Human STING is a proton channel (Live-cell pH Calibration Meaurements)

hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to GALT constructed based on designs reported in Linders et al. ACS Chem. Biol. 2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. BJ1 SEP-mRuby3 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 pH calibration experiments, cells were treated with buffers at defined pH values supplemented with nigericin and valinomycin following manufacturer’s instructions (Thermo Fisher Scientific cat. # P35379). Additional buffers at pH 7, 6, and 5 were created by titration with acid. 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 pH value: 7.5, 7, 6.5, 6, 5.5, 5, and 4.5 (except for replicate 1, which omitted pH 7). Channels are: Hoechst 34580, SEP (super-ecliptic GFP), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.