Cell culture, transfection, and imaging

Culture the HeLa-M cells at 37°C in 5% CO₂ and DMEM containing 10% FBS, 100U/ml penicillin, 100mg/mL streptomycin, and 2millimolar (mM) L-glutamine (all from Gibco).

For live-cell imaging experiments, seed the cells on glass-bottomed dishes (MatTek) at a concentration of 35,000 cells per dish and transfect after 24h 0m 0s using FuGene HD (Promega) in Opti-MEM (Gibco).

Image the cells 24h 0m 0s after transfection.

Just before imaging, remove the growth medium and replace with prewarmed live-cell imaging solution (Life Technologies).

For lysotracker experiments, incubate the cells in 50nanomolar (nM) LysoTracker Red DND-99 (ThermoFisher) in complete DMEM for 0h 30m 0s, wash twice with media, then image in live-cell imaging solution.

Perform all live-cell imaging at 37°C and 5% CO₂.

Perform spinning-disk confocal microscopy using an Andor Dragonfly system equipped with a plan apochromat objective (63×, 1.4 NA, oil) and a Zyla scientific CMOS camera.

For any given experiment, use the same exposure time, laser power, and gain for image acquisition to allow for quantitative comparison.

Generate the cells stably expressing STING-GFP as described elsewhere.

Culture the stable STING-GFP HeLa-M cells at 37°C in 5% CO₂ and DMEM containing 10% FBS, 100U/ml penicillin, 100mg/mL streptomycin, and 2millimolar (mM) Lglutamine (all from Gibco).

For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per dish according to manufacturer protocol. Image the cells 72h 0m 0s after siRNA transfection.

For experiments using cGAMP, transfect 50μg/L of cGAMP using 18µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per dish according to manufacturer protocol. Image the cells 14h 0m 0s after transfection.