ln229 cells  (ATCC)

Journal: PLOS One

Article Title: An optimized protocol for metabolic measurement in 3D tumor spheroids derived from primary and established glioblastoma cells

doi: 10.1371/journal.pone.0347569

Figure Lengend Snippet: A) Number of formed spheroids. Here, the number of single spheroids of roundish shape obtained per well for three tested methods used for the four glioblastoma cell lines and three different cell numbers is depicted. For BioFloat and magnetic bead technique all yields were equal to one and thus pooled. The 2D projected size is presented.(B, C). Graphs show the size of the formed spheroids as a function of time for the BioFloat (B) and magnetic bead (C) method. The circularity of the formed spheroids is shown as a function of time for the BioFloat (D) and magnetic bead (E) method. The relative height of spheroids for different techniques and all cell lines is presented (F). Error bars in B)-E) depict the standard error of the mean. Box plots in F) show the median (red line), the 25 and 75 percentile (blue box) and the maximal range (whiskers). Stars depict statistically significant results with p < 0.05. B) to E) For each cell line and cell number 16 spheroids were analyzed. F) n GBM10 Beads = 8, n GBM10 Biofloat = 8, n GBM4 Beads = 12, n GBM4 Biofloat = 9, n LN229 Beads = 11, n LN229 Biofloat = 8, n U138 Beads = 12, n U138 Biofloat = 8.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA), and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques:

Journal: PLOS One

Article Title: An optimized protocol for metabolic measurement in 3D tumor spheroids derived from primary and established glioblastoma cells

doi: 10.1371/journal.pone.0347569

Figure Lengend Snippet: Seahorse analyses showing a comparison between metabolic values in the different glioblastoma cell lines LN229, U138 and the primary cells GBM#4, GBM#10. Representative mitochondrial respiration measurement using the Seahorse XF Cell Mito Stress Test for U138, LN229, GBM#4 and GBM#10 spheroids generated from 30,000 cells. Oligomycin, FCCP, rotenone plus antimycin A are used as pharmacological tools to study mitochondrial respiration. Oligomycin inhibits ATP synthase, reducing oxygen consumption. Rotenone and antimycin A block the electron transport chain, preventing mitochondrial respiration and allowing measurement of non-mitochondrial oxygen consumption. Oligomycin reduces the oxygen consumption rate (OCR) through inhibition of ATP synthase. FCCP uncouples the proton gradient, maximizing oxygen consumption. Rotenone and antimycin A are electron transport chain inhibitors.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA), and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Comparison, Generated, Blocking Assay, Inhibition

Journal: PLOS One

Article Title: An optimized protocol for metabolic measurement in 3D tumor spheroids derived from primary and established glioblastoma cells

doi: 10.1371/journal.pone.0347569

Figure Lengend Snippet: (A) t-SNE clustering of spheroids from the four cell lines, showing the distinct response of LN229 compared to the other GBM cell types. Each point represents a single spheroid. Each point corresponds to the data of a single spheroid. (B) Illustration of the energetic state of the four GBM spheroid types at baseline and after FCCP injection to achieve maximal respiration. Error bars represent the standard error of the mean (SEM). The following number of spheroids was used for each group: n GBM10k = 42, n GBM4 = 44, n LN229 = 47, n U138 = 48.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA), and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Injection

Journal: PLOS One

Article Title: An optimized protocol for metabolic measurement in 3D tumor spheroids derived from primary and established glioblastoma cells

doi: 10.1371/journal.pone.0347569

Figure Lengend Snippet: (A) Graphic of the raw basal respiration data obtained from the Seahorse metabolic analysis. The data were normalized using different approaches and are shown in (B) for median normalization, (C) for median plus area normalization, and (D) for median plus cell number normalization. Error bars represent the standard error of the mean (SEM). Asterisks indicate statistically significant differences at p < 0.05. The number of samples used in each group was as follows: n GBM10 15k = 13, n GBM10 20k = 16, n GBM10 25k = 13, n GBM4 15k = 12, n GBM4 20k = 15, n GBM4 25k = 17, n LN229 15k = 13, n LN229 20k = 14, n LN229 25k = 20, n U138 15k = 16, n U138 20k = 15, n U138 25k = 17.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA), and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques:

