Journal: MedComm

Article Title: Discovery of a First‐in‐Class Murine Double Minute 2‐Recruiting Positive Transcription Elongation Factor B PROTAC Degrader With Selective Antitumor Activity

doi: 10.1002/mco2.70723

Figure Lengend Snippet: MDM2‐ and p53‐dependent activities of MDM2‐recruiting P‐TEFb PROTAC degraders. (A) Effects of siRNA‐mediated MDM2 knockdown on cellular sensitivity to dCDK9‐010. Data were presented as mean ± standard deviation (SD); n = 3. (B) Effects of MDM2 knockdown on dCDK9‐010‐mediated CDK9/Cyclin T degradation. TC‐32 cells were transfected with indicated siRNAs for 48 h, followed by treatment with 2 µM dCDK9‐010 for 8 h. (C) MDM2 mRNA levels in wild type versus TP53 ‐knockout U87 cells, with or without compound treatment (2 µM, 8 h). Data were presented as mean ± SD ( n = 3); *** p < 0.001 based on one‐way analysis of variance (ANOVA); n.s., no significance. (D) Immunoblot analysis of P‐TEFb components, p53, and MDM2 in wild‐type and TP53 ‐knockout isogenic U87 cells after compound treatment (2 µM, 8 h). (E) Viability of isogenic U87 cells treated with dCDK9‐010. Data were presented as mean ± SD ( n = 3). (F) Immunoblot analysis of P‐TEFb components, p53, and MDM2 in NCI‐H226 and TC‐32 cells after siRNA‐mediated TP53 silencing and dCDK9‐010 treatment (2 µM, 8 h). (G) Effects of TP53 or MDM4 knockdown by siRNA relative to control on dCDK9‐010 sensitivity in NCI‐H226 and TC‐32 cells. Data were presented as mean ± SD ( n = 3).

Article Snippet: HEK293T (ATCC), TC‐32 (COG Repository, USA), HCT116 (ATCC), A549 (ATCC), HEPG2 (ATCC), HT1080 (ATCC), MDA‐MB‐231 (ATCC), U87 (U‐87MG, ATCC), U251 (U251MG, ATCC), and MKN45 (ATCC) cells lines were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma‐Aldrich, Taufkirchen, Germany).

Techniques: Knockdown, Standard Deviation, Transfection, Knock-Out, Western Blot, Control

Journal: MedComm

Article Title: Discovery of a First‐in‐Class Murine Double Minute 2‐Recruiting Positive Transcription Elongation Factor B PROTAC Degrader With Selective Antitumor Activity

doi: 10.1002/mco2.70723

Figure Lengend Snippet: Selective activities of compounds 12 (dCDK9‐009) and 13 (dCDK9‐010) against TP53 wild‐type cancer cells. (A and B) Examination of dose‐dependent impacts of dCDK9‐009 and dCDK9‐010 in TP53 wild‐type HCT116, HT1080, NCI‐H460, and U87. Cells were treated with increasing concentrations of indicated compounds for 24 h. (C and D) Examination of impacts of dCDK9‐009 and dCDK9‐010 in non‐malignant HEK293T and mesenchymal stem cell line ASC52telo. Cells were treated with increasing concentrations of indicated compounds for 24 h. (E and F) Effect of dCDK9‐009 and dCDK9‐010 treatment on cellular viabilities of indicated cancer cell lines, as well as ASC52telo. IC 50 was presented as mean ± SD with three independent replicates. (G) Heatmap summarizing the IC 50 values of dCDK9‐009 and dCDK9‐010 across human cell lines tested in this study (related to E and F, Figures S3, S4, and S8).

Article Snippet: HEK293T (ATCC), TC‐32 (COG Repository, USA), HCT116 (ATCC), A549 (ATCC), HEPG2 (ATCC), HT1080 (ATCC), MDA‐MB‐231 (ATCC), U87 (U‐87MG, ATCC), U251 (U251MG, ATCC), and MKN45 (ATCC) cells lines were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma‐Aldrich, Taufkirchen, Germany).

