Downregulation of PI5K-1B by plumbagin and functional role of PI5K-1B for cell viability and ROS generation in MCF-7 human breast cancer cells.

(A) MCF-7 cells were cultured in a 6-well plate as described in Materials and Methods and then either left untreated or treated with 5 µM plumbagin for 24 h. Then, cells were harvested, and the levels of proteins in the cell lysates were analyzed by western blotting (panel A). (B) PI5K-1B knockdown in MCF-7 cancer cells was carried out a described in Materials and Methods using PI5K-1B-specific siRNA (5′-GUCCUCAAUUAGCCAGGAA(dTdT)-3′) or a control siRNA (5′-CUUACGCUGAGUACUUCGA(dTdT)-3′) for 48 h. Then, knockdown of PI5K-1B was validated by either RT-PCR (panel Ba) or western blotting (panel Bb) as described in Materials and Methods. (C) Cell viability after siRNA transfection for 48 h was measured by the WST-1 assay. Data represent the mean ± standard error (n = 3) (panel Ca). ***P<0.001 compared with the untreated samples. The cells after siRNA transfection for 48 h were stained with 4 µM DHE for 30 min, and then the ROS level was observed using a fluorescence microscope (excitation 518 nm, emission 605 nm) (panel Cb).