Fig. 3. ROS (reactive oxygen species) generation plays a vital role in AKR1C1-SQSTM1 interaction.

a Purified GST-AKR1C1 was incubated with or without 5BPSA for 30 min in 37 °C. Then NCI-H1299 cell lysates and GST beads were added into the mixtures of different groups and assessed by GST pulldown assay. b–e Cells were transfected with AKR1C1-HA or SQSTM1-Myc or their combinations and assessed by Myc IP and IB. b Cells were treated with or without 5BPSA for 10 h plus FBS starvation or not. c ROS was mimicked by 1 mM H₂O₂ in fresh DMEM medium for 2 h. d Antioxidants treatment for 1 h, including 1 mM NAC, GSH and VC were used for the rescue experiment. e Cells were treated with fresh medium (10% FBS and 90% DMEM) or with HBSS (hank’s balanced salt solution) for the indicated time.