Figure 6. Differential expression of several proteins influenced by quercetin treatment.

SHSY5Y cells were treated with different concentrations of quercetin for 16 hr and protein expression levels in total cell lysates were analyzed. (A) Representative immunoblots for differentially expressed proteins. (B) Quantitative analyses of the protein expression levels. The density of each band was normalized to the corresponding actin level and expressed as the ratio to control (no quercetin: DMSO alone). Data represent the mean ± standard deviation of at least three independent experiments. ***p<0.001, **p<0.01, *p<0.05 compared to the DMSO alone control via a one way ANOVA, followed by Dunnett’s post-hoc test. (C) HIF-1α is SUMOylated after quercetin-treatment. Lysates from SHSY5Y cells treated either with DMSO alone or quercetin at 20 μM for 16 hr were immunoprecipitated with anti-HIF-1α antibody followed by immunoblotting using anti-HIF1α, anti-SUMO-1 or anti-SUMO-2,3 antibodies. Left panel shows input and right panel shows immunoprecipitated products. Arrow shows the predicted HIF-1α band. (D) SENP3 depletion (reduction) by SENP3 shRNA down regulates HIF-1α expression in SHSY5Y cells. SHSY5Y cells were infected with SENP3/shRNA lentiviral particles, and selected under the pressure of puromycin (2 μg/ml). After established stable lines, the SENP3 and HIF-1α protein levels in the total cell lysates were analyzed by the Western blot. Actin was used as a loading control. Left, immunoblots; right, quantitative analysis. (E) SENP3-depleted cells were more resistant to OGD and no additional protection by quercetin was afforded in these cells. SHSY5Y cells with or without SENP3 depletion were subjected to OGD with or without quercetin (20 μM) for 16 hr, and LDH levels (released and total) were measured. Released LDH were shown as percentage of the total LDH.