Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: Screening of downstream targets of CD97 in GSCs (A) Schematic representation of the experimental strategy for the transcriptome analysis. Heatmap of the hierarchical clustering analysis showing the expression of 131 common downregulated genes in CD97-knockdown GSCs (right). (B) Gene set enrichment analysis (GSEA) plot for the regulation of stem cell population maintenance signature from the Gene Ontology Biological Process (GOBP) category comparing the shCtrl and shCD97 groups. NES, normalized enrichment score. (C) Bubble chart showing the top 10 biological processes of Gene Ontology terms for genes downregulated in CD97-knockdown GSCs. Size, gene number; color, −log 10 ( p value). (D) RT-qPCR of PCBP1, BSG, ARHGAP1, BZW1, BZW2, and DBN1 expression in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. β-actin was used as a loading control. All error bars represent mean ± SD ( n = 3 independent experiments). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, t test. (E) Kaplan-Meier overall survival curves for patients with GBM presenting the high and low expression of the CD97/ARHGAP1 (left), CD97/BZW1 (middle), and CD97/BZW2 (right) mRNAs according to TCGA GBM database. Log rank (Mantel-Cox) test. (F) Bubble chart showing the top 10 hallmark genes enriched in shCtrl GSCs. Size, gene number; color, −log 2 ( p value). (G) GSEA plot for the PI3K/AKT/mTOR signaling signature from the Hallmark category comparing the shCtrl and shCD97 groups. See also Figure S11 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Infection, Control

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: The membranes were incubated with primary antibodies against CD97 (Abcam), Nestin (BD Transduction Laboratories), Oct3/4 (Proteintech), Nanog (Sangon biotech), p-S6K S371 (Cell Signaling Technology), p-S6K T389 (Cell Signaling Technology), S6K (Cell Signaling Technology), p -AKT T308 (Cell Signaling Technology), p -AKT S473 (Cell Signaling Technology), AKT (Cell Signaling Technology), ARHGAP1 (Santa Cruz), BZW1 (GeneTex), BZW2 (Bethyl Laboratories), CD133 (Proteintech), CD44 (R&D Systems), SOX2 (R&D Systems), Vinculin (Sigma-Aldrich), and β-actin (Bioworld Technology) overnight at 4°C.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot