Journal: Cell Death and Differentiation

Article Title: STING directly activates autophagy to tune the innate immune response

doi: 10.1038/s41418-018-0251-z

Figure Lengend Snippet: STING-induced autophagy is independent of BECN1, ULK1 and Atg9a. a Immunoblot analysis of LC3-ΙΙ expression levels in BECN1 knockdown cells and control cells transfected with Flag-vector or increasing amounts of Flag-STING plasmid. Quantification of LC3-II expression levels in a is shown in the right panel (mean ± s.d.; from three independent experiments). ∗P < 0.05. b BECN1 knockdown cells and control cells were transfected with GFP-LC3 plasmid together with Flag-vector or Flag-STING plasmid. LC3 dot formation was examined by immunofluorescence microscopy. Scale bar: 10 µm. c The numbers of GFP-LC3 dots per cell in b were quantified (mean ± s.d.; n > 100 cells from three independent experiments). ∗∗∗P < 0.001. The inset shows immunoblot analysis of BECN1 KD HeLa cells (expressing BECN1 shRNA) and control cells (expressing scramble shRNA). d Flag-vector and Flag-STING plasmids were transfected into ULK1 wild-type and knockout cells for 24 h, followed by western blotting assays of LC3-ΙΙ conversion. Quantification of LC3-II expression levels in d is shown in the right panel (mean ± s.d.; from three independent experiments). ∗P < 0.05. e ULK1 wild-type and knockout cells were transfected with GFP-LC3 and Flag-vector or Flag-STING plasmids, and images were captured by confocal microscopy. Scale bar: 10 µm. f Quantification of the numbers of LC3 dots per cell in e (mean ± s.d.; n > 100 cells from three independent experiments). ∗∗∗P < 0.001. The inset shows immunoblot analysis of ULK1 knockout HeLa cells and wild-type cells. g Western blotting analysis of LC3-ΙΙ expression levels in Atg9a wild-type and knockout HeLa cells transfected with Flag-vector or increasing amounts of Flag-STING plasmid. Quantification of LC3-II expression levels in g is shown in the right panel (mean ± s.d.; from three independent experiments). ∗P < 0.05, ∗∗P < 0.01. h Atg9a wild-type and knockout HeLa cells were transfected with plasmids expressing GFP-LC3 and Flag-vector or Flag-STING. Formation of LC3 puncta was detected by immunofluorescence microscopy. Scale bar: 10 µm. i Quantification of the numbers of LC3 puncta per cell in h (mean ± s.d.; n > 100 cells from three independent experiments). ∗∗∗P < 0.001. The inset shows immunoblot analysis of Atg9a knockout HeLa cells and wild-type cells

Article Snippet: The mouse anti-GFP (sc-9996), the rabbit anti-ULK1 (sc-33182), anti-rabbit IgG (sc-2027), anti-mouse IgG (sc-2025), and protein G agarose beads were obtained from Santa Cruz.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, shRNA, Knock-Out, Confocal Microscopy