Fig 2: Combined treatment of BRZ and TMZ increases cell death in GBM stem-like cells. (A–C) mRNA expression of CA2 after TMZ treatment at 3 d and 5 d were detected by RT-PCR, the mRNA level of CA2 increased after TMZ stimulation (n = 3). (D) Visualization of the morphology of GSCs (Nr. 2016/175) using light microscopy after TMZ and CA inhibitor stimulation for 10 d. (E–G) Cell viability of co-treatment measured by CellTiter-Glo assay. Co-treatment with 500 µM TMZ and 400 µM ACZ decreased the viability compared to TMZ alone. However, 100 µM ACZ in combination with TMZ did not change the cell viability. Both 100 µM and 400 µM BRZ in combination with 500 µM TMZ decreased the viability significantly compared to TMZ treatment alone (n = 3). * (Red): compared to Control group; # (Purple): compared to TMZ group; & (Orange): compared to ACZ 100 µM + TMZ group; % (Black): compared to BRZ 100 µM + TMZ group; $ (Green): compared to ACZ 400 µM + TMZ group. Results were obtained from 3 independent experiments. In (A–C,E–G), data are presented as mean ± SD, student’s t-test was used to analyze (A–C), One-way ANOVA was used to analyze (E–G), *, & p < 0.05; ## p < 0.01; ***, ###, &&&, $$$ p < 0.001, ns: not significant.

Fig 3: (A) The number of differentially expressed genes in CA2-transfected T24 and UMUC3 cells compared with their respective controls (empty vector-transfected T24 and UMUC3 cells). Left: number of upregulated genes: six genes were upregulated in both CA2-transfected T24 and UMUC3 cells. Right: number of downregulated genes: three genes were downregulated in both CA2-transfected T24 and UMUC3 cells. (B) mRNA expression level of PIP5K1B was significantly increased in CA2-transfected T24 and UMUC3 cells. *p < 0.05 versus controls

Fig 4: The combination of BRZ and TMZ increased cell death in GBM stem-like cells by activating autophagy (A,B) Autophagy marker LC3 immunostaining of U251_Ctrl and U251_CA2 cells after TMZ and ACZ/BRZ stimulation for 24 h. Co-treatment with TMZ and BRZ induced the expression of LC3 puncta in U251_CA2 cells (scale bar: 50 μm). (C,D) Western Blotting of autophagy-related proteins and CA2 protein in U251_Ctrl and U251_CA2 cells with the same treatment as in (A). TMZ plus BRZ did not increase the protein expression of LC3II in U251_Ctrl cells compared to TMZ treatment alone (C) but increased in U251_CA2 cells (D) (n = 3). (E–G) Western Blotting of autophagy-related proteins and CA2 protein in GBM stem cells with the same treatment as in Figure 5E–G for 24 h stimulation. Compared with the control group, TMZ monotherapy has a tendency to increase the protein level of LC3II, but the expression of it is significantly increased in BRZ alone and TMZ combined with BRZ treatment (n = 3). Results were obtained from three independent experiments. Data are presented as mean ± SEM, One-way ANOVA was used to analyze the data, * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant.

Fig 5: Effect of combined treatment with TMZ and either ACZ or BRZ on CA2 overexpressing GBM cell lines U87 and U251. (A) Co-treatment with 500 µM TMZ and ACZ/BRZ on U87_Ctrl cells and U87_CA2 cells for 5 d. U87_CA2 cells significantly decreased cell viability compared to U87_Ctrl cells after BRZ + TMZ treatment. However, only 400 µM ACZ + TMZ decreased cell viability compared to the U87_Ctrl group (n = 3). (B) In U87_CA2 cells treated with TMZ, ACZ/BRZ, or TMZ + ACZ/BRZ, TMZ plus 400 µM ACZ decreased the viability compared to TMZ alone. However, 100 µM ACZ in combination with TMZ did not change the cell viability. Both 100 µM and 400 µM BRZ in combination with 500 µM TMZ decreased the viability significantly compared to TMZ treatment alone (n = 3). (C) Co-treatment with 30 µM TMZ and ACZ/BRZ on U251_Ctrl cells and U251_CA2 cells for 5 d. U251_CA2 cells decreased cell viability compared to U251_Ctrl cells after 400 µM BRZ + TMZ treatment. However, ACZ + TMZ did not change cell viability compared to the U251_Ctrl group (n = 3). (D) In U251_CA2 cells, ACZ/BRZ has no cytotoxic effect, TMZ plus 400 µM ACZ decreased the viability compared to TMZ alone. Both 100 µM and 400 µM BRZ in combination with 500 µM TMZ decreased the viability significantly compared to TMZ treatment alone (n = 3). * (Red): compared to Control group; # (Purple): compared to TMZ group; & (Orange): compared to ACZ 100 µM + TMZ group; % (Black): compared to BRZ 100 µM + TMZ group; $ (Green): compared to ACZ 400 µM + TMZ group. Results were obtained from 3 independent experiments. Data are presented as mean ± SD, Two-way ANOVA was used to analyze (A,C), One-way ANOVA was used to analyze (B,D), ***, $$$, &&&, ###, p < 0.001, ns: not significant.