Azd8055

About 30–40 seeds, that were imbibed as described above, were sown in 3 ml 0.5x MS in petri dishes of 35 mm diameter. Plants were kept in darkness for three days after the induction of germination by 6 hr light treatment. The medium of three day old etiolated seedlings was supplemented with 15 mM glucose and/or 0.5–2 µM AZD-8055 (Santa Cruz Biotechnology, Dallas, TX). Stock solutions of 1000x concentrated AZD-8055 were diluted in DMSO, therefore control plants were mock treated with the same volume of DMSO.

Protoplasts were isolated from leaves of four-weeks-old plants expressing GFP-ATG8 in atg5-1 using the “Tape-Arabidopsis Sandwich” method described in [71 (link)]. The isolated protoplasts were transformed using 10–20 µg of each plasmid (Table S4) using the method described in [72 (link)]. The transfected protoplasts were incubated in 24-well glass bottom plates (VWR CORN4441) for 16 h in light. The protoplasts were further treated with 5 µM AZD-8055 (364424, Santa-Cruz Biotech, Dallas TX, USA) and 0.5 µM Concanamycin A (202111A, Santa-Cruz Biotech) for 24 h, where applicable. The transformed protoplasts were imaged using CLSM800 (Carl Zeiss AG, Oberkochen BW, Germany), objective C-Apochromat 40×/1.2 W, excitation light 488 nm and 561nm and emission ranges of (515–560 nm) and (570–650 nm) for GFP and TagRFP, respectively. Images were analyzed using ZEN blue software (Carl Zeiss).