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. 2020 May 15;9:e53948. doi: 10.7554/eLife.53948

Figure 3. Kinetics of selected signalling proteins and impact of MEK and mTOR pathway inhibitors on T cell activation parameters.

(a) The percentage of cells positive for each marker is plotted against time. Results depict combined data from six biological replicates measured in two independent experiments as detailed in Supplementary file 1. Points represent the mean and error bars depict the SD. Data underlying plots are provided in Supplementary file 5. (b–c) Flow cytometry measurements of pS6[S235/236] and pERK1/2 after 2 hr of pre-treatment with DMSO vehicle control, rapamycin (Rapa) 200 nM, MEK162 (MEK) 0.5 μM, 1 μM and 5 μM, or combined rapamycin with MEK162 (R + M), followed by 4 hr stimulation with 1 μM N4 or NP68 peptides. Results are representative of 3 independent experiments. (d) The fraction of pS6+ cells in N4-stimulated conditions with the indicated inhibitor treatments versus DMSO (top). The median fluorescent intensity of pS6 among pS6+ cells in N4-stimulated conditions with the indicated inhibitor treatments (bottom). Lines represent the median. Results depict combined data from three independent experiments.

Figure 3.

Figure 3—figure supplement 1. Kinetics of total protein levels and surface markers.

Figure 3—figure supplement 1.

The median intensity of proteins measured by mass cytometry under stimulation with ligands of various potencies over time. Points represent the mean and error bars depict the SD. Results depict combined data from six biological replicates measured in two independent experiments as detailed in Supplementary file 1.
Figure 3—figure supplement 2. Kinetics of pSTAT5, pLCK and pSLP76 signalling proteins and testing the impact of IL2.

Figure 3—figure supplement 2.

(a) As Figure 3a for pSTAT5, pLCK and pSLP76. Plots depict combined data from six biological replicates measured in two independent experiments as detailed in Supplementary file 1. Points represent the mean and error bars depict the SD. Data underlying plots are provided in Supplementary file 1. (b) Flow cytometry measurements of pSTAT5, pS6[S235/236] pERK1/2, pAKT and IκBα, stimulated with peptides of various potencies for 4 hr, in the presence or absence of IL2. Results depict combined data from three biological replicates measured in 2–3 independent experiments. Bar charts represent the mean and error bars depict the SD.
Figure 3—figure supplement 3. pERK and pS6 distributions and additional MEK and mTOR inhibition data.

Figure 3—figure supplement 3.

(a) Cells were gated on pERK1/2 positive and negative populations. Within these populations, the median marker intensity was calculated at each timepoint in each condition. (b) As (a) for pS6[S235/S236]. (c) As (b) after normalization of pS6 intensity to total S6 intensity within each cell. (a–c) Plots are representative of 6 biological replicates measured in two independent experiments as detailed in Supplementary file 1. (d) As Figure 3b, measuring pERK1/2. (e) Flow cytometry measurements of pS6[S235/236] after stimulation in the presence of the indicated concentrations of rapamycin. (f) The median fluorescent intensity of CD44 among live cells in N4-stimulated conditions with the indicated inhibitor treatments. Results depict combined data from three independent experiments. Lines represent the mean. (g) 2 day proliferation assay of cells treated as in (d–f) measured using Cell Proliferation Dye eFluor450. (d,e,g) Results are representative of 3 biological replicates from 2 to 3 independent experiments.