A1 reduces the cyclin D1 protein through the endoperoxide of DHA-mediated NOXA production. (A) THP-1 and K562 cells were treated with 1.6 to 3.2 μM DHA or VEN and 0.4 to 0.8 μM A1 for 24 hours. The protein levels were determined by western blotting. The right column figures are image density analyses of 3 independent experiments. (B) THP-1 and K562 cells were treated with 3.2 μM DHA and 0.8 μM A1 for the indicated times. The protein levels were detected by western blot analysis. (C) THP-1 cells were pretreated with 25 μM Q-VD-OPh for 4 hours, followed by 3.2 μM DHA and 0.8 μM A1 treatment for 24 hours. The cyclin D1 and NOXA levels were detected using western blot analysis. (D) The DNA distribution was assessed by flow cytometry after PI staining. (E) Cell growth inhibition of THP-1 cells pretreated with Q-VD-OPh, followed by A1 and DHA for 24 hours. (F) THP-1 cells were transduced with shNC or shPMAIP1 and then treated with 0.8 μM A1 for 24 hours. (G) THP-1 cells were transduced with shNC or shBCL2L11, then treated with 0.8 μM A1 for 24 hours. (H) Raji cells that were defective in NOXA were treated with 0.2 to 0.8 μM A1 for 24 hours. (I) Jeko-1 cells that were defective in Bim were treated with 0.2 to 0.8 μM A1 for 24 hours. The relative levels of the NOXA and cyclin D1 were determined by western blotting. (J) The chemical structures of DODHA and DOA1 with the reduced endoperoxide moiety of DHA and A1. (K) The protein levels of cyclin D1 and NOXA in the THP-1 and K562 cells that were treated with DHA, A1, DODHA, and DOA1 for 24 hours. The right column figures are image density analyses of 3 independent experiments. (L) The iron chelator DFO blocks A1-induced NOXA. THP-1 cells were pretreated with 100 μM DFO for 4 hours and then treated with 3.2 μM DHA and 0.8 μM A1 for 24 hours. (M) The heme synthetic precursor and inhibitor regulate A1-induced NOXA. THP-1 cells were pretreated with 250 μM SA, 5 μM PPIX, or 250 μM SA + 5 μM PPIX for 24 hours, followed by 3.2 μM DHA and 0.8 μM A1 treatment for 24 hours. 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 < .01; ###P < .001 2-group analysis. BimEL, Bim extralong; BimL, Bim long; BimS, Bim short; Con, control; PPIX, protoporphyrin IX; SA, succinylacetone.

A1 reduces the cyclin D1 protein through the endoperoxide of DHA-mediated NOXA production. (A) THP-1 and K562 cells were treated with 1.6 to 3.2 μM DHA or VEN and 0.4 to 0.8 μM A1 for 24 hours. The protein levels were determined by western blotting. The right column figures are image density analyses of 3 independent experiments. (B) THP-1 and K562 cells were treated with 3.2 μM DHA and 0.8 μM A1 for the indicated times. The protein levels were detected by western blot analysis. (C) THP-1 cells were pretreated with 25 μM Q-VD-OPh for 4 hours, followed by 3.2 μM DHA and 0.8 μM A1 treatment for 24 hours. The cyclin D1 and NOXA levels were detected using western blot analysis. (D) The DNA distribution was assessed by flow cytometry after PI staining. (E) Cell growth inhibition of THP-1 cells pretreated with Q-VD-OPh, followed by A1 and DHA for 24 hours. (F) THP-1 cells were transduced with shNC or shPMAIP1 and then treated with 0.8 μM A1 for 24 hours. (G) THP-1 cells were transduced with shNC or shBCL2L11, then treated with 0.8 μM A1 for 24 hours. (H) Raji cells that were defective in NOXA were treated with 0.2 to 0.8 μM A1 for 24 hours. (I) Jeko-1 cells that were defective in Bim were treated with 0.2 to 0.8 μM A1 for 24 hours. The relative levels of the NOXA and cyclin D1 were determined by western blotting. (J) The chemical structures of DODHA and DOA1 with the reduced endoperoxide moiety of DHA and A1. (K) The protein levels of cyclin D1 and NOXA in the THP-1 and K562 cells that were treated with DHA, A1, DODHA, and DOA1 for 24 hours. The right column figures are image density analyses of 3 independent experiments. (L) The iron chelator DFO blocks A1-induced NOXA. THP-1 cells were pretreated with 100 μM DFO for 4 hours and then treated with 3.2 μM DHA and 0.8 μM A1 for 24 hours. (M) The heme synthetic precursor and inhibitor regulate A1-induced NOXA. THP-1 cells were pretreated with 250 μM SA, 5 μM PPIX, or 250 μM SA + 5 μM PPIX for 24 hours, followed by 3.2 μM DHA and 0.8 μM A1 treatment for 24 hours. 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 < .01; ###P < .001 2-group analysis. BimEL, Bim extralong; BimL, Bim long; BimS, Bim short; Con, control; PPIX, protoporphyrin IX; SA, succinylacetone.

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