cell lines ln229 atcc  (ATCC)

Journal: Neuroscience

Article Title: Knockdown of Girdin Induced Apoptosis of Glioblastoma Cells via the Mitochondrion Signaling Pathway.

doi: 10.1016/j.neuroscience.2022.07.025

Figure Lengend Snippet: Fig. 3. Knockdown of Girdin induced apoptosis of LN229 cells. (A) Comparison of cell survival rate in scr/LN229 and shGirdin/LN229 cells by CCK- 8 assay. Values were expressed as mean ± SD from three independent experiments, data satisfied normal distribution with equal variances (two- tailed Student’s t test and two-way ANOVA, ***P < 0.001, ****P < 0.0001). (B) The effect of knockdown of the Girdin expression on glioma cell cycle. (C) The annexin V-PE/7-ADD apoptosis detection kit was used to detect cells apoptosis with flow cytometry. Apoptotic condition of cells was shown in the images. Quantitative result of apoptosis assay was analyzed by flow cytometry. Values were expressed as mean ± SD from three independent experiments, data satisfied normal distribution with equal variances (two-tailed Student’s t test, **P < 0.01).

Article Snippet: Human glioblastoma cell line LN229 was obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 C in a 5% CO2 incubator.

Techniques: Knockdown, Comparison, CCK-8 Assay, Two Tailed Test, Expressing, Cytometry, Apoptosis Assay

Journal: Neuroscience

Article Title: Knockdown of Girdin Induced Apoptosis of Glioblastoma Cells via the Mitochondrion Signaling Pathway.

doi: 10.1016/j.neuroscience.2022.07.025

Figure Lengend Snippet: Fig. 4. The role of Girdin regulating glioma cells apoptosis was further confirmed by measuring Cytochrome C efflux from mitochondria. (A) DAPI staining condition of scr/LN229 and shGirdin/LN229 cells. Original magnification, 200. Values were expressed as mean ± SD from three independent experiments, data satisfied normal distribution with unequal variances (two-tailed Student’s t test, **P < 0.01). (B) Cells were incubated with anti-cytochrome c antibody at room temperature in the dark and Cytochrome C efflux from mitochondria was detected by flow cytometry. (C) Western blotting was performed to evaluate the expression of AKT, p-AKT and apoptosis-related proteins. Result is the representative image from three independent experiments.

Article Snippet: Human glioblastoma cell line LN229 was obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 C in a 5% CO2 incubator.

Techniques: Staining, Two Tailed Test, Incubation, Cytometry, Western Blot, Expressing

Journal: Neuroscience

Article Title: Knockdown of Girdin Induced Apoptosis of Glioblastoma Cells via the Mitochondrion Signaling Pathway.

doi: 10.1016/j.neuroscience.2022.07.025

Figure Lengend Snippet: Fig. 5. Knockdown of Girdin induced glioma cells apoptosis in vivo assay. (A) scr/LN229 and shGirdin/LN229 cells were subcutaneous injected into Nu/Nu mice respectively. The size of tumors was measured each week. The upper panel showed the representative images of hematoxylin–eosin staining in scr/LN229 and shGirdin/LN229 groups (200). The bottom showed volume of tumor xenografts in scr/LN229 mice group (n = 6) and shGirdin/LN229 mice group (n = 10), data satisfied normal distribution with equal variances (two-tailed Student’s t test, *P < 0.05, **P < 0.01). (B) Xenograft tumor tissues were obtained from subcutaneously inoculated Nu/Nu mice, and the expression of Bad and Cyt-C were detected by immunohistochemical staining after tissue section. Original magnification, 400.

Article Snippet: Human glioblastoma cell line LN229 was obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 C in a 5% CO2 incubator.

