. The Ki67 index was significantly different between U-138 MG and LN229. LN229 cells have the highest level of proliferating cells followed by U-138 MG. Application of ACEA, AM281, JWH133, and AM630 did not influence the ratio of the BrdU and Ki67 positive cells in ( c , d ) U-138 MG ( n CTL = 3, n ACEA = 3, n AM281 = 3, n JWH133 = 3, n AM630 = 3); ( e , f ) LN229 ( n CTL = 3, n ACEA = 3, n AM281 = 3, n JWH133 = 3, n AM630 = 3); and ( g , h ) U-87 MG cells ( n CTL = 3, n ACEA = 3, n AM281 = 3, n JWH133 = 3, n AM630 = 3). ( i ) Representative Western Blots of G1 and S-phase marker, proliferating nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) show no significant differences between the groups. All data were normalized to the control group.

Article Snippet: U-87 MG and LN229 cells were purchased from the American Type Culture Collection (U-87 MG: ATCC HTB-14; LN229: ATCC CRL-261, Manassas, VA, USA) and U-138 MG cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Control, Expressing, Marker, Western Blot

Journal: Cancers

Article Title: Synthetic Cannabinoids Influence the Invasion of Glioblastoma Cell Lines in a Cell- and Receptor-Dependent Manner

doi: 10.3390/cancers11020161

Figure Lengend Snippet: No changes in the ratio of death cells could be observed in U-138 MG, LN229, and U-87 MG cell lines after treatment with agonists (ACEA, 10 µM; JWH133, 10 µM) and antagonists (AM281, 1 µM; AM630, 1 µM) for CB 1 and CB 2 . Differences between the cell lines in the basal level of cell death markers were not significant. Control groups of U-138 MG, LN229 and U-87 MG cell lines were compared in the ratio of positive cells for ( a ) propidium iodide (PI) ( n U138 = 3, n LN229 = 3, n U87 = 3) and ( b ) caspase ( n U138 = 4, n LN229 = 3, n U87 = 4). Application of ACEA, AM281, JWH133 and AM630 did not influence the ratio of PI or caspase positive cells in ( c ), ( d ) U-138 MG ( n CTL = 3, n ACEA = 3, n AM281 = 3, n JWH133 = 3, n AM630 = 3); ( e ), ( f ) LN229 ( n CTL = 3, n ACEA = 3, n AM281 = 3, n JWH133 = 3, n AM630 = 3) and ( g ), (h) U-87 MG cells ( n CTL = 3, n ACEA = 3, n AM281 = 3, n JWH133 = 3, n AM630 = 3). ( i ) Representative Western Blots of caspase and GAPDH show no significant differences between the groups. All data were normalized to control group.

Article Snippet: U-87 MG and LN229 cells were purchased from the American Type Culture Collection (U-87 MG: ATCC HTB-14; LN229: ATCC CRL-261, Manassas, VA, USA) and U-138 MG cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Control, Western Blot

Journal: Cancers

Article Title: Synthetic Cannabinoids Influence the Invasion of Glioblastoma Cell Lines in a Cell- and Receptor-Dependent Manner

doi: 10.3390/cancers11020161

Figure Lengend Snippet: Activation of PI3K-Akt pathway in glioblastoma cell lines ( a ) U-138 MG, ( b ) LN229, and ( c ) U-87 MG. After 0 min, 5 min, 10 min, 30 min, 2 h, and 24 h no significant changes were observed in all examined cell lines (U-138 MG: n = 5–7, LN229: n = 5–9, U-87 MG: n = 4–7) in groups treated with agonists (ACEA, 10 µM; JWH133, 10 µM) and antagonists (AM281, 1 µM; AM630, 1 µM) for CB 1 and CB 2 .

