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. 2016 May 26;7(5):e2235. doi: 10.1038/cddis.2016.144

Figure 1.

Figure 1

EPA, but not AA, induced autophagy and inhibited Dex-induced apoptosis in mBMMSCs in vitro. mBMMSCs were treated with Dex (10−6 M) in the absence or presence of EPA or AA for 24 h. (a and b) Annexin V/PI double staining was performed to detect apoptotic cells. (c) EPA or AA was applied to the mBMMSCs at concentrations of 10, 40, and 100 μm. The cultures were incubated with Dex (10−6 M) for 24 h in the presence or absence of EPA or AA and then cell viability was determined by MTT assay. (d) Caspase-3 activity was detected by the caspase-3 assay kit in mBMMSCs that were treated with Dex (10−6 M) in the absence or presence of EPA or AA for 24 h. (e) Cell apoptosis was detected by TUNEL assay and number of TUNEL-positive cells as a percentage of the positively stained mBMMSCs. (f) Fixed cells processed for thin-section electron microscopy. Arrows indicate the autophagic vacuoles digesting organelles or cytosolic contents (magnification, × 2500). (g) Confocal microscopic analysis of mBMMSCs following immunofluorescent staining using an anti-LC3 antibody labeled the cytoskeleton with phalloidine and labeled with the nuclear marker DAPI (magnification, × 200). LC3, red; phalloidine, green; nuclei, blue. (h) Number of LC3-positive cells as a percentage of the positively stained mBMMSCs. (i and j) RT-PCR analysis of LC3 and p62 treated with certain groups. Expression of each target gene was calculated as expression relative to β-actin and represented as normalized fold expression. (k) Western blot analysis and quantification of LC3 (LC3-2/LC3-1) and p62 (p62/GAPDH) protein treated with certain groups. The data are represented as the mean±S.D. of three independent experiments. *P<0.05 compared with control cells (cells cultured in the same dosages of vehicle without Dex, EPA and AA), #P<0.05 compared with 10−6 M Dex-treated cells. BMMSCs, bone marrow-derived mesenchymal stem cells; Dex, dexamethasone; PI, propidium iodide; EPA, eicosapentaenoic acid; AA, arachidonic acid, LC3, microtubule-associated protein 1 light chain 3