Fig 2: Temozolomide (TMZ) resistance was associated with mesenchymal features, ferroptosis resilience, and elevated mitochondrial ROS state. (A) Heatmap plots present the enrichment score of functional signatures in each cell line. Cell lines with putative mesenchymal-like enrichment are marked by a red box in the top panel, and cell lines without mesenchymal features are indicated by a blue box. (B) The drug–response curve describes differential TMZ sensitivity between parental U87MG and resistant U87MG-R glioma cell lines. (C) Western blotting indicates the upregulation of CYBB, Nrf2, SOD2, mesenchymal markers (slug, vimentin, N-cadherin, CD44), and stemness marker (CD133) in TMZ-resistant U87MG-R. (D) Representative images of the tumor sphere assay and corresponding quantification are provided. Quantification bar displays the differential extent of tumor sphere generation in terms of the number and size of spheres between parental and resistant cells. (E) Representative images of the basic morphology of cell lines and migration and invasion assays are presented. Mesenchymal-like U87MG-R cells exhibited a spindle shape. Populations of migrated and invaded cells were intensified in U87MG-R. (F) Immunofluorescence images illustrate the comparative expression of the mitochondrial ROS marker MitoSOX and the mitochondrial label MitoTracker between U87MG and U87MG-R cells. Bar chart quantification of the mean fluorescence intensity of the corresponding marker and cell lines is presented. (G) Drug–response curve represents different sensitivity to erastin in U87MG and U87MG-R. (H) Bar chart compares the percentage of cell death, as shown by cells stained with propidium iodide following the induction of ferroptosis by treatment with either 150 µM tert-butyl hydroperoxide (TBHP) or 10 µM erastin with or without ferroptosis inhibitors (5 mM N-acetyl cysteine (NAC) or 2 µM SRS11-92) between U87MG and U87MG-R. Significance level: * p < 0.05; *** p < 0.001; **** p < 0.0001. Scale bar in D & E: 100 µm, F: 50 µm.

Fig 3: Cluster of glioma tumor with aberrant CYBB exhibited mesenchymal features, temozolomide (TMZ) resistance, and Nr2 pathway signature activation. (A) Representative t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of the Chinese Glioma Genome Atlas (CGGA) dataset show cluster cells grouped by the sample type (top panel) and number of clusters (bottom panel). Putative cell clusters with high CYBB expression were contained in an arbitrary border (dotted red line). (B) Array of t-SNE plots shows the expression levels of the putative main targets of this study (CYBB, NFE2L2 (or Nrf2), and SOD2). (C) t-SNE plots depict the module score, which represents the degree of activation of each gene set signature. Module scores of clusters with aberrant CYBB expression were delineated in an arbitrary border (dotted red line). (D) Scatter plot illustrates the co-association of each gene set signature of interest in terms of their module scores. Pearson’s coefficient and p value are provided in the top margin. (E) Violin plots present the comparison of module scores in each cell cluster. The median module score is labeled with a red line in each violin bar. Cluster number 0 served as control due to the presence of predominant tumoral cells with low CYBB expression. (F) Volcano plot depicts the overexpression of CYBB and SOD2 among DEGs between mesenchymal and nonmesenchymal GBM tumor cells in the TCGA-GBM dataset. (G) Venn diagram represents shared common DEGs among mesenchymal GBM, GBM with high NRF2 expression, and GBM tumor; CYBB and SOD2 are commonly expressed genes. (H) Dot plot depicts the significance of oxidoreductase genes in mesenchymal GBM. Significant genes are presented as red dots (SOD2 and CYBB), while the blue dots represent the genes below minimum threshold of log fold change. Significance level: ns: not significant, * p < 0.05.

Fig 4: Schematic illustration of how CYBB contributes to temozolomide resistance and ferroptosis resilience in mesenchymal glioblastoma. The left panel indicates that temozolomide resistance is associated with high oxidative stress and mesenchymal shift. The right panel illustrates that aberrant CYBB expression induced ROS accumulation and activated Nrf2/SOD2 circuitry, thus perturbing ferroptosis resilience upon erastin treatment.

Fig 5: Binding of CYBB to Nrf2 promotes mesenchymal features and temozolomide (TMZ) resistance. (A) Representative images of cellular morphology are presented following shRNA-mediated knockdown of CYBB (shCYBB) in U87MG-R cells. Spindle-shaped mesenchymal-like cells were less evident in shCYBB cells. Knockdown efficiency was observed through real-time quantitative reverse transcription polymerase chain reaction and is presented with a quantification bar chart. (B) Drug–response curve describes different sensitivity to TMZ treatment between scramble and shCYBB U87MG-R cells. (C) Western blotting indicated the downregulation of Nrf2, SOD2, and mesenchymal markers (CD44, Slug, Vimentin, and N-Cadherin) upon repression of CYBB. (D) Representative images of migration and invasion study between scramble and shCYBB U87MG-R cells are presented. Population of migrated and invaded cells are presented in a bar chart. (E) Images of tumor sphere study in scramble and shCYBB U87MG-R cells are presented. Generation of tumor spheres in terms of the number and size of spheres was quantified in a corresponding bar chart. (F) Immunofluorescence images illustrate the comparative expression of Nrf2 between scramble and shCYBB U87MG-R cells. Bar chart quantification of mean fluorescence intensity is presented. (G) U87MG-R cell lysate was incubated with the CYBB/p91-phox antibody or isotypic IgG. The immune complex precipitation revealed the coprecipitation of Nrf2 with CYBB/p91-phox (top panel). CYBB/p91-phox precipitation occurred and was identified using the corresponding antibody (second panel). The total cell lysate as input was immunoblotted for Nrf2 (third panel) and β-actin (bottom panel). Significance level: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale bar in A, D & E: 100 µm, F: 20 µm.