Fig. 2: PA binds to PRDX1 and inhibits its peroxidase activity.

a Global PRDX peroxidase activity in HepG2 cells after PA (250 μM) treatment for 1 hr. Veh, vehicle. PA, palmitic acid. n = 5 biologically independent samples. b ROS levels in HepG2 cells after PA treatment for 1 hr. Intracellular ROS were monitored by measuring the fluorescent intensity of dichlorofluorescin (DCF) using a fluorometer. n = 4 independent experiments. c Representative images of HKPerox-Red staining in HepG2 cells after treatment with Veh or PA at different concentrations for 1 hr. Arrows denote the signals of HKPerox-Red staining. n = 5 independent experiments. Scale bar, 50 μm. d Quantification of fluorescence intensity of images from c. n = 5 independent experiments. e Binding affinity of sodium palmitate on recombinant PRDX1 determined by SPR assay. Data were calculated from three independent experiments. f Representative images showing the increased thermal stabilization of PRDX1 after binding to PA and quantification. n = 3 independent experiments. HepG2 cells were treated with PA (250 μM) for 1 hr and then the cell lysate was collected for the thermal shift assay. In brief, the cell lysate was divided into six aliquots. One aliquot was used for input control and the other five aliquots were heated at different temperatures as indicated for 3 min. Finally, western blotting was carried out to detect PRDX1 stability. g Inhibition of recombinant WT PRDX1’s peroxidase activity by sodium palmitate at different concentrations as indicated. For more details, please see methods section. Data were calculated from three independent experiments. All the data are presented as means ± SEM. Unpaired and two-tailed Student’s t test was performed for a, b, d, and f.