Figure 3.
Environmental Cues in the Thymus are Required for the Basal ERK Activity of Thymocytes
(A) The experimental design for analyzing ERK activity in the sliced thymic lobe. The thymus isolated from Lck-EKAREV-NLS mice was mounted in agarose and sliced with a vibratome. Then, the sliced thymic lobes were observed by TPEM.
(B) Representative images of sliced thymic lobes. (Left) Fluorescence image through a BA 520-560 nm emission filter showing both thymocytes and thymic DCs. (Right) Fluorescence image through a BA 615-675 nm emission filter showing non-T cells, including thymic DCs. Scale bar, 100 μm.
(C) A representative merged image (merged fluorescence images through a BA 520-560 nm emission filter and BA 615-675 nm emission filter) showing the cortical medullary junction. Images shown in (B) were merged. The relative density of inherent autofluorescence of thymic DCs (cyan) was used to identify the cortical-medullary junction (dashed yellow line). Scale bar, 100 μm.
(D) Representative FRET/CFP ratio images of the cortex (top) and the medulla (bottom) in intensity-modulated display (IMD) mode. Scale bar, 20 μm.
(E) The FRET/CFP ratio examined within sliced thymic lobes. Dots indicate the FRET/CFP ratio in each thymocyte. Data represent the analysis of samples from three individual mice (n = 676 cells for the cortex and n = 766 cells for the medulla). See also Table S1. All data are presented as mean ± SD. p value was calculated by Student's two-sample t test.
(F) The experimental design for monitoring the ERK activity of specific subsets of isolated thymocytes in vitro (left) and thymocytes overlaid onto sliced thymic lobes (right). For the observation of isolated thymocytes in vitro, thymocytes from Lck-EKAREV-NLS mice were sorted and directly imaged by TPEM. For the observation of overlaid thymocytes, sorted subsets of thymocytes were overlaid onto sliced thymic lobes obtained from C57BL/6 mice. Thymocytes on the slices were incubated for at least 3 hr at 37°C/5% CO2 to allow cells to infiltrate the tissue. Then, the sliced thymic lobes were examined by TPEM.
(G) FRET/CFP ratios of the indicated subsets of isolated thymocytes in vitro and of thymocytes overlaid on the sliced thymic lobes. Each dot indicates the FRET/CFP ratio of each thymocyte. Data represent the analysis of samples from three individual mice for isolated thymocytes in vitro (indicated as Isolated) (n = 382 cells for DP and n = 474 cells for SP). Data are from the analysis of samples from two individual mice for thymocytes overlaid on the sliced thymic lobes (indicated as Overlaid) (n = 42 cells for DP and n = 98 cells for SP). See also Table S1. All data are presented as mean ± SD. p values were calculated by Student's two-sample t test.
(H) The level of ERK phosphorylation (pERK) analyzed by immunoblotting. The whole thymus and single-cell suspension of the thymus (isolated thymocytes) were prepared for immunoblotting (top). Then, phosphorylation of ERK was analyzed (bottom). The level of ERK is shown as a loading control.