Figure 4.

MIF secretion in starved cells is not dependent on autophagy induction. (A) RAW264.7 cells were transfected with nontargeting (scrambled) or Atg5 siRNA for 48 h and starved in HBSS for 6 h. Secreted MIF was measured by ELISA. (B and C) PMA-differentiated THP-1 cells were treated with different doses of the MTOR inhibitor Torin 1 in the presence or absence of LPS (100 ng/ml). Secretion of (B) MIF and (C) IL6 was measured by ELISA. (D) Western blot analysis of LC3-I and LC3-II in lysates from RAW264.7 cells starved in HBSS or treated with Torin 1 for the times indicated. Experiments were done in the presence or absence of bafilomycin A₁. Blot shown is representative of 3 separate experiments. (E) Immortalized bone marrow-derived macrophages (iBMM) stably expressing GFP-LC3 (green) were cultured in complete medium or HBSS for 6 h, fixed and analyzed for autophagosome formation by confocal microscopy. (F) Bar graph showing the number of GFP-LC3⁺ autophagosomes per cell. Bars represent means ± SEM from triplicates, representative of at least 3 separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001.