Fig. 6

circPSD3 interacts with HDAC1 to inhibit its nuclear translocation. (A) Schematic of the TRAP assay. (B) Relative expression levels of circPSD3 and GST in HCC-LM9 cells co-transfected with the circPSD3-MS2 and GST-MS2 vectors. (C) Venn diagram of circPSD3-interacting proteins, as identified by TRAP assay and LC-MS/MS. (D) HDAC1-specific peptide sequence identified using LC-MS/MS. (E) TRAP assay and western blotting analysis of the interaction between circPSD3 and HDAC1. (F) RIP assay of the enrichment of anti-HDAC1 on circPSD3 in HCC cells. (G) FISH and immunofluorescence analysis of the co-localisation of circPSD3 and HDAC1 in the cytoplasm of HCC cells. Scale bar = 20 μm; Scale bar (zoom) = 10 μm. (H) Relative expression levels of circPSD3 in HCC cells transfected with HDAC1 siRNA. (I) Schematic of the circPSD3 binding site on HDAC1 and FLAG-tagged HDAC1 truncated mutants. (J) Western blotting analysis of the protein expression of the negative control, wild-type, and truncated HDAC1 mutants in lysates of HEK-293T cells transfected with the indicated vectors. (K) RIP analysis of the enrichment of the negative control, wild-type, and truncated HDAC1 mutants on circPSD3 in HEK-293T cells. (L) HDAC1 nuclear localisation signal predicted using DeepLoc-1.0 software (upper) and circPSD3 binding site on HDAC1 (lower). (M) Immunofluorescence analysis of the subcellular localisation of HDAC1 in circPSD3 knockdown and control HCC cells. Scale bar = 20 μm. (N) Western blotting analysis of HDAC1 expression in the nuclear (N) and cytoplasmic (C) fractions of circPSD3 knockdown and control HCC cells. Data are shown as the mean ± SD. Statistical analyses were performed using unpaired Student’s t-tests (NS, no statistical significance; **p < 0.01; ***p < 0.001). circPSD3, circRNA pleckstrin and Sect. 7 domain containing 3; FISH, fluorescence in situ hybridisation; HCC, hepatocellular carcinoma; HDAC1, histone deacetylase 1; LC-MS/MS, liquid chromatography–tandem mass spectrometry; RIP, RNA immunoprecipitation; TRAP, MS2-tagged RNA affinity purification; siRNA, small interfering RNA