ULK1/2 Restricts the Formation of Inducible SINT-Speckles, Membraneless Organelles Controlling the Threshold of TBK1 Activation

Saul, Vera Vivian; Seibert, Markus; Krüger, Marcus; Jeratsch, Sylvia; Kracht, Michael; Schmitz, Michael Lienhard

293T, HEK-TLR4, HeLa and U2OS cells were cultured in DMEM (Gibco) containing high glucose (4.5 g/l), L-alanyl-glutamine (4 mM) and sodium pyruvate (110 mg/l) supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were grown in a humidified incubator at 37 o C under 5 % CO 2 . Transient transfections of plasmids were performed using linear polyethylenimine (PEI, Polyscience Inc.) as described previously (Saul et al., 2015) . Unless indicated otherwise, cells were treated with 0.5 mM arsenite for 1 h or were exposed to heat shock at 43 °C in a humidified cell culture incubator with a 5 % CO 2 atmosphere for 1 h. Inhibitors were incubated 1 h prior cell stimulation. Cell stimulations for the experiments displayed in Fig. 1C were done as follows: HeLa cells were treated with 0.5 M sorbitol (30, 60, 90 min), 0.5 mM arsenite (20, 40, 60 min) or 4 % (v/v) ethanol (15, 30 and 60 min). In addition, HEK-TLR4 cells were treated with 1 µg/ml LPS (1, 2, 4 h), U2OS cells with 10 ng/ml IL-1β (0.5, 1.5, 5 h) and HEK293 cells with 20 ng/ml TNFα (30, 60, 90 min).