Techniques:

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: The frequency of ILC1 is decreased, whereas ILC3 is increased, in IDH1-mutant human glioma tissues compared with the IDH1-wild-type group. A Representative flow-cytometry gating strategies for ILCs in human tonsil (control) tissue, IDH1-wildtype, and IDH1-mutant glioma tissue (FSC-A = forward-scatter area; SSC-A = side-scatter area). B Comparison of total ILC percentages in tonsil controls (n = 11), IDH1-wild-type (n = 9), and IDH1-mutant (n = 3) glioma tissues (top center); comparison of ILC1 percentages (bottom left), ILC2 percentages (bottom center), and ILC3 percentages (bottom right) across the same groups. C Comparison of mean fluorescence intensity (MFI) of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) expression on ILC1s in tonsil control, IDH1-wild-type, and IDH1-mutant groups. D Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC2s. E Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC3s. Error bars represent ± SD. Statistical significance was determined by one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Flow Cytometry, Control, Comparison, Fluorescence, Expressing

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: The frequency of ILC3 is increased in the peripheral blood of IDH1-mutant glioma patients compared with the IDH1-wild-type group. A Representative flow-cytometry gating strategies for ILCs in peripheral blood of healthy controls, IDH1-wild-type glioma patients, and IDH1-mutant glioma patients (FSC-A = forward-scatter area; SSC-A = side-scatter area). B Comparison of total ILC percentages in blood samples from healthy controls (HC, n = 20), IDH1-wild-type (n = 12), and IDH1-mutant (n = 7) groups (top center); comparison of ILC1 (bottom left), ILC2 (bottom center), and ILC3 (bottom right) frequencies across the same groups. C Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC1s in HC, IDH1-wild-type, and IDH1-mutant groups. D Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC2s. E Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC3s. Error bars represent ± SD. Statistical significance was determined by one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Flow Cytometry, Comparison

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: Co-culture of tonsil-derived ILCs with U87-MG significantly increased surface PD-1, KLRG1, and CTLA-4 compared with IDH1-mutant U87-MG. A – D Human tonsil-derived ILCs were cultured alone or co-cultured with U87-MG or IDH1-mutant U87-MG glioma cell lines, either with cytokine supplementation (recombinant human IL-2 [5 ng/mL], IL-7 [50 ng/mL], IL-12 [50 ng/mL], IL-1β [50 ng/mL], IL-23 [50 ng/mL]) or without cytokines for four days. Data points represent two independent experiments (ILC n = 3; U87-MG + ILC n = 6; IDH1-mutant U87-MG + ILC n = 6; technical replicates) A Representative flow-cytometry gating strategy for human tonsil-derived ILCs. B Flow-cytometry contour plots showing CTLA-4, KLRG1, and PD-1 surface expression percentages on ILCs. C Quantification of CTLA-4, KLRG1, and PD-1 expression percentages on ILCs. D Mean fluorescence intensity (MFI) of CTLA-4, KLRG1, and PD-1 expression on ILCs. E – F ILCs were cultured alone or exposed to glioma-conditioned medium (GCM) from U87-MG or IDH1-mutant U87-MG cell lines under the same cytokine conditions for four days. Data points represent two independent experiments (each with three technical replicates). E Percentages of CTLA-4, KLRG1, and PD-1–expressing ILCs following GCM exposure. F Corresponding MFI of CTLA-4, KLRG1, and PD-1 expression on ILCs. G – H Proliferation of CFSE-labeled tonsil ILCs co-cultured with U87-MG or IDH1-mutant U87-MG cells was analyzed after four days. G Representative flow-cytometry plots of CFSE dilution. H Quantification of proliferating CFSE-labeled ILCs from three independent experiments (each with four technical replicates). Error bars represent ± SD. Statistical analyses were performed using one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Co-Culture Assay, Derivative Assay, Mutagenesis, Cell Culture, Recombinant, Flow Cytometry, Expressing, Fluorescence, Labeling

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: IL-17 and IFN-γ production is increased in tonsil-derived ILCs exposed to glioma-conditioned medium (GCM) from U87-MG and IDH1-mutant U87-MG cell lines. A – E Tonsil-derived ILCs were cultured alone or with glioma-conditioned medium (GCM) obtained from U87-MG or IDH1-mutant U87-MG cell lines for four days, with or without cytokine supplementation (recombinant human IL-2 [5 ng/mL], IL-7 [50 ng/mL], IL-12 [50 ng/mL], IL-1β [50 ng/mL], IL-23 [50 ng/mL]). Golgi Stop was added during the final nine hours of incubation. A Percentages (left) and mean fluorescence intensity (MFI; right) of TNF-α–producing ILCs. B Percentages (left) and MFI (right) of IL-2–producing ILCs. C Percentages (left) and MFI (right) of IL-17–producing ILCs. D Percentages (left) and MFI (right) of IFN-γ–producing ILCs. E Percentages (left) and MFI (right) of GM-CSF–producing ILCs. Data represents two independent experiments (each with three technical replicates). Error bars indicate ± SEM. Statistical significance was determined by one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Mutagenesis, Cell Culture, Recombinant, Incubation, Fluorescence

