Efficacy of autophagy inhibition in Atg7^f/fCD4-Cre mice after CLP.

<p>Atg7^floxp/floxp mice (Atg7^f/f) were crossed with CD4-Cre transgenic mice to generate doubly transgenic mice (Atg7^f/fCD4-Cre) in which <i>Atg7</i> gene was specifically deleted in T cells. ATG7 protein levels in Atg7^f/f and Atg7^f/fCD4-Cre mice were detected by Western blot analysis. Splenic CD4⁺ and CD8⁺ T cells were isolated using CD4 and CD8 MicroBeads, respectively. The efficacy of protein deletion of ATG7 from CD4⁺ and CD8⁺ cells was determined in normal Atg7^f/f and Atg7^f/f/CD4-Cre mice (A). ATG7 protein levels were further detected in sham and CLP-induced Atg7^f/fCD4-Cre and Atg7^f/f mice (cells obtained at 18h after CLP) (B). Actin was used as a loading control. Autophagy was further evaluated by Cyto-ID Green (C) and acridine orange (D) staining and followed by flow cytometry analysis. Cells were initially gated on a forward- and side-sactter lymphocytes gate to exclude dead cells, monocytes and granulocytes. T cells were divided into CD4⁺CD8⁻ and CD4⁻CD8⁺. Representative histograms were gated on CD4⁺CD8⁻ and CD4⁻CD8⁺ T cells, and shown as mean fluorescence intensities (MFI) of Cyto-ID Green and acridine orange staining. Data are shown as mean ± SEM of 3 animals in each group and compared by two-tailed Student t-test. CLP: cecal ligation and puncture. †<i>P</i><0.05 vs. sham-operated Atg7^f/f mice. *<i>P</i><0.05 vs. respective sham mice. #<i>P</i><0.05 vs. CLP-induced Atg7^f/f mice.