Article Snippet: U-87 MG and LN229 cells were purchased from the American Type Culture Collection (U-87 MG: ATCC HTB-14; LN229: ATCC CRL-261, Manassas, VA, USA) and U-138 MG cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Activation Assay

Journal: Cancers

Article Title: Synthetic Cannabinoids Influence the Invasion of Glioblastoma Cell Lines in a Cell- and Receptor-Dependent Manner

doi: 10.3390/cancers11020161

Figure Lengend Snippet: Invasiveness of glioblastoma cells was analyzed in a co-culture model with murine organotypical slice cultures. ( a , b ) Treatment with AM281 (1 µM) had no significant effect on the covered area, whereas coincubation of AM281 with ACEA (10 µM) led to strong anti-invasive effect in LN229. Application of AM630 (1 µM) alone led to significant increase in invasiveness of LN229. Treatment with combination of AM630 with JWH133 reversed the JWH133 (10 µM) mediated reduction in invasiveness indicating that previously observed decrease in covered area was mediated by CB 2 receptor (LN229: n LN229 CTL = 60, n LN229 ACEA = 34, n LN229 AM281 = 33, n LN229 ACEA+AM281 = 35, n LN229 JWH133 = 18, n LN229 AM630 = 19, n LN229 JWH133+AM630 = 15). ( c , d ) Coincubation of ACEA and AM281 reversed the decrease in the covered area mediated by ACEA in U138-MG cells. Incubation with JWH133 led to an increase in the invasiveness, which was abolished by co-application with AM630 (U-138 MG: n U138 CTL = 69, n U138 ACEA = 23, n U138 AM281 = 32, n U138 ACEA+AM281 = 21, n U138 JWH133 = 32, n U138 AM630 = 24, n U138 JWH133+AM630 = 25).

Article Snippet: U-87 MG and LN229 cells were purchased from the American Type Culture Collection (U-87 MG: ATCC HTB-14; LN229: ATCC CRL-261, Manassas, VA, USA) and U-138 MG cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Co-Culture Assay, Incubation

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLND4 promotes malignancy in glioma cells. A. Expression levels (mRNA) of CLDN4 in normal tissues (n=5) and glioma tissues (n=156); data derived from TCGA database. B. Representative images of immunohistochemical staining for CLDN4 in para-carcinomatous tissues (n=15), early-stage glioma tissues (stage I-II, n=15), and late-stage glioma tissues (stage III-IV, n=15); scale bar =50 μm. C. Kaplan-Meier survival curve for high and low expression of CLDN4 in glioma patients (n=513); data from TCGA database. D. Western blotting for CLDN4 in LN229/T98G cells transfected with empty vector or a CLDN4-overexpression vector. E. Cell proliferation assay (CCK-8 assay) for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector. T98G, P<0.05, 72 hours; LN229, P<0.01, 72 hours. F. Cell migration assay (Transwell assay) for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector; scale bar =100 μm. G. Colony formation assay for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector.

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, Western Blot, Transfection, Plasmid Preparation, Over Expression, Proliferation Assay, CCK-8 Assay, Cell Migration Assay, Transwell Assay, Colony Assay

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLDN4-stimulated NNAT upregulation. A. The top 30 upregulated genes in patients with gliomas that had high CLDN4 expression levels as compared to those with gliomas having low CLDN4 expression levels; data derived from TCGA database (n=513). B. Western blotting for CLDN4 and NNAT in LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector. C. Cell proliferation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. LN229, vector vs vector + siRNA, P>0.05, OE vs OE + siRNA, P<0.05, 72 hours; T98G, vector vs vector + siRNA, P>0.05, OE vs OE + siRNA, P<0.01, 72 hours. D. Cell migration assay for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. E. Colony formation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. F. Representative images of immunohistochemical staining for CLND4 in early-stage glioma tissues (stage I-II, n=15) and late-stage glioma tissues (stage III-IV, n=15); scale bar =100 μm.

