Human STING is a proton channel (HEK293T Autophagy Experiment)

RFP-LC3 and STING-HA-expressing FIP200 KO 293T cells were seeded on Fibronectin bovine plasma coated 24-well glass-bottom plates (Greiner Bio-One) the night before stimulation. Cells were then stimulated with 20 µg/ml cGAMP (Invivogen, #tlrl-nacga23-1) with 5ug/ml digitonin (Promega, #G9441) or  1 µM  DIABZI (Invivogen, #tlrl-diabzi) with or without the addition of 10 µM C53 (Cayman, #37354)  for 1 hour. Cells were then fixed with 2% Paraformaldehyde (Electron Microscopy Sciences) in PHEM buffer (Electron Microscopy Sciences) for 30 minutes at 37°C, washed three times with PBS and quenched with freshly prepared 0.1M Glycine for 10 minutes. Cells were permeabilized in 100% methanol for 30 minutes and stained with anti-HA (Millipore, #11867423001) for 1 hour at room temperature in 3% BSA, washed 5 times, and then stained with Alexa 647 anti-rat IgG (H+L) (Thermo, A-21247) in 3% BSA for 1 hour. After five washes,  cells were incubated in 2X SSC with 200 ng/mL DAPI (Thermo Fisher) and imaged using the Nikon microscope used for organelle pH images. Images were acquired using a 60X 1.40 NA Plan Apo λ oil immersion objective (Nikon MRD01605) with Nikon type F immersion oil with the following lasers and filters: DAPI (405 nm laser, Chroma ET455/50), RFP-LC3B (561 nm laser, Chroma ET605/52), and STING-HA (640 nm laser, Chroma ET705/72), assaying five z planes per field of view with 0.625 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.

Images are maximum projections of multiple z-stacks. Channels are: DAPI, LC3B-RFP, and STING.