Doxycycline Induces Mitophagy and Suppresses Production of Interferon-ß in IPEC-J2 Cells

Xing, Yang; Liqi, Zhu; Jian, Lin; Qinghua, Yu; Qian, Yang

Based on the evidence that autophagy protects cells from apoptosis and the so-called apoptosis/autophagy paradox hypothesis (González-Polo et al., 2005; Fimia and Piacentini, 2010) , we determined whether DOX induces autophagy in IPEC-J2 cells. LC3 is a labeling protein found on the autophagosome membrane. Newly synthesized LC3 is diffusely distributed in the cytoplasm of mammalian cells. However, LC3I is transformed into LC3II when autophagy occurs, and the latter accumulates on autophagosome membranes. Therefore, the degree of autophagy can be determined by the LC3II: LC3I ratio (Tanida et al., 2004; Feng et al., 2013) . Thus, we examined expression of the LC3 autophagosome protein in IPEC-J2 cells by Western blot analysis. The LC3II: LC3I ratio increased in a dose-dependent manner during a 24-h incubation with DOX compared with the control; the exception was the 1 µg/ml DOX group, in which the LC3II: LC3I ratio increased only slightly (Figures 3A,B) . To this end, we used CCCP and the lysosomal inhibitor chloroquine (CQ) (Bjørkøy et al., 2009) as a positive-control group. Moreover, we established stable IPEC-J2 cells that expressed the green fluorescent protein (GFP)-LC3 fusion protein. DOX treatment led to the punta formation in GFP-LC3-labeled vesicles. In contrast, most cells in the control group displayed a weak and diffuse cytoplasmic GFP-LC3 fusion protein signal ( Figure 3C ). The number of GFP-LC3B puncta per cell is shown in Figure 3D . An increase in LC3II or GFP-LC3 vesicles can occur due to increased synthesis of autophagosomes and due to impaired autophagosomelysosome fusion. Therefore, a simple assessment of LC3II levels or LC3-positive vesicles cannot distinguish between the two scenarios. Thus, we used the monomeric red fluorescent protein (mRFP)-GFP-LC3 tandem reporter construct to measure autophagic flux (Kimura et al., 2007) . The green fluorescence of this tandem autophagosome reporter is attenuated in the acidic pH of the lysosome by lysosomal hydrolysis, whereas red fluorescence is not. Therefore, autophagosomes have both GFP and mRFP signals, whereas autolysosomes have only mRFP signals, because GFP is attenuated in the acidic lysosomal environment. RFP-LC3-labeled puncta structures FIGURE 3 | DOX induces complete autophagy in IPEC-J2 cells. (A, B) IPEC-J2 cells were treated with DOX, and CCCP + chloroquine (CQ) treatment was used as a positive control. LC3I, LC3II, and GAPDH expression (loading control) were analyzed by Western blot using specific antibodies. LC3II/LC3I levels normalized relative to control cells are shown. (C,D) IPEC-J2 cells stably expressing enhanced green fluorescent protein (EGFP)-LC3 were treated with DOX or CCCP+CQ and stained with DAPI. The number of GFP-LC3 puncta per cell was quantified. (E) IPEC-J2 cells stably expressing monomeric red fluorescent protein (mRFP)-EGFP-LC3 were treated with rapamycin or CQ for 12 h or treated with 50 µg/ml DOX for 12, 24, and 48 h and stained with Hoechst 33342 (blue color in the images) and observed by fluorescence microscopy. Yellow puncta (incomplete autophagic flux); red puncta (complete autophagic flux). Data are means ± standard deviations of three independent experiments. One-way analysis of variance; *P < 0.01; **P < 0.001.