Assaying VPS8D knockdown phenotypes
To measure survival rates after hypoosmotic shock, wildtype andVPS8D -KD strains (3 of each, with independent isolates of the
mutant strain) were grown to log phase in 10 ml SPP. Cell densities were
measured and equalized, and 0.5ml aliquots were diluted 1:100 in 50 ml
SPP medium with or without 1 µg/ml CdCl2. Cells were
grown for 16 hours at 30°C with agitation. Culture densities were
measured, and then cells were pelleted by centrifugation in a clinical
centrifuge for 1 minute and resuspended gently in 10 mM Tris-HCl buffer,
pH 7.4. After an overnight incubation, culture densities were once again
measured to calculate survival rates. For experiments monitoring changes
in CVC markers, the VPS8D -KD strains endogenously expressing
Dop1p-mNeon, Vma4p-mNeon or Scr7p-mNeon were grown as above in
non-inducing or inducing conditions. Cells were withdrawn and fixed for
imaging after 3, 5 and 8 (Fig. 2) or 16h (Fig. 3).