Figure 4. MBIP activates JNK pathway and regulates direct downstream targets.

30μg of a whole cell extract from (A) i393P and (B) iH157 cells with induced expression of MBIP or GFP(Vector) as control, or (C) MBIP knockdown (344SQ-Scramble control and 344SQ-shMBIP#4,#5) cells were resolved on 10% SDS PAGE gels followed by Western blotting with specific antibodies as indicated. Note that p-JNK and p-c-JUN levels are substantially higher in MBIP overexpressing cells and conversely lower in the MBIP knockdown cells when compared to the respective control cells. Western blots for β actin served as a loading control (lower panels). qRT-PCR analysis performed for the indicated JNK downstream target genes, using mRNA from (D) 393P cells with constitutive expression of MBIP (393P-MBIP or 393P-mCherry control), or (E) MBIP knockdown (344SQ-Scramble control and 344SQ-ShMBIP#4, #5) cells. (F) Western blot analysis for indicated proteins performed on whole cell extracts from i393P and iH157 cells with induced expression of MBIP or GFP (Vector) as control, which were treated with a specific JNK inhibitor (SP600125) or DMSO as control. (G) Representative images of Transwell chamber assays performed for migration and invasion of i393P or iH157 cells with induced expression of MBIP or GFP (Vector) as controls, which were treated with a specific JNK inhibitor (SP600125) or DMSO as control. The number of cells migrated/invaded through the membrane/matrix were quantified as described in Materials and Methods. (H) Representative images of 3D matrix (Matrigel+collagen) with invasive spheroids, formed by doxycycline-inducible MBIP-overexpressing i393P cells, which were treated with a specific JNK inhibitor (SP600125) or DMSO as control. **P < 0.01, ****P < 0.0001 is based on the Student t test. All results are from three independent experiments. Error bars represent SD.