A1 has enhanced NOXA and apoptosis induction abilities in THP-1 cells. (A) THP-1 cells were treated with DHA, VEN, DHA + VEN, and A1 for 24 hours. The apoptotic cells were quantified using FACS after staining with annexin V/PI. (B) THP-1 cells were treated with DHA, VEN, DHA + VEN, and A1 for 24 hours. The relative protein levels were determined by western blotting. The right column figures are image density analyses of 3 independent experiments. (C) THP-1 cells were pretreated with 25 μM Q-VD-OPh for 4 hours, followed by treatment with 0.8 μM DHA + VEN and A1 for 24 hours. The right column figures are the image density analyses of 3 independent experiments. (D) IP assay with anti-Bak (Ab-1) and anti-Bax 6A7 antibodies that detected the active forms, followed by probe detection for Bak and Bax in THP-1 cells treated with 0.8 μM DHA, VEN, DHA + VEN, and A1 for 24 hours. (E) IP assay of the binding of Bim and NOXA to Bcl-2 and Mcl-1 in THP-1 cells treated with 0.8 μM DHA, VEN, DHA + VEN, and A1 for 24 hours. (F) THP-1 cells were transduced with shNC or shPMAIP1, followed by treatment with 0.8 μM A1 for 24 hours. (G) THP-1 cells were transduced with shNC or shBCL2L11, followed by treatment with 0.8 μM A1 for 24 hours. The apoptotic cells were quantified using FACS after staining with annexin V–fluorescein isothiocyanate (mean ± SD of 3 independent experiments). The relative levels of the indicated proteins were determined by western blotting. (H) U937, Mono Mac 6, THP-1, Kasumi-1, and ME-1 cells were treated with 0.8 μM A1 for 24 hours. The relative levels of the indicated proteins were determined by western blotting. The right column figures are image density analyses of 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 when compared with the control group. ###P < .001, 2-group comparison. Bim_EL, Bim extralong; Bim_L, Bim long; Bim_S, Bim short; Con, control; shNC, short hairpin RNA negative control.