Fig. 6

ADAMTS9-AS1 is physically associated with HuR and increases the binding of HuR to ADMTS9 3’UTR. A. RNA pull-down assay by ADAMTS9-AS1 and its antisense RNA followed by silver staining of protein extract from A498 cells. A band indicated by an arrow was excised for mass spectrometry analysis. S: sense strand of ADAMTS9-AS1, AS: antisense strand of ADAMTS9-AS1. B. Western blot analysis of the specific association of HuR and ADAMTS9-AS1. Vinculin was used as the negative control. The same amount of sense and antisense ADAMTS9-AS1 transcripts confirmed by dot-blot assay. C. ADAMTS9-AS1 associated HuR was measured by qRT-PCR following CLIP HuR after ADAMTS9-AS1 overexpression or KD in A498 cells. GAPDH and CLIP IgG were used as negative controls. HuR pull-down efficiency was determined by WB. D. Schematic diagram of the full-length and truncated ADAMTS9-AS1(left). CLIP assay was used to detect the binding of HuR with full-length or fragmented ADAMTS9-AS1 after overexpression of the indicated ADAMTS9-AS1. CLIP IgG and GAPDH were used as negative controls (right). Similar expression levels of ADAMTS9-AS1 were confirmed by qRT-PCR (middle). HuR pull-down efficiency was determined by WB. E, F. The mRNA expression levels of ADAMTS9 were determined by qRT-PCR after truncated ADAMTS9-AS1 (E) and HuR (F) overexpressed or HuR KD (F) in in A498 cells. The transfection efficiency of HuR was measured by WB(F). (G). ADAMTS9 associated HuR was measured by qRT-PCR following CLIP HuR after ADAMTS9-AS1 overexpression or KD in A498 cells. GAPDH and CLIP IgG were used as negative controls. HuR pull-down efficiency was determined by WB. H-I. The expression levels (H) and the half-life (I) of ADAMTS9 mRNA of ADAMTS9 were determined by qRT-PCR after HuR KD in Control and ADAMTS9-AS1 overexpressed cells. The data derived from three independent experiments are presented as the mean ± SEM in the bar graphs (C–I). Controls were normalized to 1 (D, E, F, H, I). **P < 0.01, ***P < 0.001. N.S. not significant