Figure 4.

MG132 inhibition increases A3A specifically. Breast cancer cell lines MDA-MB-453 and BT474 were transduced with a non-specific scramble shRNA, A3A-targeting shRNA or A3B-targeting shRNA. Efficiency of shRNA knockdown lines previously published in (17). Representative images of western blot GAPDH analysis and denaturing gel of activity assays of the knockdown lines in MDA-MB-453 cells (A) or BT474 cells (B) treated with MG132 or DMSO control. Plotted percent substrate cleavage for the scramble shRNA, A3A shRNA and A3B shRNA expressing lines in MDA-MB-453 (C, n = 5) and BT474 (D, n = 3). Significant differences in A3A activity between the knockdown lines are marked with asterisks as compared by unpaired t-test. Mean is shown for all replicates and error bars represent one standard deviation. (E) Log₂ fold change in transcription initiation of APOBEC3 family genes and ACIDA during a 24 h treatment of MDA-MB-453 cells with 0.5 μM MG132 compared to DMSO control. nd, not detected. Expression relative to HPRT1 was quantified via qRT-PCR for all seven APOBEC3 proteins; only APOBEC3A showed a significant increase in expression after 24 h treatment with 0.5 μM MG132 (ratio paired t-test; P-value 0.0266). nd, not detected; S, substrate; P, product. *P ≤ 0.05 and ***P ≤ 0.001; ns, non-significant with P-value >0.05.