Figure 4. DDRGK1 regulates the UPR by targeting IRE1α.

(a). MCF7 and HepG2 cells were transfected with either control siRNA or siRNA targeting IRE1α for 72 h. The protein levels of p-PERK, PERK, p-eIF2α and eIF2α were determined by western blot. (b). Western blot analysis of IRE1α in control and DDRGK1-knockdown MCF7 cells treated with or without MG132 (20 μM, 8 h). (c). Western blot analysis of IRE1α decay in control and DDRGK1-knockdown MCF7 cells after treatment with 100 μg ml⁻¹ cycloheximide for the indicated times. The graph represents the quantification of the IRE1α protein levels. (d) MCF7 cells were transfected with either control siRNA or siRNA targeting DDRGK1 for 72 h. Before harvesting, the DDRGK1-knockdown cells were transfected with either control or IRE1α vectors for 36 h. The cells were subsequently stained with Annexin V and PI and subjected to flow cytometric analysis, followed by quantification of apoptotic cells (Annexin V⁺). All data are presented as mean±s.d. from three experiments. *P<0.05 by Student's t-test. (e) Western blot analysis of IRE1α in MCF7 cells in d.