Human STING is a proton channel (Live-cell MGAT, GALT, and LAMP1 pH Measurements Upon BafA1 and diABZI treatment)

<p>hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to MGAT, GALT, or LAMP1 constructed based on designs reported in Linders et al. <em>ACS Chem. Biol.&nbsp;</em>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&deg;C with 0.5 &micro;g/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed&nbsp; 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 &micro;M Bafilomycin A1 (Santa Cruz Biotechnology cat. #sc-201550),&nbsp; or 1 &micro;M diABZI (Invivogen, #tlrl-diabzi). 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&nbsp; 37&deg;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 &lambda; 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&nbsp; (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 &micro;m spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.</p> <p>Images are maximum projections of multiple z-stacks with each frame representing one&nbsp;timepoint: 0, 10, 20, 30, 40, 50, 60 minutes post treatment (except for replicate 1, which omitted the final timepoint). Channels are: Hoechst 34580, SEP (super-ecliptic pHluorin), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.</p>