Facsymphony a3 analyzer

VDY 5844 (Zpr1-AID cells containing the 4xHSE-GFP reporter) were transformed with low-copy, TRP1-based vectors, either empty (EV) or carrying 3ˣFLAG-tagged wild-type (WT) Zpr1 or 3ˣFLAG-tagged Zpr1 mutants (Zpr1^Zn-N: I76A, I77A, I78A, M79A; Zpr1^Zn-C: V317A, I318A, I319A, M320A; Zpr1^Zn-NC: I76A, I77A, I78A, M79A, V317A, I318A, I319A, M320A; Zpr1^aH-C: E391A, E399A; Zpr1^aH-NC: E148A, D156A, E391A, E399A). Cells were grown to saturation in SD-Trp media. Cells were next back-diluted into SD-Trp media containing 1 μM beta-estradiol and grown for ~2–3 hours until an OD600 ~0.3–0.4 was reached. Cells were diluted in half into SD-Trp media supplemented with 1 μM beta-estradiol and DMSO or 5 μM 5-Ph-IAA and grown for 2 hours. Cycloheximide was added to a final concentration of 50 μg/mL for 1 hour to arrest translation. Samples were measured using a FACSymphony A3 analyzer (BD Biosciences) using the 488 nm laser (FITC). 20,000 cells were measured for each sample and medians/histograms were collected using Bioconductor packages flowCore and flowViz.

VDY6188 was grown overnight to saturation in SC + 2% galactose media. Cells were back-diluted to an OD600 of 0.1 and grown for 4 hours (in the presence of 1 μM beta-estradiol) until an OD600 ~0.5 was reached. 0.1 OD600 units were spun down and resuspended in either SC + 2% galactose or SC + 2% glucose (for transcriptional shutoff of the TEF1/2 loci) and grown for 1 hour in a thermomixer set at 30°C and mixing at 1400 RPM. After 1 hour, cells from each sample (in either galactose-containing media or glucose-containing media) were split into three different tubes and fresh media was added to reach a final volume of 1 mL. Each tube either received DMSO, 5-Ph-IAA (5 μM final concentration), or was grown in a thermomixer set at 39°C (to induce heat shock) with shaking (1400 RPM). Samples with DMSO or 5-Ph-IAA were grown at 30°C in a thermomixer shaking at 1400 RPM. At the indicated time points, 200 μL of cells were aliquoted into a second tube and cycloheximide was added to a final concentration of 50 μg/mL to arrest translation. Aliquoted samples were grown for 1 hour at 30°C for fluorophore maturation and subsequently measured using a FACSymphony A3 analyzer (BD Biosciences) using the 488 nm laser (FITC). 10,000 cells were measured for each sample and medians/histograms were collected using Bioconductor packages flowCore and flowViz.