Fig. 3.

CircXRN2 prevents LATS1 from SPOP-mediated degradation by binding to its degron sequence. a We contrasted different fragments of the LATS1 protein to test the exact region interacting with circXRN2. A schematic illustration of the depletion map of LATS1 protein. b Fragment 2 and full-length LATS1 protein interacted with circXRN2, as determined by RIP assay in 293T cells. c, d CircXRN2 enhanced the expression level of LATS1 at the protein level but not the mRNA level. e Cycloheximide (CHX) was used to block protein synthesis. LATS1 protein levels were detected by western blotting in control and circXRN2-overexpressing bladder cancer cells. f Downregulation of LATS1 protein induced by circXRN2 knockdown was reversed by treatment with bortezomib (500 nM, an inhibitor of the proteasome). g Cells were pretreated with bortezomib (500 nM) and NEM (10 μM, an inhibitor of deubiquitinating enzyme) for 8 h. The ubiquitinated LATS1 protein levels in the control group and circXRN2-overexpressing group were detected by IP assay. h A mass spectrometer was used to detect the proteins interacting with LATS1. i LATS1 protein levels were detected by immunoblot analysis in cells transfected with incremental doses of SPOP plasmid. j Downregulation of LATS1 protein could be rescued by depletion of SPOP. k SPOP increased the ubiquitination of LATS1 protein in T24 and TCCSUP cells. l Schematic illustration of the sequence alignment of the LATS1 protein with the SPOP-binding degron among known substrates. m Flag-LATS1-containing wild-type or mutant SBCs and HA-SPOP were transfected into 293T cells. Western blotting indicated that mutation of SBC1 led to remarkable blockade of LATS1 degradation mediated by SPOP, while depletion of SBC2 had little effect. n Co-IP results showed that wild-type LATS1 could bind to SPOP, but the interaction of SBC1-mutant LATS1 with SPOP was almost completely diminished. Western blotting was performed to determine the expression levels of Flag-LATS1 and HA-SPOP. o The interactions between LATS1 and SPOP in control and circXRN2 knockdown cells were verified by Co-IP assay. All the data are presented as the mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01