Figure 5
Kaempferol, but not kaempferide, reduced the intracellular aggregates via the activation of autophagy. (A) Induction of LC-3-II by kaempferol, but not kaempferide. N2a cells were transfected with SOD1G85R, and then incubated with 3 µM kaempferol or 15 µM kaempferide for 24 h. The lysates were analyzed by immunoblotting with anti-LC-3 and anti-β-actin antibodies. (B) Relative levels of LC-3-II/LC-3-I normalized by the expression of β-actin were quantified, based on the band density of SOD1WT. (C–E) Immunoblotting analysis of autophagy. N2a cells expressing SOD1G85R were treated with kaempferol for 24 h with and without 3-MA. The lysates were analyzed by immunoblotting with antibodies for LC-3, p62 and β-actin. Relative levels of LC-3-II/LC-3-I and p62 normalized by the expression of β-actin were quantified, based on the band density of SOD1WT. (F) Representative fluorescent microscopy images of N2a cells expressing mCherry-SOD1G85R incubated for 24 h with 3 µM kaempferol with and without 3-MA. (G) Quantified data of intracellular SOD1 aggregates are expressed as mean ± S.E.M from three independent experiments. In each experiment, at least 200 cells were counted. (H) N2a cells expressing SOD1G85R was incubated for 24 h with 3 µM kaempferol with and without 3-MA. Cell viability was determined. Data is expressed as mean ± S.E.M from three independent experiments. *p < 0.05; **p < 0.01. ***p < 0.001.
