Supplementary Figure 1. BMS98662 enhances human T cell activation in vitro in a concentration-dependent manner. Jurkat T cells were activated with anti-cd3 and anti-cd28 antibody in the presence of titrated concentrations of BMS98662. Upregulation of the activation marker, CD69, was assessed by flow cytometry. Representative histograms comparing surface CD69 levels are shown. In the left panel, Jurkat cells that are stimulated (black line) are compared to unstimulated Jurkat cells (grey line). In the middle panel, activated Jurkat cells treated with vehicle control (grey line) are compared to those treated with BMS98662 at a concentration of.2 μm (black line). In the right panel, activated Jurkat cells treated with vehicle control (grey line) are compared to those treated with BMS98662 at a concentration of 2 μm (black line). Supplementary Figure 2. BMS98662 inhibits the growth of BRAF mutant tumor cells in vitro. The human BRAF mutant tumor cell line, SK-MEL-19, was cultured in the presence of increasing concentrations of BMS98662. The number of cells was quantified daily for 3 days of culture. These numbers were used to calculate an area under the curve (AUC) presented in Figure 1. Supplementary Figure 3. BMS98662 enhances murine T cell activation in vitro in a concentration-dependent manner. (A,B) Purified, CFSE labeled murine CD8 + or CD4 + T cells were activated with a combination of anti-cd3 and anti-cd28 antibody in the presence of indicated concentrations of BMS98662. The percentage of proliferated cells, reflected by CFSE dilution, was quantified by flow cytometric analysis four days later. (C) Purified murine CD4 + or CD8 + T cells were activated in the presence of indicated concentrations of
BMS98662. The percentage of cells that upregulated CD69 were quantified after 48 hours in culture. In all experiments, samples were treated and analyzed in triplicate and error bars represent standard error. Supplementary Figure 4. Detection of ERK phosphorylation in activated T cells. The top panel depicts the schema of T cell activation for the detection of phosphorylated ERK after stimulation with anti-cd3 antibody. The bottom panels demonstrate the detection of indicated molecules (perk, total ERK, pmek, total MEK) in Jurkat cells at indicated timepoints after stimulation. The line graphs plot the mean fluorescence intensity (MFI) of each molecule detected by flow cytometry and the western blots below each graph demonstrate the detection of the same molecule at each timepoint. Supplementary Figure 5. Gating strategies for OT-1 and ex vivo phosphoflow. (A) The gating of OT-1 cells by size (left panel) and by detection of a CD3 + tetramer + population (right panel) is shown. (B) The gating strategy for the detection of CD4 and CD8 T cells from mouse splenocytes. (C,D) Representative samples demonstrating the detection of perk or total in splenocytes activated with anti-cd3 ex vivo. Supplementary Figure 6. PLX472 enhances human T cell activation in vitro in a concentration-dependent manner. (A) Jurkat T cells were activated with anti-cd3 and anti- CD28 antibody in the presence of titrated concentrations of PLX472. Upregulation of the activation marker, CD69, was assessed by flow cytometry. MFI represents the median fluorescence intensity, reflecting the level of expression of CD69. One experiment representative
of three independent experiments is shown here. (B) Jurkat T cells were activated with anti-cd3 antibody in the presence of titrated concentrations of PLX472, as described in detail in supplemental materials and in Figure S1. Levels of phosphorylated ERK (perk) or total ERK were measured prior to activation (-) or 15 minutes after activation (+) by western blotting.
Supplementary Figure 1.
Supplementary Figure 2. Tumor Growth in the presence of BMS98662 Number of Cells ( x 1^6) 15 1 5 vehicle.2.5.2.5 2 5 2 1 2 3 Days in Culture
Supplementary Figure 3.
Supplementary Figure 4. 2 15 1 5 perk% 2 1 1 perk ERK% pmek% MEK 8 8 15 6 6 1 4 4 5 2 2 MEK% 2 5 1 3 6 9 12 18 24 ERK Ti ( i ) 2 5 1 3 6 9 12 18 24 2 5 1 3 6 9 12 18 24 MEK 2 5 1 3 6 9 12 18 24 MFI Time post s mula on (min)
Supplementary Figure 5. A. SSC-A PE (Tetramer) FSC-A Pacific Blue (CD3)
Supplementary Figure 6.