Journal: PLOS One

Article Title: An optimized protocol for metabolic measurement in 3D tumor spheroids derived from primary and established glioblastoma cells

doi: 10.1371/journal.pone.0347569

Figure Lengend Snippet: Seahorse analyses showing the differences in the maximal respiration values of glioblastoma cell lines LN229, U138 and the primary cells GBM#4, GBM#10 after normalization of spheroids yielded from different cell seeding densities. (A) Graphic of the raw maximal respiration data obtained from the Seahorse metabolic analysis. The data were normalized using different approaches and are shown in (B) for median normalization, (C) for median plus area normalization, and (D) for median plus cell number normalization. Error bars represent the standard error of the mean (SEM). Asterisks indicate statistically significant differences at p < 0.05. The number of samples used in each group was as follows: n GBM10 15k = 13, n GBM10 20k = 16, n GBM10 25k = 13, n GBM4 15k = 12, n GBM4 20k = 15, n GBM4 25k = 17, n LN229 15k = 13, n LN229 20k = 14, n LN229 25k = 20, n U138 15k = 16, n U138 20k = 15, n U138 25k = 17.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA), and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques:

Journal: Translational Oncology

Article Title: CD38 overexpression drives glioblastoma progression via L1CAM/ICAM1/JAK-STAT-Driven tumor microenvironment rewiring

doi: 10.1016/j.tranon.2026.102758

Figure Lengend Snippet: A. Evaluation of CD38 expression following human and mouse GBM cell lines (A) and (B) overexpression of CD38 in T98G and Ln229 (human GBM cell lines) using western blot and fluorescent microscope imaging. (C) Significant overexpression of CD38 in transduced Gl-261 cells compared to wild type Gl-261 cells. B Kaplan-Meier overall survival analysis indicated significantly decreased in CD38-OE GBM- bearing mice (26.45 days) versus CD38-WT GBM- bearing mice (35.73 days) (p < 0.0001; n = 11 per group).

Article Snippet: Both T98G and LN229 human glioblastoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Over Expression, Western Blot, Microscopy, Imaging

Journal: Translational Oncology

Article Title: CD38 overexpression drives glioblastoma progression via L1CAM/ICAM1/JAK-STAT-Driven tumor microenvironment rewiring

doi: 10.1016/j.tranon.2026.102758

Figure Lengend Snippet: A. Comparison of doubling time among CD38-OE, CD38-WT, and CD38-Knockdown human GBM cells (T98G and LN229) and CD38-OE, CD38-WT GL-261 cells displayed non-significant changes. (n = 3, triplicate). One-way ANOVA, no significant differences. B Western blot quantification of CKIs (p16, p21, p27) in GL-261 cells (n = 3). One-way ANOVA, no significant differences.

Article Snippet: Both T98G and LN229 human glioblastoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Comparison, Knockdown, Western Blot

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 prohibits the stress-mediated formation of ub + /p62 + aggresomes. (A and B) LN229 cells were transfected with HA or HA-SMURF2 and treated with DMSO; MG132 (10 μM, 12 h); H 2 O 2 (200 μM, 2 h); or LPS (100 ng/mL, 12 h). Representative immunofluorescence (IF) images of the colocalization of ub and p62. Nuclei stained with DAPI (A). Quantification the percentage of cells with ub + /p62 + puncta (B). (C – E) HEK293T cells were transfected with HA or HA-SMURF2 and subsequently treated with MG132 (10 μM, 12 h) or H 2 O 2 (200 μM, 2 h). Detergent-soluble and detergent-insoluble fractions were analyzed by western blotting with indicated antibodies (C and D). Quantification of the relative intensity of ub and p62 levels in the detergent-insoluble fractions shown in C and D (E). (F) LN229 cells were transfected with HA or HA-SMURF2 and treated with DMSO; MG132 (10 μM, 12 h); H 2 O 2 (200 μM, 2 h); or LPS (100 ng/mL, 12 h). Representative IF images of the colocalization of Proteostat and p62. Nuclei stained with DAPI. (G) The proposed model suggests that SMURF2 inhibits the formation of ub + /p62 + aggresomes/ALIS under stress conditions. Data were presented as the mean ± SD from three independent experiments. NS: not significant, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Scale bar: 10 μm. Short Exp: short exposure; Long Exp: long exposure.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Immunofluorescence, Staining, Western Blot

