Extended Data Fig. 6: The binding of PDNPA to Nur77 generated hindrance to impede the interaction between Nur77 and Akt.
(a) PDNPA inhibited TGF-β-mediated induction of a myofibroblast-like morphology (top) and α-SMA expression (bottom) in LX-2 cells in a Nur77-dependent manner. Scale bar, 100 μm, 3 times experiments were repeated independently with similar results. (b) Comparison of the effect of PDNPA and Csn-B on TGF-β-induced HK1 palmitoylation and secretion. TGF-β (2 ng/mL), PDNPA (10 μM) or Csn-B (10 μM) were used to treat LX-2 cells for 36 hours. (c) Effect of PDNPA on Nur77 expression and HK1 secretion detected in primary HSCs. (d) Nur77 protein levels were detected in primary HSCs and hepatocytes isolated from control mice and CCl4-induced liver fibrosis mice treated with or without PDNPA. (e) PDNPA had no effect on TGF-β-induced Akt activity. (f) Detection of the interaction between the Nur77 LBD and Akt in the presence of PDNPA upon transfection in LX-2 cells (left) and in an in vitro GST pulldown assay (right). (g) A docking model showing that Akt and p38 bind the same pocket of the Nur77 LBD. (h) Crystal structures of the S441W and L437W D594E mutants shows that the introduction of bulkier residues prevents PDNPA binding. (i) A docking model indicates that F395A, D481A, T564A and T567A impair the interaction between the Nur77 LBD and Akt. Tubulin was used as a protein loading control. All western blots are repeated three times and one of them is shown.
