Figure 1. BRG1 is repressed by Nrg1p and its over-expression leads to increased invasion of solid medium.

A. Quantitative real-time PCR analysis identified BRG1 as a target of Nrg1p-mediated repression. Expression of BRG1 is derepressed in the nrg1Δ strain (BCa23-3) compared to a wild-type strain (SC5314) in yeast conditions (YPD 28°C 6 h, left panel). Conversely, BRG1 expression is repressed when NRG1 is over-expressed in our tet-NRG1 strain (SSY50-B, No DOX) in hypha-inducing conditions (RPMI-1640 37°C 3 h, middle panel). Levels were normalized by ACT1 and expressed relative to the wild-type strain (left panel) or to the doxycycline-containing culture (right panel) therefore those results are 1.0. Error bars represent the standard error of the mean.

B. Cells from an overnight culture were washed in sterile PBS, diluted 1:20 into fresh, pre-warmed YPD+10% FBS and incubated with shaking at 37°C for 3 h. The brg1Δ strain (ICY326) fails to form hyphae.

C. A copy of BRG1 under the control of the tetO promoter was integrated at the RPS1 locus in a wild-type background (THE1-CIp10) and in our tet-NRG1 strain (SSY50-B) to construct strains ICY171 and ICY175, respectively. Cells were streaked on YPD plates with or without doxycycline (DOX) and the plates incubated at 30°C for 48 hours. The plates were photographed prior to (upper panels) and after (lower panels) surface cells were removed by gentle washing under running water. Cells which grew solely on the surface of the medium washed away while cells that had invaded the agar remained.

D. Cells from an overnight culture were washed in sterile PBS, counted and spotted onto solid Lee medium (buffered to pH 4 or 7) in the presence or absence of doxycycline and incubated at 30°C or 37°C for 2 days. A wrinkled colony morphology indicates the presence of filamentous cells.