Figure 4.

MCPIP1 destabilizes the mRNAs of anti-apoptotic genes via the PIN domain. MDA-MB-231/Tet-On cells were treated with 500 ng/ml of Dox for 36 hours to induce MCPIP1 expression and then ActD and DRB added for different times. The remained anti-apoptotic gene transcripts, bcl2l1 (A), bcl3 (B), birc3 (C), and relb (D), were measured by qRT-PCR. HEK293 cells were co-transfected with luciferase reporter constructs containing full-length 3’UTRs of the anti-apoptotic genes and either MCPIP1 or control vector. Luciferase activities were measured in cell lysates after 36 h. β-actin 3’UTR was used as negative control. Results shown represent the mean ± SD of four independent experiments (*: p< 0.05). (F) Schematic representation of the RNase and zinc finger domains in MCPIP1, and their mutant, D141N and ΔZF. (G) HEK293 cells were co-transfected with luciferase reporters containing 3’UTRs of the anti-apoptotic genes and WT MCPIP1 as well as the two mutants. After 36 h, luciferase activities were measured in cell lysates and compared to empty vector transfected cells. (H) MDA-MB-231 cells were transiently transfected with MCPIP1 and two mutants, respectively. 36 h later, total RNA was extracted to measure the mRNA expression of different genes as indicated by qRT-PCR. (I) MDA-MB-231 cells were transiently transfected with MCPIP1 and two mutants. 48 h later, floating dead cells were counted in each group. n=3. (J) MDA-MB-231 cells were transiently transfected with MCPIP1 and two mutants. 48 h later, whole cell lysates were collected to measure total PARP1 and cleaved PARP1 by western blot.