Metaxpress image analysis software

Images were acquired at 10× in a Pico high-content imager (Molecular Devices, San Jose, CA, USA) and stitched to create one image containing three fields of view across the well of a 384-well plate. Stitched images were then analyzed with a custom neurite-tracing script written in the MetaXpress image-analysis software (Molecular Devices, San Jose, CA, USA, version 6.6.2.46). In brief, neurite detection was set to be ≥ 3 times the intensity of the background, to be in the width range of 0–5 μm, and to be at least 2 μm long to be counted. All calculated lengths were summed across the stitched fields, covering approximately 50% of the area of a well of a 384-well plate. The analyst was blinded to the conditions.

Streptavidin (Jackson) was conjugated with biotinylated Ro09–0198 (provided by Dr. M. Umeda, Kyoto Univ., Japan) and separated from free biotinylated Ro09–0198 using a Sephadex G-25 gel filtration column (GE Healthcare). For staining of cell surface PE, cells were incubated with 50 μg/ml SA-Bio-Ro for 30 min in α-MEM containing 0.2% (w/v) fatty acid-free bovine serum albumin (BSA) (Sigma). The cells were washed with 20 mM HEPES, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 2.2 mM CaCl₂, 0.8 mM MgCl₂ and 13.8 mM glucose, and then fixed with 4% (w/v) paraformaldehyde in PBS for 30 min, followed by a 5-min permeabilisation with 0.1% (v/v) Triton X-100 in PBS. Immunostaining was performed with a FITC-conjugated anti-Streptavidin antibody (Vector Laboratories). F-actin was stained using Alexa Fluor 594-conjugated phalloidin (Molecular Probes). PS on the cell surface was detected with an annexin V-Cy5 apoptosis detection kit (BioVision) in accordance with the manufacturer’s protocol. Fluorescence microscopy was performed using a DSU-IX81 microscope (Olympus). The images were photographed with a C10600 digital camera (Hamamatsu Photonics). In some experiments, immunofluorescence intensity of cells cultured in 96-well plates was quantified with an ImageXpress Micro XL HCS (Molecular Devices) and MetaXpress Image Analysis software (Molecular Devices).

Cells were seeded in Greiner 96‐well microtiter plates (Greiner Bio‐One), fixed in 4% paraformaldehyde for 10 min, and then incubated in 0.5% Triton X‐100 in PBS for 10 min at room temperature as previously reported 7. After the cells were blocked in 5% fetal bovine serum/TPBS for 1 h at room temperature, they were incubated overnight at 4 °C with primary antibodies, including mouse anti‐TG2 (1 : 200, ab2386; Abcam, Cambridge, MA, USA), rabbit anti‐nuclear factor erythroid‐2‐related factor 2 (Nrf2) (1 : 400, ab62352; Abcam), mouse anti‐Ki67 (1 : 200, 350502; BioLegend, San Diego, CA, USA), or control rabbit/mouse IgG. The cells were then washed and stained with fluorochrome (FITC/TRITC)‐conjugated secondary antibodies (1 : 500; Invitrogen, Eugene, OR, USA) for 20 min at room temperature. Cell nuclei were visualized using DAPI (1 : 2000; Wako Industries). Cellular fluorescence signals were detected using an ImageXpressMICRO High‐Content Screening System (Molecular Devices, Sunnyvale, CA, USA). Morphological analysis was performed using the ‘Multi‐Wavelength Cell Scoring Application Module’ in MetaXpress Image Analysis software (Molecular Devices).

Intracellular TG2 transamidase activity was measured as previously described [34 (link)]. Briefly, the cells were seeded in Greiner 96-well microtiter plates (Greiner Bio-One, Kremsmuenter, Austria) and incubated with 0.2 mM 5BAPA and 0.1 mM aminoguanidine overnight at 37 °C. The cells were treated with increasing concentrations of ACR in serum-free media for 4 h and then fixed with a 10% formaldehyde solution, permeabilized, blocked, and stained with streptavidin-tetramethylrhodamine isothiocyanate (TRITC, Jackson ImmunoResearch Laboratories). Intracellular TG2 transamidase activity was then detected as a fluorescence signal from TRITC and analyzed using an ImageXpressMICRO High Content Screening System (Molecular Devices, Sunnyvale, CA, USA). Morphological analysis was performed using a Multi-Wavelength Cell Scoring Application Module in MetaXpress Image Analysis software (Molecular Devices).