S1876

Separately, solutions containing 5% sucrose (S9378, Sigma), 1.125% sodium chloride (S3014, Sigma), 6.25% sorbitol (S1876, Sigma), 1% Tween 80 (BP338, Fisher Scientific), and 2% gelatin (G1393, Sigma) were prepared (w/v) in deionized water. A bivalent mixture of measles and rubella virus was prepared and combined with each vaccine-relevant matrix, resulting in samples of virus plus 0.38% sucrose, 0.90% sodium chloride, 5% sorbitol, 0.125% Tween 80, or 1.60% gelatin. A control was prepared by diluting in PBS (P3813, Sigma). Each sample was lysed using MR Lysis Buffer for 30 min, further diluted in MR Blocking Buffer to 6x LLOQ, with each added to 4 replicate arrays. A standard curve was prepared by serially diluting the PBS-control sample in MR Blocking Buffer after lysis. All slides were processed and imaged as described previously, and samples were quantified against the relevant standard curve and the average concentration of each virus in each sample determined. A student’s T-test evaluated the statistical significance of each sample relative to the control.

To induce SGs, arsenite (S7400; Sigma-Aldrich; 500 µM), sorbitol (S1876; Sigma-Aldrich; 0.5 M), or hippuristanol (1 µM [Bordeleau et al., 2006 (link)]) was added, and the cells were incubated at 37°C for 1 h. To allow for faster stress recovery, cells were incubated with 100 µM arsenite for 1 h. For ribopuromycinylation assays, cells were incubated with puromycin (P8833; Sigma-Aldrich; 10 mg/ml) at 37°C for 5 min prior to fixation.

A. fumigatus (Fresenius, 1863) strain AF293 [13 (link)] served as the wild type strain in this study. The racA null mutant (ΔracA strain; ΔracA::Aspergillus parasiticus pyrG pyrG1) was generated in our previous study [9 (link)]. Since the racA complement strain exhibited a normal phenotype with respect to conidiation [9 (link)], genetically demonstrating that the conidiation defects in the ΔracA strain were due to deletion of the racA gene, we didn’t include the complement strain in this study. All fungal strains were maintained on glucose minimal media (GMM, 1% glucose) plates including 1.5% (w/v) agar as previous described [14 (link), 15 (link)]. Fungal colonies usually were maintained in the dark at 30°C, but were also grown at 25°C or 37°C for temperature shifting test(s). Osmotic stress was created by amending GMM with a sorbitol solution (Sigma, S-1876) to a final concentration of 1 M. Carbon starvation stress was induced by subtracting glucose from GMM. Plates were placed in the incubator upside down to prevent condensation on the lid and accumulation of carbon dioxide in the culture, which may affect conidiogenesis.