Journal: Cancer research
Article Title: MCPIP1 selectively destabilizes transcripts associated with an anti-apoptotic gene expression program in breast cancer cells that can elicit complete tumor regression
Figure Lengend Snippet: MCPIP1 selectively inhibits the mRNA expression of anti-apoptotic genes. (A) Clustergram of the apoptosis related genes affected differentially in MDA-MB-231/Tet-On cells at 32 hours after adding Dox (500 ng/ml). MCPIP1: cells with Dox; Control: cells without Dox. MDA-MB-231/Tet-On cells were induced to express MCPIP1 by Dox (500 ng/ml) for different times. Then total RNA was collected to detect mRNA levels of selected anti-apoptotic genes by qRT-PCR, including bcl2l1 (B), bric3 (C), bcl3 (D), and relb (E). The mRNA levels of pro-apoptotic genes in the above cells were measured by qRT-PCR, including bad (F), ripk2 (G), fas (H), and dedd2 (I). MDA-MB-231/Tet-On cells were induced to express GFP/MCPIP1 fusion protein by Dox (500 ng/ml) for 36 h, followed by lysate extraction, immunoprecipitation with anti-GFP antibody or IgG, and RNA extraction. RT-PCR was performed to detect anti-apoptotic gene transcripts (J) and pro-apoptotic gene transcripts (K). IL-6 transcript was used as positive control and GAPDH served as negative control.
Article Snippet: Total RNA was extracted with Trizol from MCPIP1-expressed and -unexpressed MDA-MB-231 cells, purified using the RNA Cleanup Kit (Fisher Scientific), reverse-transcribed to cDNA, and added to each well of the PCR Array plates combined with TaqMan Master Mix according to the manufacturer's instructions.
Techniques: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Immunoprecipitation, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control