Fig. 5: CircNUP54 interacts with the RBP HuR in HCC.

A RIP-qPCR analysis showed that circNUP54 was not enriched with the AGO2 antibody. B qRT-PCR results of RNA pull-down products using a biotin-labeled circNUP54 probe and control probe in circNUP54-overexpressing HCC cells. C Silver staining of circNUP54 pull-downs. D HuR was found to interact with circNUP54 by overlapping MS and target RBPs predicted using the RBP suite and RPBDP databases. E WB results verifying HuR enrichment using the circNUP54 probe. F RIP-qPCR showing precipitation of circNUP54 with anti-HuR. G Schematic representation of circNUP54 overexpression plasmids with predicted binding regions truncated (top panel). WB was used to detect HuR pulled-down by the circNUP54 probe in the lysates of Huh7 and SNU449 cells after transfection with wild-type or truncated circNUP54 overexpression plasmids (bottom panel). H Plasmids expressing FLAG-labeled FL or RRM-truncated HuR were transfected into circNUP54-overexpressing Huh7 cells, and then the RIP products pulled by anti-FLAG were analyzed by qRT-PCR to evaluate the enrichment efficiency of circNUP54. I 293T cells transfected with FL or RRM-truncated HuR plasmids were subjected to RNA pull-down using a circNUP54 probe. WB detected the pulled-down HuR by anti-FLAG. J, K HuR expression remained unchanged, as detected using RT-qPCR and WB after silencing circNUP54. Data presented as means ± SD of three independent experiments. ***p < 0.001, ****p < 0.0001 (Student’s t-test).