Fig. 5
STAT3 stabilises TGFβR1 protein expression by promoting the transcription of USP15 (A, B) Immunoblotting and RT‒qPCR analysis were performed to examine the protein or mRNA expression levels of core components of the TGF-β pathway in Huh-7 cells. (C) Immunoblotting analysis was performed to examine the protein expression levels of STAT3 and p-STAT3 in Huh-7 cells. (D) Huh-7 cells were transfected with shNC or shRNA targeting STAT3. After 48 h, the cells were treated with the proteasome inhibitor MG132 (10 μM, 6 h) or the lysosome inhibitor CQ (20 μM, 6 h) or 3-MA (4 mM, 6 h). Immunoblotting analysis was used to examine TGFβR1 expression. (E) Huh-7 cells transfected with the indicated plasmids were treated with CHX (50 μg/ml) for the indicated time intervals. The cells were harvested, and the expression of TGFβR1 was measured by immunoblotting. The level of protein expression was quantified by densitometry and plotted. β-actin was used as the internal reference, and the protein expression of TGFβR1 was normalised to that at the t = 0 time point. (F, G) Ubiquitination assays were performed in Huh-7 cells overexpressing TGFβR1 (His-tagged), Ub (Myc-tagged), and shSTAT3/Flag-STAT3 in Huh-7 cells. After 48 h, the cells were treated with MG132 (10 µM) for 6 h, the cell lysates were collected, and immunoprecipitation was performed to analyse the ubiquitination of TGFβR1. (H) STAT3 expression in liver cancer was positively correlated with USP15 expression according to the analysis of the GEPIA online dataset. (I-J) RT‒qPCR and immunoblotting analyses were performed to examine the mRNA or protein expression levels of DUBs. (K) JASPAR was used to identify STAT3-binding sites in the transcriptional regulatory region of USP15, and chromatin immunoprecipitation analysis was conducted to elucidate the binding of STAT3 to the transcriptional regulatory sequences of USP15. (L–M) Huh-7 cells were cotransfected with Ub-Myc or shUSP15/USP15-HA/USP15C298A and treated with MG132 (10 µM) for 6 h after 48 h of transfection. Immunoprecipitation was performed to analyse the ubiquitination of TGFβR1. (N) HEK293T cells were cotransfected with the indicated plasmids and treated with MG132 (10 µM) for 6 h after 48 h of transfection. Immunoprecipitation was performed to analyse the ubiquitination of TGFβR1. The data are presented as the mean ± SDs. ****p < 0.0001.
