Figure 6. NQO1 residing in the mitochondrial IMS drives IB-DNQ redox cycling, promoting mitochondrial oxidative damage, Cyto c release, and activation of Casp3-mediated mitochondrial apoptotic pathway.

A-B, NQO1^med and NQO1^lo (A) and NQO1^hi (B) TNBC cells were examined for NQO1, SOD1, SDHA and Skp1 expression in the mitochondrial and cytosol fraction. C, SUM159 cells labeled with specific antibodies against NQO1 and Cyto c were examined by confocal microscopy. Scale bar: 120 μm. D-E, Vari08 and SUM149 cells treated with 100 nM of IB-DNQ for 0–8h (D) or with vehicle, IB-DNQ (100 nM), ATN224 (5 μM), IB-DNQ+ATN224 for 16h (E) were processed and examined by TEM. Bars: 200 (D) and 500 (E) nm. F, SUM159 cells were treated with vehicle or IB-DNQ for 2h and measured for oxygen consumption based on fluorescent intensity (RFU) of an oxygen-bleaching fluorescent dye in the media covered by mineral oil at 1.5 min intervals for 90 min. ****: p<0.0001 (n=3, two-way ANOVA). G, ATP levels in the lysates of SUM159 cells treated with vehicle or IB-DNQ for 2h were measured and normalized by protein concentrations. ****: p<0.0001 with unpaired student’s t-test (n=3). H-I, Vari068 and SUM149 cells treated with IB-DNQ (100 nM) for 0–12h (H) or IB-DNQ and ATN224 alone or in combination for 16h (I) were used to isolate mitochondrial and cytosol fractions and examine Cyto c, SDHA and Skp1 expression. J-K, Lysates of Vari068 (J) and SUM149 (K) cells treated with IB-DNQ alone or IB-DNQ plus ATN224 for 20h were subjected to immunoblotting to examine the expression of apoptosis-related proteins. L, A model illustrating the mechanisms of action for IB-DNQ mediated pro-oxidant therapy targeting TNBC cells including CSCs.