Fig. 4

circRHOT1 associates with TIP60. a A specific biotin-labeled circRHOT1 probe was added to HCC lysates, and the precipitated proteins were resolved by SDS-PAGE, followed by silver staining. Next, the differential band in the circRHOT1 probe lane was identified by mass spectrometry (MS). The anti-sense of circRHOT1 serves as control. As shown, TIP60 was identified. b RNA pulldown was performed to verify the interaction between TIP60 and circRHOT1 using TIP60-overexpressing HCC cells. Biotin-labeled circRHOT1 or anti-sense was used to precipitate Flag-TIP60. c The specific biotin-labeled circRHOT1 probe (5′-TCTTCTGCCCGGGGAGGAACCTGCTGTCTTTGTCTGTTCTTTCAT-3′) did not precipitate TIP60 in circRHOT1-knockout HCC cells. d RNA IP results indicated that TIP60 enriched circRHOT1 in HCC cells. e CircRHOT1 co-localized with TIP60 in HCC cells as shown by RNA fluorescence in situ hybridization (FISH). Scale bar, 10 μm. f RNA EMSA assay indicated that TIP60 interacts with circRHOT1 directly. Linear circRHOT1 was labeled with biotin. All the data are representative of at least three independent experiments and presented as the means ± SD