Fig. 4. PRX2 inhibits 6-OHDA-induced activation of ASK1 and JNK/p38 signaling pathways in MN9D cells.

(A) Neuronal differentiated MN9D cells were infected with Lenti-PRX2, the empty vector (Lenti), or Lenti-GFP (not shown) for 3 days, and then challenged with 6-OHDA (50 µM). At the indicated time points, cell extracts were subjected to immunoblotting against p-JNK, total JNK (t-JNK), p-p38, or total p38 (t-p38). The fold changes of p-JNK and p-p38 over the vehicle controls are illustrated in the graphs. *p<0.05 vs. empty vector control (Lenti); data are from 3 experiments. (B) MN9D cells were infected for 3 days with lentiviral vectors containing shRNA targeting PRX2 (PRX2t) or the scramble control sequence (PRX2sc), or the empty vector (Lenti), and immunoblotting was performed at 2 and 8 h after 6-OHDA (50 µM). The fold changes of p-JNK and p-p38 over the vehicle controls are illustrated in the graphs. *p<0.05 vs. empty vector control (Lenti) or PRX2sc-infected cells; data are from 3 experiments. (C) 6-OHDA induces ASK1 phosphorylation in MN9D cells in a time-dependent manner. The graph illustrates the temporal profile of p-ASK1 increases after 6-OHDA (50 µM) exposure. *p<0.05; **p<0.01 vs. vehicle control; data are from 3 experiments. (D) Under the experimental conditions described in (A), cell extracts were processed for immunoblotting against p-ASK1 or ASK1 kinase activity assay. The fold changes of p-ASK1 and ASK1 activity over vehicle control are illustrated in the graphs. *p<0.05 vs. Lenti-GFP; data are from 3 experiments. (E) MN9D cells were infected for 3 days with Lenti-GFP or both Lenti-GFP and Lenti-PRX2, and then challenged with 6-OHDA (50 µM). Representative triple-label immunofluorescent images show increased cytosolic p-ASK1 (b, red; b1, yellow, merged with GFP) and nuclear p-c-Jun (e, red; e1, pink, merged with DAPI) immunofluorescence after 6-OHDA; infection with Lenti-PRX2 abolishes the p-ASK1 (c and c1) and p-c-Jun (f and f1) immunofluorescence after 6-OHDA.