Quercetin activates the cell wall integrity signaling pathway.
(A) Phospho-Slt2 protein levels. S. cerevisiae BY4741 cells were grown to exponential phase and treated with 300 µM of quercetin or equal volume of DMSO for the indicated time periods. Proteins were isolated, separated by SDS-PAGE, blotted into a membrane and incubated with anti-phospho-p44/42 antibody that detects dually phosphorylated Slt2p or with anti-actin antibody (loading control), as described in Materials and Methods. A representative blot is shown. (B) Rlm1p-transcription factor activity. Exponentially growing S. cerevisiae BY4741 cells transformed with a pRLM1-LacZ reporter construct were treated with 300 µM of quercetin or equal volume of DMSO (control) at the indicated times. β-galactosidase activity was determined as described in Materials and Methods and expressed as percentage of control cells. (C) Resistance to zymolyase digestion. S. cerevisiae BY4741 cells were treated with 300 µM of quercetin or equal volume of DMSO for 15 min and incubated with 0.25 U/ml zymolyase at 37°C. Cell lysis was determined spectrophotometrically at 600 nm over time. (D) Hydrogen peroxide resistance of slt2Δ cells was assessed as described in legend to Figure 4D. Values are mean ± SD of at least three independent assays. **p<0.01; *p<0.05 (unpaired t-test).