Figure 5.

TIG reverses BTZ-dependent mitochondrial ROS increase without increased expression of superoxide antioxidants. (a) Analysis of superoxide production in mitochondria by MitoSOX Red flow cytometry assessment in KMS20 (left) and KMS28BM (right) cell lines. Cells were incubated with their EC50 of BTZ only (0) or with it plus increasing concentrations of TIG, as shown. C, control: vehicle-treated cells. Plots show means ± SEM of the data for 24 h (in blue) and 48 h (in red) culture in each condition. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. (b) SOD2 protein levels analyzed by western blot in KMS20 (top) and KMS28BM (bottom) cells after 48 h drug incubation as in (a). On the left panels, quantification of SOD2 levels normalized with beta actin, analysis using ImageJ Fiji win64 software (n = 3). Results are expressed using box and whiskers diagrams, where lines represent the median value, boxes the 25th and 75th percentiles, and whiskers mark maximum and minimum values; p values are shown. On the right, representative western blots for each cell line (KMS20 on the top and KMS28BM on the bottom) showing SOD2 and beta actin (β-ACTIN) control, as indicated. MW: molecular weight marker. C, control: vehicle-treated cells; 0, BTZ EC50 (12.5 nM); 20, 12.5 nM BTZ plus 20 μM TIG, and 50, 12.5 nM BTZ with 50 μM TIG. (c–e) mRNA expression of genes encoding antioxidant proteins SOD1, NFE2L2, and G6PD, respectively, analyzed by RT-qPCR as in Figure 4c. KMS20 cells are shown in the top panels and studies on KMS28BM in the bottom ones. Culture conditions for 48 h drug exposure and statistical significance as in (a). Plots show means ± SEM of 3 independent experiments.