Figure 2

Creation of a tetracycline inducible MR1GFP construct. (A) The “on” kinetics of TET-MR1GFP in BEAS-2B measured by flow cytometry. The percentage of GFP positive cells and mean GFP intensity of the GFP positive population normalized to a percentage of the maximum are shown. Data are representative of 2 independent experiments. (B) The “on” kinetics of TET-MR1GFP in BEAS-2B measured by western blot. Cropped image is shown (full length blot is presented in Supplementary Fig. S3). Data are representative of 2 independent experiments. (C) The “off” kinetics of TET-MR1GFP in BEAS-2B measured by flow cytometry. The percentage of GFP positive cells and mean GFP intensity of the GFP positive population normalized to a percentage of the maximum are shown. Data are representative of 2 independent experiments. (D,E) BEAS-2B cells transfected with TET-MR1GFP and incubated with doxycycline to induce preformed MR1 were treated with 6-FP or control, after the doxycycline was removed, for 16 hours. Cells were then analyzed by fluorescence microscopy (D) or flow cytometry (E). (D) Green: MR1GFP; blue: nucleus. Scale bars = 10 µm. Images are representative of 3 independent experiments. (E) Data are representative of 2 independent experiments. (F) IFN-γ ELISPOT assay results of BEAS-2B transfected with TET-MR1GFP using Msmeg supernatant as the antigen. Non-transfected cells ((−)TET/(−)Dox) and A549 MR1^−/− served as controls. Data are representative of 2 independent experiments. The difference in Log EC₅₀ was statistically significant between the BEAS-2B curves (P < 0.0001).