Figure 1.

Lack of Atg5 mRNA and ATG5 protein expression as well as a functional defect in autophagy in neutrophils of Atg5^NΔ mice. (a) Quantitative real-time PCR. Freshly purified immature and mature bone marrow-derived neutrophil samples from WT and Atg5^NΔ mice were analyzed for Atg5 expression. S18 was used as a reference gene to normalize the expression of Atg5. Values are means±S.E.M. of two independent experiments. (b) Immunoblotting. Freshly purified mature bone marrow-derived neutrophils of WT and Atg5^NΔ mice were analyzed for ATG5 protein expression. The neutrophil-depleted bone marrow samples were lysed and immunoblotted next to the neutrophil lysates to demonstrate the cell specificity of the ATG5 knockout. If present, ATG5 protein is seen as a conjugate with ATG12. The accumulation of p62 confirms the attenuated autophagic activity in ATG5-deficient neutrophils. The results are representative of three independent experiments. The quantification of the ATG5 signal was performed using the Odyssey Fc Imaging System (LI-COR Biosciences GmbH, Bad Homburg, Germany) and the image Studio software. (c) Confocal microscopy. Freshly purified mature bone marrow-derived neutrophils of WT and Atg5^NΔ mice were cultured in the presence and absence of rapamycin for 1 h and stained with AUTOdot autophagy visualization dye. The dye is specific for autophagic vacuoles that can be detected in rapamycin-treated WT neutrophils as bright white dots. In contrast, rapamycin had no detectable effect on neutrophils derived from Atg5^NΔ mice. Bars, 10 μm. (Right) Statistical analysis of three independent confocal microscopy experiments. Values are means±S.E.M.