Fig. 3. TAS4464 increases NOXA expression and decreases c-FLIP expression at the mRNA transcriptional level.

A HL-60 and THP-1 cells were treated with 0.1 μmol L⁻¹ TAS4464 for 1, 4, 8, 16. and 24 h, and total protein was extracted. Immunoblotting for neddylated Ubc12 and cullin1 was performed with the anti-NEDD8 antibody. Parallelly, immunoblotting for Bcl-2 family proteins in the intrinsic apoptotic pathway and death receptor signal-related proteins in the extrinsic apoptotic pathway was performed with the indicated antibodies. B qRT-PCR was performed to compare the mRNA levels of PMAIP1 and CFLAR in cells treated with 0.1 μmol L⁻¹ TAS4464 and control cells (0 h) at the indicated time points (hours). PMAIP1 and CFLAR mRNA expression levels were normalized to 18S rRNA expression levels. Data are presented as the mean ± SEM values from three independent experiments.