Eight well culture slides

Astroglia were plated at an approximate concentration of 20,000 cells per well in eight-well culture slides (Corning Inc., Corning, NY, USA) and cultured in medium including 100 µM INI-0602 for 40–60 min at 37 °C. After the treatment, the medium was aspirated, and cells were washed with PBS, fixed with 4% PFA for 5 min, and permeabilized with 0.05% Triton X-100 in PBS for 15 min. The cells were then incubated with primary antibodies against Cx43, GFAP, EAAT1, and EAAT2 (comprehensive antibody information is provided in Supplementary Table 1) in 0.05% Triton X-100 in PBS with 5% goat serum for 1 h at 37 °C. Next, the cells were rinsed before being incubated with DAPI and Alexa 488-, 546-, or 594-coupled secondary antibodies for 30 min at 37 °C. Images were acquired using a confocal laser microscope system (Nikon A1) or a conventional fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

CRL5946 cells were seeded on eight-well culture slides (Corning Life Sciences) with each well 10⁴ cells and treated with cisplatin 2 ug/ml or γ-ray radiation 7.5 Gy. After incubating for 96 h, the medium was removed and the slide was washed twice with cold PBS. The viable cells were fixed with 4% paraformaldehyde for 10 min. The slides were stained with anti-human GITRL antibody (Clone:109101, R&D SYSTEMS), anti-mouse IgG Antibody-AF488 conjugated (Invitrogen), anti-human GITR antibody-PE conjugated (Clone#621, Biolegend Inc.), and DAPI (Cell Signaling) following the commercial instructions. The fluorescence images of whole sides were captured by Nikon A1R+ system, which is built onto a Nikon Eclipse TI-E inverted microscope with Nikon NIS Elements C for acquisition and analysis of images.