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: D-2-HG levels are comparable in-patient plasma but elevated in glioma-conditioned medium (GCM) from IDH1-mutant U87-MG + ILC co-cultures compared with U87-MG + ILC. A Quantification of D-2-HG (OD₄₅₀) in plasma samples from healthy controls (HC, n = 12), IDH1-wild-type glioma patients (n = 12), and IDH1-mutant glioma patients (n = 7). B Quantification of D-2-HG (OD₄₅₀) in glioma-conditioned medium (GCM) collected from cultures of ILCs alone, U87-MG, IDH1-mutant U87-MG cell lines, and tonsil ILCs co-cultured with U87-MG or IDH1-mutant U87-MG cells under cytokine-supplemented (recombinant human IL-2 [5 ng/mL], IL-7 [50 ng/mL], IL-12 [50 ng/mL], IL-1β [50 ng/mL], IL-23 [50 ng/mL]) and cytokine-free conditions for four days. Data points represent two independent experiments (ILC n = 4, U87-MG + ILC n = 2, IDH1-mutant U87-MG + ILC n = 2 technical replicates). Error bars indicate ± SEM. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Clinical Proteomics, Mutagenesis, Cell Culture, Recombinant

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Lis1 and CD133 gene expression in neurosphere-like U87 cells. (A) Exposure of U87 glioma cells to stem-conditioned medium (SM) induces phenotypic modifications resulting in neurospheres after five days, in both regular U87 cells (left) and shLis1-U87 cells (right). The expression of Lis1 (B) and CD133 (D) is induced in U87 cells exposed to SM, with the highest level at day 5 of incubation. (C) As expected, silencing Lis1 gene in U87 cells (shLis-U87) inhibits its induction in cells incubated with SM. CD133 induction is almost abrogated in shLis-U87 cells (E) compared with U87 cells (D). Lis1 is highly expressed in CD133+ cells isolated from U87 cell line and primary glioblastoma (HTC1 and HTC2) cell cultures in normal culture medium (CM) or SM (F) . Data show enrichment up to 60 fold in Lis1 expression in CD133 + fractions for cells grown in CM and up to 32 fold in cells incubated in SM. Lis1 expression in CD133+ fraction isolated from U87 cells grown in CM is 32 times higher than that of CD133- fraction (U87 columns); in CD133+ fraction isolated from U87 incubated in SM Lis1 expression is 35 times higher than in CD133 negative cells from the same culture. The negative control is represented by CD133+ cells isolated from shLis-U87 incubated in CM or SM for which Lis1 was not increased as compared with CD133- cells (shLis-U87/CM and SM columns).

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Gene Expression, Expressing, Incubation, Isolation, Negative Control

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Proliferation of irradiated U87 and shLis-U87 cells. Cells having Lis1 silenced or not were irradiated with X-ray doses from 5 to 50 Gy. Cells seeded at a density of 1x10 4 cells/well, in quadruplicates or triplicates in E-plates and placed in xCelligence RTCA instrument, were followed-up for 100 hours (A) . Alternatively, irradiated or not irradiated cells were seeded in 24-well plates and the DNA amount per well was determined using Hoechst 33342 (B) . Both methods showed that irradiated U87 cells recovered better their proliferative capacity than shLis-U87 cells.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Irradiation

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Cell adhesion, migration and proliferation of CD133 + cells isolated from U87 and shLis-U87 cells . CD133+ cells were isolated from control U87 and shLis-U87 cells. The purity of the fraction is revealed by higher CD133 expression (assessed by RT-PCR) in CD133+ fraction as compared with CD133- fraction (A) . CD133+ cells isolated from control U87 (blue circles) or from shLis-U87 (red squares) cultures were subjected to functional assays using xCELLigence Real-Time Cell Analysis instruments. The data representing the recorded cell index at different times show that CD133+ cells isolated from shLis-U87 culture present two times lower (B) adherence to the surface, (C) migratory potential and (D) proliferative rate, as compared with the those isolated from control U87 culture.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Migration, Isolation, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, Cell Analysis

