FIGURE 4.

PCAT6 interacts with IGF2BP2 to play oncogenic roles in PCa. (A) Nuclear‐cytoplasmic fractionation assays revealing PCAT6 expression in the cytoplasm and nucleus of PCa cells. U6 and GAPDH were used as positive controls in the nucleus and cytoplasm, respectively. Error bars represent the mean ± SD of triplicate experiments. (B) RNA FISH showing the subcellular localization of PCAT6 in PCa cells. Scar bar, 100 μm. (C) RNA pull‐down assay was performed using PCAT6 sense and antisense RNAs incubated with cell extracts of PC‐3 cells, followed by silver staining. A red arrow indicates IGF2BP2. (D) The interaction between PCAT6 and IGF2BP2 was confirmed by RNA pull‐down and western blotting. GAPDH served as the negative control. (E) RIP was performed using anti‐IGF2BP2 and control IgG antibodies, followed by RT‐qPCR to examine the enrichment of PCAT6 and GAPDH. GAPDH served as the negative control. Error bars represent the mean ± SD of triplicate experiments. (F) Secondary structure of PCAT6 predicted by RNAfold Website. (G) Serial deletions of PCAT6 were used in the RNA pull‐down assays to identify the core regions of PCAT6 that were required for the physical interaction with IGF2BP2. (H) Schematic structures showing RNA‐binding domains within IGF2BP2 protein and a summary of IGF2BP2 variants used in this study. Blue boxes are RRM domains, brown boxes are wild‐type KH domains with GxxG core, and gray boxes are inactive KH domains with GxxG to GEEG conversions (upper panels). RIP was performed using anti‐Flag and control IgG antibodies, followed by RT‐qPCR to examine the enrichment of PCAT6 (down panels). Error bars represent the mean ± SD of triplicate experiments. (I) Western blotting analysis of IGF2BP2 expression in the indicated group. GAPDH served as the loading control. All experiments were performed in biological triplicate. Statistical analyses were performed by χ ² test (A) and unpaired Student's t‐test (E, H). * p < 0.05