Fig. 2: Unique regulation of UPF3B in activating the UPR signaling pathway.

A The levels of phosphorylated IRE1α were evaluated following treatment with shRNA of UPF1, UPF2 and UPF3B in HEK293T cells, respectively. Negative control is the vector pLKO.1-TRC containing non-hairpin insert. The cells were then harvested and subjected to western blotting analysis, and the relative phosphorylation of IRE1α was statistically analyzed (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. B The expression levels of ER stress-related proteins were evaluated in overexpressed UPF3B cells. HEK293T cells were transfected with pCMV or pCMV-UPF3B for 24 h. The cells were then harvested and subjected to western blotting analysis. C–E Flow cytometry analysis apoptosis of shUPF3B and overexpressed UPF3B cells under Tg (2 μM for 3 h or 12 h) treatments. Data statistical analyses were performed on Annexin-V + /PI+ double positive cells and Annexin-V+ single positive cells (n = 3). UPF3B interacts with IRE1α. Co-IP analysis of the interaction between IRE1α and UPF3B in HEK293T (F) and U2OS cells (G). The cells lysates were subjected to immunoprecipitation (IP) and western blot analysis with the indicated antibodies, IgG was used as a negative control in IP assay. (H) Up: the localization of IRE1α and UPF3B was evaluated by an immunofluorescence assay. U2OS cells were fixed and stained with anti-Flag antibody (red), anti-Myc antibody (green) and DAPI (blue). Down: the localization of UPF3B and ER. U2OS cells were fixed and stained with anti-UPF3B antibody (green), ER tracker (red) and DAPI (blue). Scale bar, 10 μm. The results are the means ± SEMs of at three independent experiments. Statistical significance was defined as *p < 0.05, **p < 0.01 or ***p < 0.001.