Fig. 6: USP8 maintains YAP protein stability by interacting with YAP in TNBC.

A Immunofluorescence staining showed the localization of USP8 (green) and YAP (red) in BT549 cells. DAPI (blue) was used to stain the nuclei, and the scale bars indicate 10 μm. B An endogenous association between USP8 and YAP was demonstrated through immunoprecipitation analysis. To investigate endogenous interaction between USP8 and YAP, BT549 cell lysates were precipitated using anti-YAP or anti-USP8 antibodies, and the resulting precipitate was analyzed through immunoblotting. C Diagram illustrating the protein structure of UPS8 and its deletion mutants (residues 1–313, 314–714, 715–1118) utilized in the co-immunoprecipitation assay. D A schematic diagram of the YAP protein structure and its deletion mutants (residues 1–171, 1–292, 172–504, 293–504) used for the CO-IP assay. E Co-immunoprecipitation revealed that the interaction of the WW domain of YAP with USP8. F Co-immunoprecipitation demonstrated that YAP interaction necessitates the USP domain of USP8. G Surface diagram of the docking model and their interfacing residues between USP8 and YAP protein (YAP, blue; USP8, yellow; hydrogen bond interaction, dotted line). H The two sites of USP8 are mutated to alanine, and the table shows the predicted binding sites of the two proteins. I, L Western blot analysis showed that the expression level of YAP protein in BT549 and MDA-MB-231 cells. Cells were transfected with siControl or siUSP8 and then treated with MG132. J, K, M, N Western blotting was used to detect the protein levels of USP8 and YAP. For the specified time, BT549 and MDA-MB-231 cells received treatment with 100 μM cyclohexylamine (CHX). The YAP protein expression was measured with ImageJ software and illustrated in the right panel. O, P Evaluation of YAP half-life in BT549 cells following transfection with the specified plasmids. The right panel graphically displays the YAP protein expression as estimated by ImageJ software.