Techniques: Knockdown, In Vivo, Injection, Staining, Two Tailed Test, Expressing, Immunohistochemical staining

Journal: Cancer Communications

Article Title: The E3 ubiquitin ligase HUWE1 acts through the N‐Myc‐DLL1‐NOTCH1 signaling axis to suppress glioblastoma progression

doi: 10.1002/cac2.12334

Figure Lengend Snippet: HUWE1 suppresses GBM progression in vitro and in vivo. (A) A volcano plot of all assayed probes shows the distribution of differentially expressed genes based on the RNA‐seq data of HUWE1‐ KD and control LN229 cells. (B) Genes with upregulated expression in HUWE1‐ KD cells were enriched for GO biological process and molecular function categories. (C) Differentially expressed genes obtained from TCGA, Rembrandt and Gravendeel databases were divided into the low expression group and high expression group based on the median expression of HUWE1 and were enriched for the cancer cell invasion and glioma invasion gene sets. (D, E) Representative images of Transwell migration and invasion assays of HUWE1‐ KD cells and NC cells. (F) Growth curves show the relative growth rates of HUWE1‐ KD cells and NC cells tested by CCK‐8 assays. (G) In vivo bioluminescent images and quantification of xenograft tumors in mouse brains injected with HUWE1‐ KD and NC cells. (H) Representative H&E staining images of xenograft tumors in the mouse brains. (I) Kaplan‐Meier survival analysis of the mice bearing HUWE1‐ KD and NC cell‐derived xenograft tumors. (J) Representative IHC staining images of HUWE1 and Ki67 in the indicated xenografts. (K) Representative H&E staining images of the indicated xenografts and regions of the normal brain. Data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001. Abbreviations: GO, gene ontology; TCGA, The Cancer Genome Atlas; NES, normalized enrichment score; KD, knockdown; NC, negative control; IHC, immunohistochemistry; GO, Gene Ontology; TCGA, The Cancer Genome Atlas; SD, Standard Deviation

Article Snippet: The human GBM cell lines LN229 (ATCC‐CRL‐2611) and T98G (ATCC‐CRL‐1690) have been authenticated in a previous study [ ].

Techniques: In Vitro, In Vivo, RNA Sequencing, Control, Expressing, Migration, CCK-8 Assay, Injection, Staining, Derivative Assay, Immunohistochemistry, Knockdown, Negative Control, Standard Deviation

Journal: Cancer Communications

Article Title: The E3 ubiquitin ligase HUWE1 acts through the N‐Myc‐DLL1‐NOTCH1 signaling axis to suppress glioblastoma progression

doi: 10.1002/cac2.12334

Figure Lengend Snippet: HUWE1 is essential for the degradation of N‐Myc in GBM cells. (A) Venn diagram shows the intersection among significantly altered genes in the mass spectrometry (MS)‐detected genes with upregulated expression ( n = 2051) in HUWE1‐ KD cells, MS data of anti‐HUWE1 coimmunoprecipitation ( n = 567), and the Ubibrowser platform‐predicted substrates of HUWE1 with a high or moderate confidence level ( n = 700). (B) Western blotting analyses of N‐Myc and HUWE1 in GBM patient specimens and normal tissues. (C) Representative immunofluorescence staining of HUWE1 (in red), N‐Myc (in green) and Hoechst (in blue) in LN229 and GBM2 cells. Scale bar = 5 μm. (D) PLAs of HUWE1/N‐Myc complexes and HUWE1/P53 complexes in situ in LN229 and GBM2 cells. PLAs of P53/MDM2 was set as positive control. Scale bar = 5 μm. (E) Co‐immunoprecipitation analyses of the interaction between endogenic HUWE1 and N‐Myc in GBM cells. (F) Schematic diagram of the HUWE1 recognition motif on the HLH and MYC_N regions of N‐Myc. (G) Western blotting analyses of N‐Myc in GBM cells treated with CHX, MG132 and heclin alone or together at the indicated time. (H) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in HA‐Ub‐overexpressing GBM cells treated with or without heclin. (I) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in HA‐Ub‐overexpressing GBM cells infected with or without the sh HUWE1 vector. (J) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in GBM cells transfected with mutant ubiquitin plasmids at the K6, K11, K48, and K63 sites. (K) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in GBM cells transfected with wild‐type or K6‐, K48‐ and K6‐K48‐mutant ubiquitin plasmids. Data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001. Abbreviations: MS, mass spectrometry; IP, immunoprecipitation; IB, immunoblot; DMSO, Dimethyl sulfoxide; Ub, ubiquitin; CHX, cycloheximide; Ub, ubiquitin; KD, knockdown; NC, negative control; PLA, Proximity ligation assay; SD, Standard Deviation

Article Snippet: The human GBM cell lines LN229 (ATCC‐CRL‐2611) and T98G (ATCC‐CRL‐1690) have been authenticated in a previous study [ ].

