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. 2023 Dec 18;14(12):839. doi: 10.1038/s41419-023-06300-2

Fig. 4. STUB1 interacts with GPX4 to promote GPX4 ubiquitination in gastrointestinal stromal tumor (GIST).

Fig. 4

A Localization of STUB1 (green) and GPX4 (red) in GIST-T1 and 882 cells by confocal laser scanning immunofluorescence. The nucleus is labeled by DAPI (blue). Colocalization of STUB1 (green) and GPX4 (red) in cells appears yellow in the merged images. B Western blot analysis of STUB1 in GIST-T1 and 882 cells transfected with STUB1 overexpression plasmids or control plasmid. C Western blotting was performed to assess the level of GPX4 in GIST-T1 cells transfected with GPX4 overexpression plasmids or control plasmid, and treated with IM (50 nM for T1 and 200 nM for 882) in the absence or presence of MG132 (5 μM) for 24 h. D STUB1 overexpression inhibited the stability of the GPX4 protein in GIST-T1 cells. The GPX4 protein levels were analyzed by western blotting after the cells were treated with CHX (100 μg/ml) for 0, 2, 4 or 8 h. E STUB1 overexpression promoted the ubiquitination of GPX4 protein in GIST-T1 cells. The ubiquitination of GPX4 protein was measured by CO-IP using an anti-ubiquitin antibody. F Direct interaction between STUB1 and GPX4 in GIST-T1 and 882 cells. Protein-protein interactions in GIST-T1 and 882 cells were validated by a CO-IP assay using an anti-STUB1 and an anti-GPX4 antibody. An antibody targeting IgG served as the negative control. G The representative images of proximity ligation assay (PLA). GIST-T1 and 882 cells were treated with IM (50 nM for T1 and 200 nM for 882) or a control vehicle for 24 h and then incubated with the anti-STUB1 and anti-GPX4 antibodies and detected by DuoLink probe (Olink Bioscience, Uppsala, Sweden). MG132 (5 μM) was added for stabilizing GPX4. The red dots indicate the positive PLA signal. H The prediction of the lysine site of ubiquitination of GPX4. I The effects of the wild-type and different mutation sites of GPX4 on ubiquitin modification. J The effect of STUB1 on the ubiquitination modification of the K191 site of GPX4. K Computational molecular docking simulation analyses of STUB1 and GPX4. Scale bar, 20 μm in A, 10 μm in G. Experiments were independently repeated three times. Significance denoted by: ns not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.