Wortmannin

Five day old Col-0 seedlings were incubated in 7.5 µM wortmannin (A. G. Scientific, USA) solution in deionised water at 22°C, constant light for 6 hr prior to death-inducing treatment. wortmannin stock (1 mM) was prepared in dimethyl sulfoxide (DMSO) and stored at −20°C. Solvent controls (0.075% DMSO v/v) were also prepared. Subsequent cell death inducing treatments and incubation prior to the root hair assay were also performed in the presence of 7.5 µM wortmannin/0.075% DMSO.

Embryos were collected every 3 h on apple juice agar plates, and incubated at 25°C for 24 h; hatching first instar larvae were transferred into 2.0 mL Eppendorf tubes with food containing 0.05% (v/v) ethanol (vehicle control), 1 μM rapamycin (AG Scientific), 0.2 or 2 μM Wortmannin (AG Scientific; R-1018) or 5 mM 3-methyladenine (Calbioche^® m, Merck Millipore, 189490), and dissected as third instar larvae. 10 μM Chloroquine was added to primary cultures for 2 h before fixation and staining with anti-mCherry. For Westerns, 5 third instar larval brains or 3 heads per treatment per lane were collected, frozen on dry ice, and ground with a pestle in RIPA lysis buffer (Thermo Fisher Scientific) with protease inhibitor cocktail. Rabbit anti-Bchs [for details see Lim and Kraut (2009) (link)] or Rabbit anti-pS6K (Cell Signaling) were diluted 1:6000 and 1:1000, respectively, incubated on blots overnight at 4C, and detected with HRP-coupled secondary and SuperSignal West Pico plus reagent (Thermo Fisher Scientific).