Journal: Biomedicines

Article Title: The Sesquiterpene Lactone Cynaropicrin Manifests Strong Cytotoxicity in Glioblastoma Cells U-87 MG by Induction of Oxidative Stress

doi: 10.3390/biomedicines10071583

Figure Lengend Snippet: Cytotoxic effects of Cyn on human glioblastoma U-87 MG cells. ( A ) Molecular structure of SL Cynaropicrin (IUPAC name: ([(3 a R,4S,6 a R,8S,9 a R,9 b R)-8-hydroxy-3,6,9-trimethylidene-2-oxo-3 a ,4,5,6 a ,7,8,9 a ,9 b -octahydroazuleno[4,5- b ]furan-4-yl] 2-(hydroxymethyl)prop-2-enoate). ( B ) Determination of IC50 values on U-87 MG cells using GraphPad Prism 7 software after 24, 48 and 72 h of incubation with Cyn 0.01, 0.1, 1, 10, 25, 50 and 100 µM and DMSO 0.1% as the vehicle control. ( C ) Dose and time-dependent reduction of the U-87 MG cell number under daily exposure to Cyn 4, 8 and 10 µM for 24, 48 and 72 h. ( D ) Morphological change of 4, 8 and 10 µM Cyn-treated U-87 MG cells for 24, 48 and 72 h. White arrows indicate round-shaped cells with a loss of filaments and the presence of cell shrinkage. ( E ) Cytotoxic effects of daily exposure to Cyn 4, 8 and 10 µM for 24, 48 and 72 h on the U-87 MG cell metabolism. ( F ) Effect of Cyn 4 µM on the U-87 MG clonogenic potential. For all the experiments, values are the mean ± SEM of 3 individual determinations. One-way ANOVA test, p -value < 0.05. According to GraphPad Prism 7 software, **** p -value < 0.0001 (extremely significant).

Article Snippet: Human continuous glioblastoma cell line U-87 MG was purchased from the Sigma Aldrich Collection (LGC Promochem, Teddington, UK).

Techniques: Software, Incubation, Control

Journal: Biomedicines

Article Title: The Sesquiterpene Lactone Cynaropicrin Manifests Strong Cytotoxicity in Glioblastoma Cells U-87 MG by Induction of Oxidative Stress

doi: 10.3390/biomedicines10071583

Figure Lengend Snippet: Cyn-induced oxidative stress in human glioblastoma U-87 MG cells. ( A ) Pretreatment of U-87 MG cells with NAC 3 mM for 4 h, followed by incubation for 24 h with Cyn 0.01, 0.1, 1, 10, 25, 50 and 100 μM. DMSO 0.1% was used as the vehicle control. ( B ) Preincubation with NAC 3 mM for 4 h preserved U-87 MG from Cyn-induced morphological change, since the cells maintained their protrusions rather than appeared round-shaped (white arrows). ( C ) Quantitative analysis of ROS generation in U-87 MG cells under 2 and 6 h of treatment with Cyn 8 and 25 μM. DMSO 0.1% was used as the vehicle control. ( D ) Qualitative analysis of ROS production after 2 h of exposure to Cyn 8 and 25 μM. ( E ) Immunofluorescence staining of NRF2 in U-87 MG treated for 24 h with Cyn 25 µM, which showed a nuclear localization with respect to the control cells treated with 0.1% DMSO (white arrows). Magnification 20×. Data were analyzed by a Student’s t -test, p -value < 0.05. According to GraphPad Prism 7 software, * p -values from 0.01 to 0.05 (significant) and ** p -values from 0.001 to 0.01 (very significant).

Article Snippet: Human continuous glioblastoma cell line U-87 MG was purchased from the Sigma Aldrich Collection (LGC Promochem, Teddington, UK).

Techniques: Incubation, Control, Immunofluorescence, Staining, Software

Journal: Biomedicines

Article Title: The Sesquiterpene Lactone Cynaropicrin Manifests Strong Cytotoxicity in Glioblastoma Cells U-87 MG by Induction of Oxidative Stress

doi: 10.3390/biomedicines10071583

Figure Lengend Snippet: Cytotoxic effects of Cyn on patient-derived glioblastoma cell lines NULU and ZAR. ( A ) Cytotoxic effects of daily exposure to Cyn 4, 8 and 10 µM for 24, 48 and 72 h on NULU ( A ) and ZAR ( B ) cell metabolism. DMSO 0.1% was used as the vehicle control. ( C ) Morphological changes for the 4, 8 and 10 µM Cyn-treated NULU and ZAR cell lines for 24, 48 and 72 h. Data were analyzed by the Student’s t -test, p -value < 0.05. According to GraphPad Prism 7 software, * p -value 0.01–0.05 was considered statistically significant, ** p -value 0.001–0.01 very significant, *** p -value 0.0001–0.001 extremely significant and while **** p -values < 0.0001 were extremely significant. Taking together, these results clearly indicate the potentialities of Cyn as adjuvant therapy to conventional chemotherapy TMZ in IDH-mutant and wild-type glioblastoma.

Article Snippet: Human continuous glioblastoma cell line U-87 MG was purchased from the Sigma Aldrich Collection (LGC Promochem, Teddington, UK).

Techniques: Derivative Assay, Control, Software, Adjuvant, Mutagenesis