Figure 1.

Peucedanocoumarin IV (PCiv) inhibits β23 aggregations and cytotoxicity in SH-SY5Y cells. (A) Scheme showing the detailed experimental steps in the organic synthesis of racemic PCiv and racemic PCiii (rac-PCiii, and rac-PCiv). Both PCiii and PCiv used in this study are racemic forms. (B) Trypan blue exclusion cell viability assessment in SH-SY5Y cells with Tet-Off-induced β23 expressions. β23 was expressed for 24 h using the Tet-off system. Further expression of β23 was halted by doxycycline (Dox) treatment with simultaneous treatment of the PCiii, PCiv, or their synthetic intermediate, trans-khellatone (t-khellatone, 1 μM 24 h). Cell viability was assessed 24 h after doxycycline treatment (n = 5 per group). (C) Synthetic PCiv’s protective effect on Tet-Off-expressed β23 toxicity in SH-SY5Y cells compared with peucedanocoumarin III (PCiii) determined by trypan blue exclusion assay. (D) The degradation of β23 monitored by Western blot at 37 h after doxycycline (200 ng/mL) treatment. The representative Western blot image of HA (β23) in SH-SY5Y cells treated with synthetic PCiii, synthetic PCiv (1 μM, 37 h), or DMSO as a vehicle. (E) Quantification of HA (β23) expression in SH-SY5Y cells from the indicated experimental groups in panel D (n = 3 experiments per group). (F) Representative immunofluorescence of steady state HA (β23) expression in SH-SY5Y cells expressing Tet-Off regulatable reporter mCherry and HA-tagged β23. HA-β23 was expressed for 24 h using the Tet-off system. Further expression of β23 was halted by Dox treatment in the presence of PCiii or PCiv (1 μM, 37 h). (G) Quantification of HA (β23) immunofluorescence signal in mCherry positive SH-SY5Y cells in each experimental group (n = 32 cells from three separate experiments per group). The data are expressed as means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, ANOVA test followed by Tukey’s post hoc analysis. Two-way ANOVA was used in panel C.