Human STING is a proton channel (Live-cell MGAT Super-Resolution Experiment)

hTERT-immortalized BJ1 cells (ATCC CRL-2522) expressing SEP-mRuby3 targeted to cis/medial Golgi (MGAT) were transduced with pXPR023 (lentiCRISPRv2) expressing an sgRNA targeting STING and selected with 0.1 µg/mL puromycin for 5 days. Cells were then transduced with blasticidin-STING-miRFP680 and selected using 10 µg/mL blasticidin HCl for 5 days. Cells were plated in 96-well glass-bottom plates (Greiner Bio-One) at 6,000 cells/well. After 48 hours, cells were 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) and stimulated with 1 µM diABZI (Invivogen, #tlrl-diabzi). All images were acquired using an LSM980 with Airyscan2 (Zeiss) with 37°C with 5% CO2 incubation. 8 z-stacks were acquired with 0.15 µm z-step.  Images were acquired using a 63X 1.40 NA DIC M27 objective with Immersol 518F 37°C oil. Acquired images were Airyscan processed and then analyzed as described in the image analysis section.

Each frame represens one timepoint imaged every 5 minutes post diABZI treatment. Channels are: SEP (super-ecliptic pHluorin), mRuby3, and STING-miRFP680.