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. ACS Chem. Biol. 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. 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.