Techniques: Mass Spectrometry, Expressing, Western Blot, Immunofluorescence, Staining, In Situ, Positive Control, Immunoprecipitation, In Vivo, Ubiquitin Proteomics, Infection, Plasmid Preparation, Transfection, Mutagenesis, Knockdown, Negative Control, Proximity Ligation Assay, Standard Deviation

Journal: Cancer Communications

Article Title: The E3 ubiquitin ligase HUWE1 acts through the N‐Myc‐DLL1‐NOTCH1 signaling axis to suppress glioblastoma progression

doi: 10.1002/cac2.12334

Figure Lengend Snippet: The rAAV dual‐vector delivery Cas9‐HUWE1 system significantly inhibits tumor growth in a GBM xenograft model. (A) Schematic description of the rAAV dual‐vector delivering dCas9‐HUWE1 system. The SadCas9‐VP64 gene and three sgRNAs of HUWE1 were packaged into two separate rAAV vectors. sgRNA NC was packaged into an empty vector and served as a control. In this dual‐vector system, the hGFAP promoter was used to guarantee transgene expression in LN229 cells in xenografts. (B) Schematic diagram of rAAV dCas9‐HUWE1 system treatment in LN229 xenografts. Ten days after tumor implantation, the rAAV9‐HUWE1 vector, the rAAV9‐control vector, and PBS were administered to the tumor‐bearing mice through orthotopic and tail vein injection. Tumor growth was monitored with an in vivo bioluminescence imaging system at the indicated time intervals. (C) In vivo bioluminescent images of the LN229 xenografts treated with orthotopic/tail vein injection of the rAAV9‐HUWE1 vector and the corresponding control. (D) Kaplan‐Meier survival analysis of the tumor‐bearing mice treated with rAAV9 vectors in different ways. (E) IHC staining images of HUWE1, Flag, N1ICD and Ki67 in xenograft tumors in the mouse brains. Scale bar = 50 μm. Abbreviations: sgRNA, single‐guide RNA; rAAV, adeno‐associated virus; IHC, immunohistochemistry; rAAV, adeno‐associated virus; hGFAP, human glial fibrillary acidic protein

Article Snippet: The human GBM cell lines LN229 (ATCC‐CRL‐2611) and T98G (ATCC‐CRL‐1690) have been authenticated in a previous study [ ].

Techniques: Plasmid Preparation, Control, Expressing, Tumor Implantation, Injection, In Vivo, Imaging, Immunohistochemistry, Virus

Journal: Cell chemical biology

Article Title: A systems chemoproteomic analysis of acyl-CoA/protein interaction networks

