Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs.

(A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (D−F) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 μm. This figure is accompanied by Figure 1—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.14023.003

Figure 1—figure supplement 1. Tregs activate autophagy in response to cytokine deprivation.

(A) Z-projected confocal field views of activated Tregs cultured without IL-2 for T6 and T15 hr and input populations (T0) fixed, permeabilized and stained for LC3 (green) and Hoechst 33342 (blue). (B) Apoptotic damage induced activated WT or Notch1^-/- Tregs cultured with or without IL-2. Scale bar 5 μm.

DOI: http://dx.doi.org/10.7554/eLife.14023.004