Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

doi: 10.4049/jimmunol.1001822

Figure Lengend Snippet: Purified T cells were added to glass coverslips coated with anti-CD3 antibody or anti-H-2Kb antibody, incubated for 20 min, and fixed with 2% paraformaldehyde. Cells were surface stained with anti-CD4 and intracellularly stained with anti-STIM-1 antibody. Z-stack images were captured at 1 μm intervals using 100x oil objective. The best focused layers were used to quantitate the fluorescence intensity of STIM-1 in resting wildtype and Atg7f/fLck-Cre CD4+ cells. More than 30 individual cells were analysed. For the analysis of STIM-1 punctae, one layer was selected from 3D deconvolution images, and the layers on either side of the selected layer were subtracted. The size and intensity of STIM-1 punctae were measured using MetaMorph 7.6 software. Fifty wildtype or Atg7f/fLck-Cre CD4+ cells were randomly selected for analysis. (A). Representative images of endogenous of STIM-1 and STIM-1 punctae in wildtype and Atg7f/fLck-Cre CD4+ cells stimulated by anti-CD3 mAb or thapsigargin. Images were obtained with Zeiss ApoTome system using AxioVision software with a 63x oil objective. (B). Fluorescence intensity of STIM-1 in resting wildtype and Atg7f/fLck-Cre CD4+ cells. (C). Area of STIM-1 punctae in anti-CD3 antibody activated wildtype and Atg7f/fLck-Cre CD4+ cells (p=0.6). (D). Fluorescence intensity of STIM-1 punctae in anti-CD3 antibody activated CD4 cells (p=3.5×10-15). (E). Area of STIM-1 punctae in wildtype and Atg7f/fLck-Cre CD4+ cells stimulated with thapsigargin (p=0.03). The LN cells from wildtype or Atg7f/fLck-Cre mice were stimulated with 100nM thapsigargin for 20min. The cells were fixed and stained intracellularly with STIM-1 antibody. The images of STIM-1 punctae were captured and quantitated using the same method as described above. (F). Fluorescence intensity of STIM-1 punctae in wildtype and Atg7f/fLck-Cre CD4+ cells stimulated with thapsigargin (p=0.16). (G). Protein expressions of STIM-1 and Orai1 in Atg7-deficient T cells. The Western blot was repeated three times using purified T cells from three different pairs of wildtype and Atg7f/fLck-Cre mice. The numbers represents the ratios of intensity of STIM-1 or Orai1 bands to the intensity of actin bands. Normalized Western blot intensities were quantified and shown in the right panel. STIM-1 (mean±SD, *p=0.0069). Orai1 (mean±SD, p=0.47). (H). IL-2 production by autophagy-deficient T cells. Purified naïve CD4+ T cells were stimulated with plate-bound anti-CD3 (5μg/ml) or anti-CD3 plus anti-CD28 (2μg/ml) over night. IL-2 in the cell culture supernatants was measured by ELISA. (*p=0.0057, **p=0.00049).

Article Snippet: Every cell was tracked and analyzed manually frame by frame using MetaMorph 7.6 software.

Techniques: Purification, Incubation, Staining, Fluorescence, Software, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay