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. 2016 Mar 24;7:11023. doi: 10.1038/ncomms11023

Figure 8. Cytosolic FoxO1 associates with Atg7 for autophagy induction in iNKs.

Figure 8

(a) mRNA was extracted from NKP and iNK cells followed by RT-PCR. Relative mRNA expression was normalized to that of NKPs. (b) NK cells were subjected to Western blotting with the indicated antibodies. (c) Autophagosomes of iNKs were isolated and analysed by immunoblotting. LC3-II, autophagosome marker. EEA1, endosome marker. LAMP1, lysosomes marker. Calreticulin, ER marker. H3, nuclear marker. Total, total cell lysates. AP, autophagosomes. (d) FoxO1 interacts with Atg7 in iNK cells. NKP or iNK cells from FoxO1flox/flox;NKp46-Cre mice cells were infected with lentivirus encoding WT or Atg7 binding domain truncated FoxO1 (ΔBD-FoxO1), and subjected to immunoprecipitation with anti-FoxO1 antibody. (e) NKPs were isolated from GFP-LC3 mice and infected with lentivirus encoding WT or ΔBD-FoxO1 or vector, and performed in vitro development assay followed by immunostaining of iNK cells. GFP-LC3 MFI of each cell was analysed and shown as means±s.d. **P<0.01. Scale bar, 5 μm. (f) Autophagic flux was impaired in ΔBD-FoxO1 NK cells. GFP-LC3 MFI of the indicated iNK cells treated as above was analysed by flow cytometry, and shown as means±s.d. **P<0.01. All data represent at least three independent experiments. For a,e and f, a two-way analysis of variance post hoc Bonferroni test was used.