doi: 10.1016/j.chembiol.2019.11.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal MultiMab Malonyl-Lysine antibody Cell Signaling Technologies Cat# 14942 Anti-FLAG (DYKDDDDK) Tag Antibody (HRP-linked) Cell Signaling Technologies Cat# 2044 Anti-rabbit IgG antibody (HRP-linked) Cell Signaling Technologies Cat # 7074 Bacterial and Virus Strains n/a Biological Samples Cell pellets from HeLa S3 epithelial suspension cell line Cell Culture Company Cat # HA48 Chemicals, Peptides, and Recombinant Proteins NHS-Activated Sepharose 4 Fast Flow resin GE Healthcare Cat# 71-5000-14 AD Amine-functionalized Lys-CoA-Ahx Montgomery et al,, 2016 n/a U- 13 C6-glucose Cambridge Isotope Laboratories Cat# CLM-1396 U- 13 C2-acetate Cambridge Isotope Laboratories Cat# CLM-440 acetyl-CoA Sigma-Aldrich Cat# A2056 coenzyme A Sigma-Aldrich Cat# butyryl-CoA Sigma-Aldrich Cat# B1508 crotonyl-CoA Sigma-Aldrich Cat# 28007 palmitoyl-CoA Sigma-Aldrich Cat# P9716 acetic-CoA This study n/a Critical Commercial Assays Anti-FLAG IP kit Sigma-Aldrich Cat# FLAGIPT1-1KT TRIzol reagent ThermoFisher Scientific Cat#15596026 oligo-(dT) 25 Dynabeads ThermoFisher Scientific Cat# 61005 Qubit Broad Sensitivity Protein Assay Kit ThermoFisher Scientific Cat# Q33211 Deposited Data Proteomic datasets for CATNIP (Lys-CoA) pulldowns and competitions ProteomeXchange Consortium; PRIDE PXD013157 Experimental Models: Cell Lines HeLa S3 cells ATCC CCL-2.2 HEK-293T cells ATCC CRL-3216 LN229 ACLY knockout cells Zhao et al. 2016 n/a LN229 ACLY wild-type cells Zhao et al. 2016 n/a Experimental Models: Organisms/Strains Oligonucleotides Recombinant DNA Software and Algorithms DTASelect v.1.9/ CONTRAST for protein detection Tabb et al.,2002 NSAF7 Zhang, et al., 2010 QSPEC/QPROT for the statistical analysis of protein differentiation Choi, et al., 2015 http://http//sourceforge.net/p/qprot/ tSNE and k-means algorithms for proteins separation and clustering Van der Maaten, et al., 2008 R source TDA software for the topological analysis Lum et al., 2013 . https://platform.ayasdi.com/workbench (AYASDI Inc., Menlo Park CA) DAVID gene ontology for functional enrichment DAVID tool http://david.abcc.ncifcrf.gov/ ConsensusPathDB http://cpdb.molgen.mpg.de/CPDB/rlFrame Other Open in a separate window KEY RESOURCES TABLE.

Techniques: Virus, Suspension, Cell Culture, Recombinant, Knock-Out, Software, Functional Assay

Journal: OncoTargets and therapy

Article Title: Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

doi: 10.2147/OTT.S67998

Figure Lengend Snippet: ZINC69391 inhibited Rac1 activation on LN229 glioma cells. Notes: ( A ) Blockade of Rac1–Dock180 interaction. Dock180 was affinity-precipitated with bacterially expressed Rac1 immobilized in glutathione agarose beads in the presence of varying concentrations of ZINC69391. Western blot analysis was carried out with anti-Dock180 antibody. The experiment was repeated three times. Densitometric values are shown below (arbitrary units). ( B ) Concentration-dependent Rac1 inhibition by ZINC69391 in glioma cells. Serum-starved LN229 cells were treated for 1 hour with different ZINC69391 concentrations and stimulated with epidermal growth factor (EGF) (100 ng/mL) for 15 minutes. Densitometric values are shown below (arbitrary units). ( C ) Concentration-dependent inhibition of Pak1 phosphorylation using the same experimental procedure as before. Densitometric values are shown below (arbitrary units).

Article Snippet: Human glioblastoma multiforme cell lines LN229 (ATCC CRL-2611) and U-87 MG (ATCC HTB-14) were obtained from ATCC.

Techniques: Activation Assay, Western Blot, Concentration Assay, Inhibition, Phospho-proteomics

Journal: OncoTargets and therapy

Article Title: Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

doi: 10.2147/OTT.S67998

Figure Lengend Snippet: ZINC69391 affected cell proliferation and cell cycle progression of glioma cells. Notes: ( A ) ZINC69391 inhibited cell proliferation. LN229 and U-87 MG cells were treated for 72 hours with different concentrations of ZINC69391. Cell viability was measured using MTT assay. ( B ) ZINC69391 arrested cell cycle progression in G1 phase. LN229 cells were synchronized and treated for 48 hours with ZINC69391 with different concentrations. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry to estimate the percentage of cells in sub-G0 phase, G1 phase, S phase, and G2/M phase. Bars, standard error of the mean. * P <0.05, ** P <0.01 determined by analysis of variance contrasted. Dunnett’s multiple comparison test versus control in each phase.

