14 mm microwell

For all imaging experiments, embryos were collected for 1 h at 25°C, and then aged for 45 min to 1 h. Before imaging, embryos were dechorionated by hand, mounted on a strip of glue painted on a 35 mm glass bottom Petri dish with 14 mm micro-well (MatTek) and desiccated for 1 min at 25°C. Embryos were then covered with Voltalef^® oil (ARKEMA).

For embryo collections, 25% cranberry-raspberry juice plates (2% sucrose and 1.8% agar with a drop of yeast suspension) were used. Embryos for imaging experiments were collected for 1h at 25°C, and aged at 25°C for ∼45–60 min. Embryos were dechorionated by hand, mounted on a strip of glue on a 35-mm glass-bottom Petri dish with 14 mm micro-well (MatTek), and were left to desiccate for 1 min at 25°C. After desiccation, the embryos were covered with Voltalef grade H10S oil (Arkema). Embryos for dsRNA injection experiments were treated in the same way except that the desiccation period was increased to 5-6 min. Embryos were injected with dsRNA at a needle concentration of 0.6–0.8 mg/ml.

Cells were cultured in 35-mm glass-bottom dishes with a 14-mm microwell (MatTek). After 48 h, cells were stimulated with IFN-γ or in combination with TNF-α for 24 h. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Samples were incubated overnight at 4 °C with anti-PD-L1 (5H1) followed by goat anti-mouse secondary Alexa Fluor 488 (Thermo Scientific) and DAPI was used to stain the nucleus. Images were acquired with Zeiss Zen software using a Zeiss LSM 880 on a ×40 oil immersion objective lens.

Flies were kept at 25°C or 18°C on Drosophila culture medium (0.77% agar, 6.9% maize, 0.8% soya, 1.4% yeast, 6.9% malt, 1.9% molasses, 0.5% propionic acid, 0.03% ortho-phosphoric acid and 0.3% nipagin). Stocks were kept in 8 cm x 2.5 cm plastic vials or 0.25-pint plastic bottles. Embryos were collected on cranberry-raspberry juice plates (25% cranberry-raspberry juice, 2% sucrose and 1.8% agar) supplemented with fresh yeast. Standard fly handling techniques were employed (Roberts, 1998 ). In vivo studies were performed using 1.5–2 hr-old syncytial blastoderm stage embryos. After 0–1 hr collections at 25°C, embryos were aged at 25°C for 30–60 min. When injecting mRNA, embryos were collected for 20 min, injected, and imaged after 120–150 min at 21°C (but always within the syncytial blastoderm stage of development). Prior to injection or imaging, embryos were dechorionated on double-sided tape and mounted on a strip of glue onto a 35-mm glass-bottom petri dish with a 14 mm micro-well (MatTek). After desiccation for 1 min (non-injection experiments) or 3 min (pre-mRNA injection) at 25°C, embryos were covered in Voltalef oil (ARKEMA). Live imaging was performed using either the spinning disk confocal or the 3D-SIM systems described below.

HeLa-mCherry-EGFP-LC3 cells were seeded into the Glass Bottom Culture Dishes (35 mm petri dish, 14 mm Microwell, MatTek Corporation) 24 hours before the experiment. Cells were treated with 5 mM ammonium chloride for 2 hours at 37°C, 5% CO₂ incubator. Subsequently, cell culture medium was replaced with PBS alone or PBS containing 5 mM ammonium chloride to better visualize the cells. The live cells were then subjected to live cell imaging by the Leica microscope using the GFP and N2.1 channels.

Drosophila melanogaster w⁶⁷ flies (a wild-type line carrying a point mutation in the white gene) were used as a WT stock in all experiments and yw flies were used as the parental stock in the generation of transgenic lines. Balancer chromosomes and markers used have been described previously (Flybase, USA). Flies were kept at 25°C or 18°C on Drosophila culture medium (0.77% agar, 6.9% maize, 0.8% soya, 1.4% yeast, 6.9% malt, 1.9% molasses, 0.5% propionic acid, 0.03% ortho-phosphoric acid and 0.3% nipagin). Stocks were kept in 8 cm x 2.5 cm plastic vials or 0.25-pint plastic bottles. Embryos were collected on cranberry-raspberry juice plates (25% cranberry-raspberry juice, 2% sucrose and 1.8% agar) supplemented with fresh yeast. Standard fly handling techniques were employed (Roberts, 1998 ). In vivo studies were performed using 1.5-2 hr-old embryos (syncytial blastoderm stage). After 0-1 hr collections at 25°C, embryos were aged at 25°C for 1 hr. When injecting mRNA, embryos were collected for 30 min and aged for 1.5 hr after mRNA injection. Prior to injection or imaging, embryos were dechorionated by using double-sided tape onto a slide and mounted on a strip of glue onto a 35 mm glass bottom petri dish with a 14 mm micro-well (MatTek). After desiccation for 1 min at 25°C, embryos were covered in Voltalef oil (ARKEMA).