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Expressing, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Over Expression, Cell Migration Assay, Immunohistochemical staining, Staining

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: The CLDN4/NNAT axis modulates glioma progression through Wnt signaling. (A and B) The results of the KEGG enrichment analysis for the major signaling pathways involved in glioma progression in 513 glioma patients divided into CLDN4-high/low (A) or NNAT-high/low (B) groups. (C) Western blotting for Wnt1, Wnt2, and Wnt3A in LN229 cells transfected with empty vector or CLDN4-overexpression vector. (D) Western blotting for Wnt3A in LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. (E) Cell proliferation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM). LN229, vector vs vector + PP, P>0.05, OE vs OE + PP, P<0.05, 72 hours; T98G, vector vs vector + PP, P>0.05, OE vs OE + PP, P<0.05, 72 hours. (F) Cell migration assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM). (G) Colony formation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM).

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Protein-Protein interactions, Western Blot, Transfection, Plasmid Preparation, Over Expression, Migration

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLDN4 facilitates glioma growth in vivo. A. Western blotting for CLDN4 in primary glioma cells derived from 6 patients. B. Representative images of PDOs derived from CLDN4-high and -low glioma tissues; scale bar =50 μm. C. Western blotting for Wnt3A and NNAT in PDOs derived from CLDN4-high and -low glioma tissues. D. Representative images for immunofluorescence staining for Ki67 in PDOs derived from CLDN4-high and -low glioma tissues; scale bar =50 μm. E. Tumor volumes of subcutaneous tumors formed by LN229 cells transfected with empty vector or CLDN4-overexpression vector (n=5 in each group). F. Kaplan-Meier survival curve for mice with tumors formed by LN229 cells transfected with empty vector or CLDN4-overexpression vector (n=5 in each group).

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: In Vivo, Western Blot, Derivative Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Over Expression

Journal: Frontiers in Oncology

Article Title: GNG12 as A Novel Molecular Marker for the Diagnosis and Treatment of Glioma

doi: 10.3389/fonc.2022.726556

Figure Lengend Snippet: Effects on proliferation and migration of glioma cells by down-regulating GNG12 expression levels. (A) Detection of knockdown efficiency of different siRNA sequences in the glioma cell line LN229. (B) CCK-8 assay to compare the effect on cell proliferation after down-regulation of GNG12. (C) Immunofluorescence assay comparing the positive rate of Ki-67 after transfection with siRNA-NC and siRNA-KD (Magnification: *400). (D) Immunofluorescence assay to analyze the results. (E) Cell scratch healing assay using LN229 cell line to compare the migration distance between GNG12-NC group and GNG12-KD group (Magnification: *200). (F) Results of statistical analysis of cell scratching experiments. (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

Article Snippet: Human glioma cell (LN229) were purchased from Wuhan Procell Biotechnology (Wuhan, China).

Techniques: Migration, Expressing, Knockdown, CCK-8 Assay, Immunofluorescence, Transfection

Journal: Frontiers in Pharmacology

Article Title: MFAP4 is a novel prognostic biomarker in glioma correlating with immunotherapy resistance and ferroptosis

doi: 10.3389/fphar.2025.1551863

Figure Lengend Snippet: MFAP4 regulates proliferation, migration and invasion of GBM cells. (A, B) Down expressed MFAP4 in U251 and LN229 cells was confirmed by Western blotting (A) and qRT-PCR (B) . (C) CCK-8 assay was used to detect the proliferation of U251 and LN229 cells. (D) Colony formation assay was used to test the proliferative capacity of U251 and LN229 cells. (E) Wound-healing assay was used to test the migratory capacity of U251 and LN229 cells. (F) Transwell assay used to detect the invasive ability of U251 and LN229 cells.

Article Snippet: The U251 and LN229 cell line was acquired from Servicebio Biotechnology Co. (Wuhan, China).

