Fig. 5: TIALD down-regulated AURKA expression via lysosomal degradation.

A Western blot showing AURKA interaction with TIALD. RNA pull-down assay was performed in SMMC-7721 and SNU449 cells using biotin-labeled TIALD RNA probe transcribed in vitro, and detected using western blots. B RIP-qPCR analysis of the interaction of AURKA and TIALD in SMMC-7721 and SUN449 cells. Enrichment of TIALD with AURKA antibody and IgG control was measured by qPCR. C RNA pulldown assays were performed with a series of truncated TIALD variants and followed by immunoblotting with the AURKA antibody. D RIP-qPCR analysis were performed using AURKA antibody and IgG control, and enrichment of different TIALD variants was detected by qPCR. E The expression of AURKA in TIALD over-expression SNU449 cells and TIALD knock-down SMMC-7721 cells was detected by western blot. F The mRNA level of AURKA in TIALD over-expression SNU449 cells and TIALD knock-down SMMC-7721 cells was detected by qPCR. G Effect of CHX on AURKA degradation. TIALD over-expression SNU449 cells and TIALD knock-down SMMC-7721 cells were treated with CHX (100 μg/ml) for the indicated times. The expression of AURKA was determined by western blotting and the levels of AURKA protein were quantified by densitometry. H TIALD induced AURKA lysosome degradation. TIALD was over-expressed in SNU449 cells and treated with MG132 (20 μM, 3 h) or chloroquine (50 μm, 12 h). The expression of AURKA was analyzed by western blot. I TIALD triggers AURKA lysosome localization. TIALD over-expressed SNU449 cells were immune-stained using AURKA antibody followed by Alexa fluor 488 conjugated secondary antibody. Lysosome was marked using Lysotracker. Stained cells were visualized under confocal microscope.