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. 2007 Jan 22;204(1):25–31. doi: 10.1084/jem.20061303

Figure 1.

Figure 1.

Atg5−/− thymocytes are reduced in number but develop normally. (A) Rag-2−/− DN thymocytes, sorted DP, CD4+ SP, CD8+ SP, and peripheral T cell subsets from C57BL/6 mice were analyzed for the expression of autophagy genes by semiquantitative RT-PCR. Both sorted CD4+ and CD8+ peripheral T cells were activated in vitro for 2 d with anti-CD3 and subjected to RT-PCR analysis. (B) Transmission electron microscopy of peripheral T cells. Freshly isolated (F) and anti-CD3–activated (A) CD4+ and CD8+ T cells (n = 50) were cross sectioned and analyzed for the presence of autophagosomes (arrows). Activated cells were cultured in vitro for 2 d with anti-CD3. (C) T cell immunoblot for LC3. Purified T cells from C57BL/6 mice were either immediately lysed or amino acid starved in vitro for 4 h in a balanced salt solution, or stimulated with plate-bound anti-CD3 with or without hIL-2 (100 U/ml) for 16 h. The lysates were probed for LC3 processing. Actin serves as a loading control. (D) Total thymocyte number in Atg5−/− and wild-type chimeras. Circles and squares represent individual mice. P = 0.04. (E) FACS profile of Atg5−/− thymocytes. Total thymocytes were stained with anti-CD4 and anti-CD8. DN thymocytes were pregated on CD3−CD4−CD8− cells. (F) FACS analysis of thymocyte apoptosis. Thymocytes were stained with CD4, CD8, 7-AAD, and annexin V. Numbers indicate the percentage of cells in each region.