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. 2016 May 10;12(6):907–916. doi: 10.1080/15548627.2016.1164358

Figure 1.

Figure 1.

Loss of autophagy enhances MIF secretion by human and mouse macrophages. (A and B) PMA-differentiated THP-1 cells were treated with 3-methyladenine (3-MA) in the presence or absence of (A) LPS (100 ng/ml) or (B) heat-killed Escherichia coli (HK E. coli, multiplicity of infection 1:1) for 6 h and MIF measured in the culture medium by ELISA. (C-E) Undifferentiated THP-1 cells were treated with 3-MA and HK E. coli for 6 h and secreted (A) MIF, (B) IL1B and (C) TNF measured by ELISA. (F) Western blot analysis of ACTB/β-actin and ATG5 protein in lysates from RAW264.7 cells transfected with siRNA against Atg5 or nontargeting (scrambled, scr) siRNA for 48 h. (G and H) RAW264.7 cells were transfected with scrambled or Atg5 siRNA for 48 h then treated with LPS for 6 h. Secreted (G) MIF and (H) TNF were measured by ELISA. Bars represent means ± SEM of triplicates, representative of at least 3 experiments. (I) Bone marrow-derived macrophages from atg7wt/wt× LysMcre (WT) and atg7fl/fl× LysMcre (atg7−/−) mice were treated with LPS (100 ng/ml) or imiquimod (10 μg/ml) for 6 h and secreted MIF measured by ELISA. Bars represent means ± SEM for 3 mice. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001.