Article Snippet: Human glioblastoma multiforme cell lines LN229 (ATCC CRL-2611) and U-87 MG (ATCC HTB-14) were obtained from ATCC.

Techniques: MTT Assay, Staining, Flow Cytometry, Comparison, Control

Journal: OncoTargets and therapy

Article Title: Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

doi: 10.2147/OTT.S67998

Figure Lengend Snippet: ZINC69391 triggered apoptosis on malignant glioma cells. Notes: ( A ) Apoptosis of LN229 cells after 10 μM and 50 μM treatment for 6 hours using annexin V staining. Representative micrographs taken at 200×. ( B ) Late apoptosis was evaluated at 10 μM and 50 μM for 6 hours using TUNEL assay. Representative micrographs taken at 100×. ( C ) Quantification of apoptotic cells per field using TUNEL assay. Bars, standard error of the mean, * P <0.05, ** P <0.01 determined by analysis of variance cont. Dunnett’s multiple comparison test versus control.

Article Snippet: Human glioblastoma multiforme cell lines LN229 (ATCC CRL-2611) and U-87 MG (ATCC HTB-14) were obtained from ATCC.

Techniques: Staining, TUNEL Assay, Comparison, Control

Journal: OncoTargets and therapy

Article Title: Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

doi: 10.2147/OTT.S67998

Figure Lengend Snippet: ZINC69391 inhibited glioma cell migration and invasion modulating actin cytoskeleton reorganization. Notes: ( A ) LN229 cells were seeded in uncoated transwell chambers with or without ZINC69391, incubated for 18 hours, and quantified. Bars, standard error of the mean. * P <0.05; *** P <0.001 determined by analysis of variance cont. Dunnett’s multiple comparison test. ( B ) LN229 cells were seeded in Matrigel-coated transwell chambers with or without ZINC69391, incubated for 48 hours, and quantified. Bars, standard error of the mean. * P <0.05; ** P <0.01; *** P <0.001 determined by analysis of variance cont. Dunnett’s multiple comparison test. ( C ) Representative micrographs taken at 1,000× showing inhibition of epidermal growth factor (EGF)-induced actin reorganization by ZINC69391 in LN229 cells. Cells were grown on cover slips, serum-starved for 16 hours (untreated panel), and treated for 1 hour with ZINC69391. After 15 minutes stimulation with EGF (100 ng/mL) (EGF control), cells were fixed and actin filaments were visualized with AlexaFluor555-phalloidin.

Article Snippet: Human glioblastoma multiforme cell lines LN229 (ATCC CRL-2611) and U-87 MG (ATCC HTB-14) were obtained from ATCC.

Techniques: Migration, Incubation, Comparison, Inhibition, Control

Journal: OncoTargets and therapy

Article Title: Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

doi: 10.2147/OTT.S67998

Figure Lengend Snippet: 1A-116 analog is a more potent Rac1 inhibitor. Notes: ( A ) LN229 and U-87 MG cells were treated for 72 hours with different concentrations of ZINC69391 and 1A-116. Cell viability was measured using MTT assay. ( B ) LN229 were transiently transfected with Rac1 small interfering (si)RNA or control siRNA. Cells were treated for 72 hours with different concentrations of 1A-116 analog and cell viability was measured using MTT assay. ( C ) LN229 cells were seeded in Matrigel-coated transwell chambers with or without ZINC69391, incubated for 48 hours, and quantified. ** P <0.01, *** P <0.001. Determined by analysis of variance contrasted with Dunnett’s multiple comparison test.

Article Snippet: Human glioblastoma multiforme cell lines LN229 (ATCC CRL-2611) and U-87 MG (ATCC HTB-14) were obtained from ATCC.

Techniques: MTT Assay, Transfection, Control, Incubation, Comparison