Attovision software

The BD Pathway 855 High Content Bioimager (BD Biosciences; San Jose, CA) was used for automated image acquisition. Acquired images were processed using AttoVision software (BD Biosciences; San Jose, CA). The Bioimager was equipped with a 488/10 nm enhanced GFP (EGFP) excitation filter, a 380/10 nm DAPI excitation filter, a 515LP nm EGFP emission filter, and a 435LP nm DAPI emission filter. Images were acquired in the DAPI and GFP channels of each well using a 10 X dry objective. The plates were exposed 0.066 ms (Gain 0) to acquire DAPI images and 0.85 ms (Gain 30) for GFP images. Cells were stained with DAPI to facilitate microscope autofocus and to aid in the image segmentation. An image algorithm based on a local threshold was applied to allow for the cell nucleus segmentation. Our segmentation strategy assumes that the cell’s cytoplasm surrounds the nucleus. Consequently, cytoplasmic fluorescence intensity is calculated from all the pixels within a circumferential ring surrounding the nuclear ring mask. The width of the ring was defined to be small enough to avoid ambiguities due to irregular cell shape. Based on the definition of cell compartments, the nuclear and cytoplasmic levels of GFP fluorescence were quantified.

Cells (1 × 10⁵ cells/well) were seeded on a slide and cultured for 24 hr. The cells were treated with DMSO, rapamycin or honokiol, and then fixed in methanol for 20 min. The slides were incubated for 30 min. in 0.1% Triton X‐100 in phosphate‐buffered saline. Anti‐LC3 polyclonal antibody (Medical & Biological Laboratories, Naka‐ku, Nagoya, Japan) was added on the slide and left overnight at 4°C. The fluorescent change in the cells was captured and analysed by high‐content image analyzer, BD pathway 435 (BD Biosciences, San Jose, CA). The percentage of cells with LC3 puncta formation and the average number of LC3 puncta per cell were analysed by BD Attovision software (BD Biosciences).

The GFP-p65 and TNF-α promoter-mCherry reporters were imaged in the RAW G9 reporter cell clone using a BD Pathway 855 bioimager (BD biosciences). BD AttoVision software was used to automatically identify and quantify DAPI-stained cell nuclei, GFP-p65 and mCherry fluorescence. GFP located within the area of the nuclear staining (eroded by 2 pixels) was defined as nuclear NF-κB, while GFP within a 2-pixel-wide ring around the nuclear staining was defined as cytosolic NF-κB. For determination of NF-κB translocation, the ratio of nuclear to cytoplasmic GFP-p65 intensity was calculated using BD Image Data Explorer software. For mCherry expression, nuclear mCherry was quantified using the same method as for NF-κB. Background was subtracted and average intensity was used as a measure of TNF-α promoter activity. Live cell imaging of RAW G9 cells expressing GFP-p65 and tnf promoter driven mCherry for Supplementary Videos 1–3 was carried out as previously described19 (link).

Cells were fixed with 4% paraformaldehyde for 15 min and stained according to a procedure previously described^{51 (link)} using mouse monoclonal anti-glucagon antibody (Sigma-Aldrich Sweden AB, Stockholm, Sweden) and secondary goat anti-mouse IgG–Alexa 647 polyclonal antibody (Invitrogen, Stockholm, Sweden). Cells were covered with Vectashield mounting medium containing 1.5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Immunkemi F&D AB, Järfalla, Sweden) and examined with a BD Pathway 855 High-Content Bioimager (BD Biosciences, Rockville, MD, USA) with an Olympus UPlanSApo 10×/0.40 objective. Segmentation of cells based on nucleic DAPI fluorescence staining and subsequent immunofluorescence intensity analysis was performed with the BD Attovision software. Classification and counting of cells was done with the FlowJo Software (Tree Star Inc., Ashland, OR, USA).

Hoechst 33258 and rhodamine-phalloidin–stained cysts in 384-well plates were imaged using a BD Pathway 855 (BD Biosciences, Breda, Netherlands) automated inverted wide-field microscope using a 4× Olympus objective. Images were obtained using BD Attovision software (BD Biosciences) accompanying the microscope, which was used to image focal planes throughout the gel at intervals of 50 µm. The gel was imaged through its entire depth (z axis), requiring around 25 images per well; each image captured approximately 75% of the well area (Suppl. Fig. S3A). High-resolution confocal images were made using a 20× objective on a Nikon Ti Eclipse confocal laser microscope (561 and 408 nm lasers). Confocal images were exported using NIS Elements Viewer (Nikon Instruments Europe B.V., Amsterdam, Netherlands).