Fig 2: ADO is upregulated in glioblastoma stem-like cells.a Sphere formation derived from LN229 ADO-Cas9 and control cells. Scale bars = 200 µm. b Analysis of correlations between ADO and a series of stemness markers in two TCGA cohorts (TCGA-LGG and TCGA-GBM), which were retrieved from GDC data portal (https://portal.gdc.cancer.gov/). c, d Sox2 and Oct4 expression was detected by western blotting and immunofluorescence under confocal fluorescence microscopy in U118 and ADO-OE U118 cells, respectively. Scale bars = 25 µm. e Sox2, Oct4, and ADO co-staining in glioma patients sections with high levels of ADO, Sox2, and Oct4 were located within the nucleus. Scale bars = 25 µm. All results are representative of three independent experiments.

Fig 3: ADO functions as an oncogene in glioma.a ADO and CDO1 protein levels in 9 glioma cell lines. b, c siRNA of ADO (870, 954) corresponding to a decrease in clonal growth and invasion of LN229 cells. d overexpression (OE) ADO in U118 and SF767 cells corresponding to clonal growth. e ADO expression levels in 108 glioma patient samples and 18 normal brain sections obtained from intracranial aneurysms and peritumor. Immunoreactive score (IRS) based on staining intensity and distribution was then used to evaluate ADO expression in low-grade gliomas (LGG) and GBMs (f). Scale bars = 100 μm and 50 μm. g ADO is highly expressed in GBM in a cohort consisting of 180 glioma tissues, localized to the cytoplasm, and negatively correlated with the overall survival rate and disease-free survival of glioma patients (h). All results are representative of three independent experiments.

Fig 4: ADO promotes NF-?B pathway activation.a, b RNA-seq analysis in three paired experiments: ADO knockdown and controls under both 2D and 3D (LN229-siADO-derived spheroids) cultural conditions, and U118 ADO-OE cells and control groups. c Venn diagram representing overlapping candidates between three pairs of RNA-seq. d Phosphorylated p65 (p-p65) and total p65 proteins levels were analyzed in LN229 ADO-Cas9 cells and e U118 ADO-OE cells and their respective controls by immunoblotting. f ADO and p65 co-staining in U118 ADO-OE cells. Scale bars = 50 µm. g LN229 cells were treated with TNFa (2 µM) or NF-?B inhibitor BAY11-7082 (2 µM) to examine the effects of co-treatment of hypotaurine with ADO-Cas9 or BAY11-7082 on p-p65 and total p65 protein levels. Data represent three independent experiments with similar results.

Fig 5: Inhibiting ADO attenuates glioblastoma growth in vivo.a LN229 ADO-Cas9 and control cells were injected into an orthotopical mouse xenograft model to analyze for in vivo tumor growth. Scale bars = 100 µm and 500 µm. b CCL20 and CCR6 expression were evaluated by immunohistochemistry in the xenograft tumors. Scale bars = 50 µm and 200 µm. Data represent three independent experiments with similar results.