Fig. 8.

circIL4R facilitates CRC progression through the circIL4R/miR-761/TRIM29/PHLPP1 axis. a and b CCK-8 and colony formation assays indicated that the inhibitory effect of circIL4R knockdown on the proliferation of HCT116 cells was reversed by TRIM29 overexpression, whereas the stimulatory effect of circIL4R overexpression on the proliferation of LoVo cells was reversed by TRIM29 knockdown. c and d Representative images and quantification of Transwell and wound healing assays showed that the inhibitory effect of circIL4R knockdown on the migration and invasion of HCT116 cells was reversed by TRIM29 overexpression, whereas the stimulatory effect of circIL4R overexpression on the migration and invasion of LoVo cells was reversed by TRIM29 knockdown. e Western blots showed that the reduction in p-AKT in HCT116 cells transfected with circIL4R siRNAs was reversed by TRIM29 overexpression, whereas the increase in p-AKT in LoVo cells transfected with circIL4R was reversed by TRIM29 knockdown. f The protein levels of PTEN, PP2A and PHLPP1 in CRC cells transfected with TRIM29 siRNAs or overexpression plasmids were detected by western blot. g The mRNA level of PHLPP1 in CRC cells transfected with TRIM29 siRNAs or overexpression plasmids was detected by qRT-PCR, respectively. h The protein level of p-AKT in HCT116 cells transfected with siTRIM29, either alone or with siPHLPP1 (left) and in LoVo cells transfected with the TRIM29 overexpression plasmid, either alone or with the PHLPP1 overexpression plasmid (right). i The interaction between TRIM29 and PHLPP1 in HCT116 cells was detected by immunoprecipitation and western blot assays. j The effect of TRIM29 overexpression on the half-life of PHLPP1 protein in HCT116 cells was detected by western blot. The cells were treated with 100 μg/ml CHX, a protein synthesis inhibitor, and harvested at the indicated time points. k HCT116 cells were treated with MG132 (10 μM), and harvested at the indicated times to measure total protein levels via western blot assays. The results showed that TRIM29 overexpression decreased the protein level of PHLPP1 in a proteasome-dependent manner. l The effect of TRIM29 overexpression or knockdown on the ubiquitination of PHLPP1 in HCT116 cells was assessed by western blot. *P < 0.05, **P < 0.01, ***P < 0.001