Figure 2.

Gene expression following doxycycline removal and the downstream effects of IFN-γ. (A) Flow cytometric analysis of mCherry expression in DFT1.Tet/IFN-γ with and without doxycycline. DFT1.WT cells treated with and without 5 ng/ml IFN-γ for 24 h (B,C) or 72 h (D) were used as positive and negative controls for β₂-m and PD-L1 upregulation, respectively. (B) mRNA expression of IFN-γ, β₂-m and PD-L1 analyzed by RT-PCR. Results are shown for DFT1.Tet/IFN-γ cells cultured with and without doxycycline for 5 days, and GAPDH was used as a reference gene. NTC, no template control. Marker shows 250 bp-size band. (C) β₂-m upregulation on DFT1.Tet/IFN-γ following doxycycline removal. Cells were stained with mouse anti-devil β₂-m antibody followed by goat anti-mouse IgG conjugated to AlexaFluor 488 and were analyzed by flow cytometry. (D) PD-L1 upregulation on DFT1.Tet/IFN-γ following doxycycline removal. Cells were stained with mouse anti-devil PD-L1 antibody conjugated to DyLight 650 and were analyzed by flow cytometry. For both flow cytometric analyses, antibody staining of cells was performed in triplicate and dead cells were excluded by DAPI staining. The results shown are representative of n = 3 replicates/treatment.