Additional file 5 of Hydroxychloroquine synergizes with the PI3K inhibitor BKM120 to exhibit antitumor efficacy independent of autophagy

Additional file 5: Figure S5. The effect of HCQ on ROS production and the synergetic effect with BKM120 on proliferation inhibition in SKOV-3 and MKN-1 cells. (A) Cell viability of SKOV-3 and MKN-1 cells after treatment with BKM120 or HCQ alone or in combination for 72 h. (B) The drug combination was analyzed using CalcuSyn software and the resulting CI-Fa plots are shown. (C) SKOV-3 and MKN-1 cells were treated with various concentrations of HCQ for 48 h and then subjected to apoptosis analysis. (D) FACS quantification of total apoptotic cell population. (E) Western blot analysis of PARP in SKOV-3 and MKN-1 cells treated with HCQ for 48 h. ROS levels (F) in SKOV-3 and MKN-1 cells were determined after treatment with 40 μM HCQ for 6 and 48 h, respectively, which were quantified as in (G). ROS level (H) in MKN-1 cells after transfected with Atg5 siRNA alone or in combination with BKM120 for 48 h was determined, and quantified as in (I). (J) Quantification of NRF2 expression after correction with the β-actin control. Graphs are shown as mean ± SD from three independent experiments; ns: p > 0.05; *p < 0.05; **p < 0.01.