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. Author manuscript; available in PMC: 2013 Aug 24.
Published in final edited form as: Immunity. 2012 Aug 24;37(2):223–234. doi: 10.1016/j.immuni.2012.04.015

Fig. 6. IL-1β induces autophagy in macrophages.

Fig. 6

A. RAW264.7 macrophages were transiently transfected with EGFP-LC3 and treated with 10 ng/ml murine IL- 1β for 2 h, and assayed for LC3 puncta formation by confocal microscopy (only puncta ≥ 1µm were scored as positive). B. RAW264.7 macrophages transfected with mRFP-GFP-LC3 tandem probe, treated with 10 ng/ml IL-1β for 2 h were scored for number (per transfected cell) of RFP+GFP+ puncta (R+G+; early autophagosomes), RFP+GFP− (R+Gβ− ; autolysosomes), and total LC3 puncta. C. Immunoblot analysis of endogenous LC3 conversion to lipidated form (LC3-II) in RAW 264.7 cells upon treatment with 10 ng/ml IL-1β for 2 h, in the absence or presence of bafilomycin A1. Graph, ratio of LC3-II to actin intensity in immunoblots from bafilomycin A1-treated samples. D. RAW264.7 murine macrophages were co-transfected with tandem mRFP-GFP-LC3 probe and expression constructs containing either wild-type MyD88 (MyD88-WT) or a dominant-negative mutant of MyD88 (MyD88-DN). Following stimulation with 10 ng/ml IL-1β for 2 h, LC3 puncta were quantified as in B. E. Induction of autophagy in response to IL-1β is abrogated in bone marrow-derived macrophages (BMM) from MyD88-deficient (Myd88−/−) mice, measured by ratios of LC3-II band relative to actin following treatments of BMMs and immunoblotting of cellular extracts. F. Proteolysis of stable proteins (radiolabeled by a pulsechase protocol) upon stimulation of RAW264.7 cells with 10 ng/ml IL-1β for 2 h (Full + IL-1β) relative to control (Full) or starvation-induced autophagy (Starve).. Data, means ± se, except in E where data are means ± sd (n≥3; †, p≥0.05; *, p<0.05; **, p<0.01; ANOVA).