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 nuclear translocation facilitates the degradation of NRF2 within the nucleus. (A and B) HEK293T cells were first transfected with HA-NRF2-WT. Subsequently, cells were transfected either with Flag or Flag-SMURF2 (A), or with SMURF2 siRNA or scrambled siRNA oligos for 48 h (B). Cells were then treated with H 2 O 2 (200 μM, 2 h) or LPS (100 ng/mL, 12 h). Following treatment, subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (C and D) LN229 cells were transfected with HA-SMURF2, then treated with PBS, H 2 O 2 (200 μM, 2 h) (C) or LPS (100 ng/mL, 12 h) (D). Representative IF images showing the subcellular localization of HA-SMURF2 are presented; nuclei were stained with DAPI. (E and F) HEK293T cells were treated with PBS, H 2 O 2 (200 μM, 2 h) (E), or LPS (100 ng/mL, 12 h) (F). Subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (G) Schematic depiction of NRF2 NLS1/2−Mut , NRF2 NES1/2−Mut , and SMURF2 NLS−Mut . (H–K) HEK293T cells were first transfected with HA-NRF2 NLS2−Mut (H and I) or HA-NRF2 NES2−Mut (J and K). Subsequently, cells were transfected either with Flag or Flag-SMURF2, then treated with H 2 O 2 (200 μM, 2 h) (H and J) or LPS (100 ng/mL, 12 h) (I and K). Following treatment, subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (L) Schematic of SMURF2 domains with NLS indicated (single-letter code). SMURF2 NLS−Mut denotes deletion of NLS residues (Top). LN229 cells transfected with Flag-SMURF2-WT or Flag-SMURF2 NLS−Mut were treated with DMSO, MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h). Representative IF images showing subcellular localization of Flag-SMURF2 or Flag-SMURF2 NLS−Mut ; nuclei were stained with DAPI (Bottom). (M) HEK293T cells were first transfected with HA-NRF2-WT. Subsequently, cells were transfected either with Flag or Flag-SMURF2 NLS−Mut , then treated with H 2 O 2 (200 μM, 2 h) or LPS (100 ng/mL, 12 h). Following treatment, subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (N) The proposed model indicates that SMURF2 specifically degrades NRF2 in the nucleus. Scale bar: 5 μm, Scale bar: 10 μm. Data were presented in three independent experiments.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Translocation Assay, Transfection, Fractionation, Western Blot, Staining

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 promotes cell apoptosis through NRF2 inactivation. (A) LN229 cells were transfected with HA, HA-SMURF2, Myc-NRF2 or HA-SMURF2 + Myc-NRF2 and subsequent treatment with MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h). Representative IF images of the colocalization of ub and p62. Nuclei stained with DAPI. (B and C) HEK293T cells were transfected with empty vector or Myc-NRF2, followed by Flag or Flag-SMURF2 expression. Western blotting was performed using indicated antibodies (B). qRT-PCR was performed using primers specific for indicated genes (C). The fold change in expression in Flag-SMURF2 overexpressing samples was calculated relative to control samples. (D) HEK293T cells were first transfected with NRF2 siRNA for 48 h, followed by transfection with either Myc-NRF2-WT or Myc-NRF2-K555R, and subsequently transfected with Flag or Flag-SMURF2.Western blotting was performed using indicated antibodies. (E) LN229 cells were transfected with SMURF2 siRNA or scramble siRNA oligos for 48 h, followed by treatment with PBS or H 2 O 2 (200 μM, 2 h). Cells were then stained with 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) and the ROS level was detected by flow cytometry. (F) Quantification of relative ROS fluorescence intensity in LN229 cells with SMURF2 knockdown or HA-SMURF2 overexpression. (G and H) LN229 cells were transfected with SMURF2 siRNA or scramble siRNA oligos for 48 h or transfected with HA or HA-SMURF2, followed by treatment with PBS or H 2 O 2 (200 μM, 2 h). Cells were then stained with Annexin-V/propidium iodide (PI), and apoptotic cells were detected by flow cytometry (G). Quantification of apoptosis in LN229 cells with SMURF2 knockdown or HA-SMURF2 overexpression (H). (I) HEK293T cells were transfection with Flag or Flag-SMURF2, and subsequent treatment with or without MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/ml, 12 h). Western blotting was performed using indicated antibodies. (J and K) HEK293T cells were transfected with empty vector and Myc-NRF2 (J) or Myc-NRF2-WT, Myc-NRF2-K555R (K), followed by Flag or Flag-SMURF2 expression. Cells were subsequently treated with MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h). Western blotting was performed using indicated antibodies. (L) The shSMURF2 and shPLKO cells were treated with or without LPS (100 ng/mL, 12 h) and cultured for 14 days. Colony formation assay was performed for cells. (M) The representative images of shSMURF2 and shPLKO patient-derived cells formed tumors in nude mice with or without LPS (10 mg/kg). (N) The graph showed the quantified data of tumor weight. (O) Immunohistochemistry (IHC) analysis of tumor tissue slides with antibodies against Ki67. Nucleus was stained by hematoxylin. Scale bar, 50 μm (P) The proposed model indicates that SMURF2 promotes cell apoptosis through NRF2 inactivation. Data were presented as the mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Scale bar: 10 μm, Scale bar, 50 μm.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Staining, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence, Knockdown, Over Expression, Cell Culture, Colony Assay, Derivative Assay, Immunohistochemistry