Author response image 1. Physiological effects of ART2 deletion.
(A) Growth of isogenic WT (art2Δ + pART2) and art2Δ cells in synthetic auxotrophy medium (YNB) at 26°C. Cultures were grown exponentially for 24 hours and then inoculated to OD600nm = 0.1 at t=0h. Culture density was followed by OD600nm. Mean ± standard deviation from n = 3 independent experiments. (B) Mean cell diameter (± standard deviation) measured with CASY TTT cell counter (Omni Life Sciences, Bremen, Germany) of isogenic WT (art2Δ + pART2) and art2Δ cells grown exponentially for >24 hours and then starved for amino acids and nitrogen for the indicated time. (C) Isogenic WT (art2Δ + pART2), art2Δ, atg8Δ (art2Δ, atg8Δ + pART2) and art2Δatg8Δ cells grown exponentially for >24 hours and then starved for amino acids and nitrogen for the indicated time. Cells were stained with propidium iodide (PI, 3 μg/ml) for ten minutes, washed, and PI-positive cells were counted on an Attune TM NxT Cytometer (Life Technologies). 50000 cells were counted for each genotype and replicate. WT and art2Δ: mean ± standard deviation from n = 3 independent experiments. For the atg8Δ and art2Δatg8Δ cells two independent experiments are shown. (D) Isogenic WT (art2Δ + pART2), art2Δ, atg8Δ (art2Δatg8Δ + pART2) and art2Δatg8Δ cells were grown exponentially for >24 hours and then starved for amino acids and nitrogen (-N) for the indicated time. Survival was assessed by colony forming ability. Serial dilutions of the cultures were spotted on YPD rich medium. (E) Isogenic WT (art2Δ + pART2), art2Δ, atg8Δ (art2Δatg8Δ + pART2) and art2Δatg8Δ cells were grown exponentially for >24 hours and then starved for amino acids and nitrogen (-N) for 21 (upper panel) and 15 days (lower panel). Starved cells were re-inoculated into fresh auxotrophic selection medium (YNB) to OD600nm = 0.1 at t = 0h. Culture density was followed by OD600nm.