Techniques: Migration, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Assay

Journal: Radiation Oncology (London, England)

Article Title: Targeting α ν β 3 and α ν β 5 inhibits photon-induced hypermigration of malignant glioma cells

doi: 10.1186/1748-717X-6-132

Figure Lengend Snippet: Photon-induced stimulation of integrin expression . A: FACS analysis of α ν β 3 (upper row) and α ν β 5 (lower row) expression with (red) and without (black) photon irradiation with single doses of 2 Gy (left) and 10 Gy (right). B: Graphical analysis of integrin α ν β 3 (gray bars) and α ν β 5 (black bars) expression with (bars) and without (red line) irradiation with single photon doses of 2, 5, and 10 Gy in U87 (upper chart) and Ln229 (lower chart) glioma cells. Display of mean value ± standard deviation (SD); statistical analysis using Student's t -test (*, indicating significance p < 0.05)

Article Snippet: Ln229 glioma cells were purchased from LGC Promochem (ATTC CRL-2611), and kept at 37°C and 5% CO 2 in DMEM (FG0415 Biochrom AG) supplemented with 1% Penicillin/Streptomycin and 10% FCS.

Techniques: Expressing, Irradiation, Standard Deviation

Journal: Radiation Oncology (London, England)

Article Title: Targeting α ν β 3 and α ν β 5 inhibits photon-induced hypermigration of malignant glioma cells

doi: 10.1186/1748-717X-6-132

Figure Lengend Snippet: Inhibiton of Ln229 glioma cell migration by integrin inhibition . Quantitative analysis of Vn-based transmigration of Ln229 glioma cells following single photon doses of 0, 2, and 10 Gy without and with the addition of 50 ng/ml anti-α ν β 3 - and -α ν β 5 -antibodies and corresponding isotype controls. Display of mean value ± standard deviation (SD); statistical analysis using Student's t -test (*, indicating significance p < 0.05)

Article Snippet: Ln229 glioma cells were purchased from LGC Promochem (ATTC CRL-2611), and kept at 37°C and 5% CO 2 in DMEM (FG0415 Biochrom AG) supplemented with 1% Penicillin/Streptomycin and 10% FCS.

Techniques: Migration, Inhibition, Transmigration Assay, Standard Deviation

Journal: Frontiers in Oncology

Article Title: System analysis based on the migration- and invasion-related gene sets identifies the infiltration-related genes of glioma

doi: 10.3389/fonc.2023.1075716

Figure Lengend Snippet: EMP3 silencing attenuates the migration and invasion of glioma cells. (A) The expression of EMP3 in different cell lines in the CCLE datasets. (B–D) Western blot and qPCR display EMP3 express highly in U118 and A172. (E–G) The growth curve and plate cloning experiment display that EMP3 silencing has little effect on the cell proliferation of U118 and A172. (H, I) EMP3 knockdown inhibits the migration and invasion of glioma cells. ( J–O , 20×) qPCR displays EMP3 silencing attenuates the expression of MMP2 and MMP9 expression (ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: The normal human astroglia (NHA), which were purchased from the Chongqing Golden Magpie Technology Development Co., and U251, LN229, A172, U118, and U87 cells, which were purchased from the China Center for Type Culture Collection (Shanghai, China), were cultured in the DMEM medium supplemented with 10% FBS (HyClone, UT, USA) and 1% penicillin–streptomycin (Beyotime Biotechnology, Jiangsu, China).

Techniques: Migration, Expressing, Western Blot, Cloning, Knockdown

Journal: Frontiers in Oncology

Article Title: System analysis based on the migration- and invasion-related gene sets identifies the infiltration-related genes of glioma

doi: 10.3389/fonc.2023.1075716

Figure Lengend Snippet: EMP3 silencing suppressed EMT marker expression. (A) Western blot analysis of U118 transfected with indicated siRNAs targeting EMP3 (siEMP3#1, siEMP3#2, and siEMP3#3) or siNC. (B) Western blot analysis of A172 transfected with indicated siRNAs targeting EMP3 (siEMP3#1, siEMP3#2, and siEMP3#3) or siNC. (p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: The normal human astroglia (NHA), which were purchased from the Chongqing Golden Magpie Technology Development Co., and U251, LN229, A172, U118, and U87 cells, which were purchased from the China Center for Type Culture Collection (Shanghai, China), were cultured in the DMEM medium supplemented with 10% FBS (HyClone, UT, USA) and 1% penicillin–streptomycin (Beyotime Biotechnology, Jiangsu, China).

Techniques: Marker, Expressing, Western Blot, Transfection