Extended Data Fig. 2: PI3K inhibitors did not inhibit STING degradation.

a, MEFs were incubated with DMEM or EBSS for 2 h. For the inhibition of PI3K (phosphatidylinositol-3 kinase), wortmannin (1 µM) or 3-methyladenine (1 µM) was added to the medium. Cells were fixed, permeabilized, and immunostained with anti-LC3 antibody. b, Sting^−/− MEFs stably expressing mRuby3-STING were treated with DMXAA (25 µg ml⁻¹) for 0 or 12 h in the presence of wortmannin (1 µM) or 3-methyladenine (1 µM). Cells were fixed and imaged. c, MEFs were treated with DMXAA (25 µg ml⁻¹) or HT-DNA (4 µg ml⁻¹) for 12 h in the presence of wortmannin (1 µM) or 3-methyladenine (1 µ M). Cell lysates were then prepared and analyzed by western blotting. d, Atg5 Tet-off cells were cultured with or without Doxycycline (Dox) (10 ng ml⁻¹). Cells were stimulated with HT-DNA (4 µg ml⁻¹) for the indicated times in the presence of wortmannin (1 µM). Cell lysates were then prepared and analyzed by western blotting. e, Sting^−/− MEFs stably expressing mRuby3-STING (magenta) and Lamp1-EGFP (green) were treated with DMXAA (25 µg ml⁻¹) or HT-DNA (4 µg ml⁻¹) for 0 or 12 h. For the inhibition of lysosomal proteolysis, E64d (30 µg ml⁻¹) and pepstatin A (40 µg ml⁻¹) were added to the medium. For the inhibition of PI3K, wortmannin (1 µM) or 3-methyladenine (1 µM) was added to the medium. Cells were fixed and imaged by Airyscan super-resolution microscopy. Scale bars, 10 µm in (a, b, and e), 500 nm in the magnified images in (e). Unprocessed blots are available in source data.

Source data