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

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

Transmission electron microscopy (TEM) is used to confirm the double-membrane structure of autophagosomes containing undigested cytoplasm or organelles (Mizushima, 2004) . We used TEM to observe whether DOX induced mitochondrial and autophagosome damage. As shown in Figure 4A , oval mitochondria in the control group had the complete crista structure. However, mitochondria became swollen and had few cristae in IPEC-J2 cells after a 24-h incubation with DOX. Double-membrane vesicles enclosing the mitochondria were also observed. Dysfunctional mitochondria number was shown ( Figure 4B) . We speculated that this structural change was caused by mitophagy. It has been reported that mitophagy clears the accumulation of damaged mitochondria, which is important for mitochondrial turnover (Hara et al., 2014) . For further evidence, we constructed the multi-functional pLVX-EGFP-LC3B-IRES-mito-mCherry lentiviral vector and transfected it into IPEC-J2 cells to detect mitophagy induced FIGURE 4 | DOX induces complete mitophagy. (A) Transmission electron microscopic pictures after 24 h with or without DOX (1 or 100 µg/ml). Arrows at the ctrl point are normal mitochondria with clear cristae, and arrows in the DOX treatment picture indicate abnormal mitochondria without clear cristae. Images in the black box were enlarged 2.5 times and placed in the lower right corner of each picture. Scale bars = 10 µm. (B) Dysfunctional mitochondria number in per total area is shown. (C) IPEC-J2 cells were treated with DOX for 24 h or with CCCP + chloroquine (CQ) as a positive control. Cell nuclei were stained with DAPI, and fluorescence signals were visualized by confocal immunofluorescence microscopy. Green fluorescence indicates autophagosomes, and red fluorescence indicates mitochondria. Yellow fluorescence indicates co-localized autophagosomes and mitochondria. Higher-magnification images represent the regions enclosed in black squares. (D) IPEC-J2 cells stably expressing monomeric red fluorescent protein (mRFP)-enhanced green fluorescent protein (EGFP)-Bcl-xL were treated with CCCP, CCCP + CQ, or DOX + CQ for 24 h or treated with DOX alone for 12, 24, or 48 h. These cells were stained with DAPI and observed by confocal fluorescence microscopy. Data are means ± standard deviations of three independent experiments. One-way analysis of variance; **P < 0.001. by DOX. Obvious GFP-LC3B and mito-mCherry were clearly co-localized in IPEC-J2 cells after a 24-h DOX treatment at all concentrations examined ( Figure 4C) . However, the complete of mitophagic flux was unknown. For this reason, we used a sensitive dual-fluorescence reporter expressing mRFP-GFPmito fused in-frame to a mitochondrial targeting sequence (Bcl-xL transmembrane structure) to monitor mitophagy and lysosomes for degradation. Many bright red color-labeled mitochondria were present as puncta after different incubation durations with DOX ( Figure 4D) . In contrast, treatment with CQ restored the expression of green fluorescence and resulted in yellow color-labeled mito in CCCP-or DOX-treated cells ( Figure 4D ).