Fig. 4

JUN as a downstream target of RRP9 in BC. A, Hierarchical clustering analysis of differentially expressed genes according to RNA-Seq. B-C, mRNA and protein expression levels of JUN in the shCtrl and shRRP9 groups were assessed by qRT‒PCR and Western blot, respectively. D, Proliferation assays were performed to further confirm the expression of candidate genes. E, Correlation between RRP9 expression and JUN expression in patients with BC. F, Co-IP assay confirmed the interaction between RRP9 and JUN. G, Decrease of JUN stability by RRP9 knockdown demonstrated by CHX assay. H, Acceleration of JUN degradation by RRP9 knockdown. I, Ubiquitination assay showed that JUN ubiquitination was increased by RRP9 depletion. J, The E3 ubiquitin ligases of JUN were predicted with UbiBrowser (http://ubibrowser.ncpsb.org.cn/ubibrowser/). The query substrate is positioned at the centre of the canvas, which is surrounded by the predicted E3 ligases. The colours and labels of the nodes reflect the type of E3 ligase. The edge width, node size, and edge shade are adjusted based on the confidence score. The node size, edge colour intensity, and edge width are proportionate to the confidence score. R denotes RING, H denotes HECT, F denotes F-box, U denotes UBOX, and S denotes SOCS. K-L, co-IP assays was performed to determine the specific interactions of MDM2 with RRP9 and JUN. M, Western blot was used to assess JUN levels after 0, 4, and 8 h of CHX treatment in modified MDA-MB-231 cells. N, Cells were treated with MG132 to inhibit the proteasome, after which the protein level of JUN was detected. O, Western blot analysis of the endogenous ubiquitination of JUN in MDA-MB-231 cells upon MDM2 overexpression. *P < 0.05, **P < 0.01 and ***P < 0.001