Figure 3.

KP372-1 causes Ca²⁺ releasing and AKT hyperactivation to enhance the lethality of PARP inhibitor. (A) Relative survival assay in A549 NSCLC cells treated with BAPTA (5 μM) under conditions of KP372-1 ± rucaparib. Cells were pre-treated ± rucaparib (15 µM) for 1 h, then added ± BAPTA (5 µM, 1 h), and then exposed to KP372-1 ± rucaparib ± BAPTA for 2 h, followed by washing and replacing media, cell viability was assessed 7 days later. (B, C), PAR and γH2AX alterations were assessed and quantified in A549 NSCLC (B) and MCF-7 cells (C). (D, E) A549 cells were pre-treated ± rucaparib (15 µM, 2 h), then exposed to KP372-1 ± rucaparib for 2 h, followed by washing and replacing media, cells were collected at indicated time points and assessed for: (D) Levels of pAKT^s473 and total AKT (t-AKT), and bottom panel showed quantification of pAKT^s473; (E) Fluorescence image of pAKT^s473. (F) Cells were treated as (B, C) then pAKT^s473 levels alterations were assessed and quantified in A549 NSCLC and MCF-7 cells. (G) MCF-7 scramble and siAKT1/2 cells were treated as (D) then cells were collected at 2 h and relative H₂O₂ in MCF-7 cells were determined. Results were separately repeated at least three times, AKT knockdown efficiency for (E) was confirmed by Western blot in (G). All error bars are means ± SD. (A) *** P < 0.001, comparing each data point with KP372-1 alone treatments (grey color) (t tests). (F), *** P < 0.001 and ns: no significant, comparing each group with control (DMSO) treatments (t tests).