Human STING is a proton channel (Live-cell MGAT STING WT or S53L pH Measurement upon STING agonist treatment with or without C53)

hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to MGAT constructed based on designs reported in Linders et al. <em>ACS Chem. Biol. </em>2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. Cells then were transduced with pXPR023 (lentiCRISPRv2) expressing an sgRNA targeting STING and selected with 0.1 µg/mL puromycin for 5 days. Finally, cells were transduced with blasticidin-STING-HA (WT or S53L) and selected using 10 µg/mL blasticidin HCl for 5 days.<strong> </strong> BJ1 SEP-mRuby3 STING-HA (WT or S53L) cells were plated in 24-well glass-bottom plates (Greiner Bio-One) at 40,000 cells/well. After 48 hours, cells were stained for 45 minutes at 37°C with 0.5 µg/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed and incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061). For time-course experiments, cells were stimulated with 1 µM diABZI (Invivogen, #tlrl-diabzi) for 1 hr with or without the addition of 10 µM C53 (Cayman, #37354). All images were acquired using a Ti2-E inverted epifluorescence microscope (Nikon) with automated XYZ stage control, hardware autofocus, and a Yokogawa CSU-W1 confocal spinning disk unit with Zyla 4.2 PLUS sCMOS camera. An Okolab cage incubator was set to 37°C with 5% CO2. 405, 488, 561, and 640 nm laser lines were used for fluorescence illumination and all hardware was controlled using NIS elements software. Images were acquired using a 40X 0.95 NA CFI Plan Apo λ objective (Nikon MRD70470) with the following lasers and filters: Hoechst (405 nm laser, Chroma Multi LED set #89401), superecliptic pHluorin (488 nm laser, Chroma Multi LED set #89401), and mRuby3 (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection. Images are maximum projections of multiple z-stacks with each frame representing one timepoint: 0, 10, 20, 30, 40, 50, 60 minutes post treatment. Channels are: Hoechst 34580, SEP (super-ecliptic pHluorin), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.