Fig. 3

Oncogenic properties of TOX2 in NKTL cells. A NKYS and HANK1 cells were infected with either scramble shRNA, or TOX2-sh1 or TOX2-sh2 tagged with green fluorescent protein (GFP) for 3 days, then subjected to mRNA and protein extraction. Quantitative RT-PCR (upper panel) and immunoblotting analysis (lower panel) of TOX2 transcript and protein level in these populations. Three independent experiments were conducted. For qRT-PCR analysis, data were normalized to GAPDH level (internal control) for each sample and are expressed as the fold change vs scramble control population (mean ± SD). *p < 0.05. For immunoblotting analysis, GAPDH and β-actin were used as loading controls in NKYS cells and HANK1 cells, respectively. Representative blotting images were shown. B Flow cytometric analysis of the percentage of GFP + cells post-infection of NKYS and HANK1 cells. The quantification started at day 3 post-infection at 2-day intervals up to day 11. The percentage of GFP + cells at day 5, 7, 9, 11 was normalized to day 3, respectively. Two sets of cell culture medium with or without human IL-2 (10 ng/ml) were used. Each data point was representative of three biological replicates (mean ± SD). *p < 0.05; **p < 0.01. Representative FACS plots show NKYS cells infected with TOX2-sh1 lentivirus at day 3 and day 11. C Cell cycle analysis of NKYS and HANK1 cells infected with either scramble shRNA or TOX2-sh1 or TOX2-sh2 lentivirus. These cell cycle experiments were triplicated and presented in mean ± SD. *p < 0.05. D Quantitative RT-PCR of TOX2 gene expression in NKYS cells transduced with either empty vector (EV) or FLAG-TOX2 overexpression vector. These data show mean ± SD of 3 independent experiments. **p < 0.01 (left panel). Western blot analysis of TOX2 protein level in EV-NKYS cells and FLAG-TOX2-NKYS cells. GAPDH was used as loading control (right panel). E Quantification of the percentage of GFP + subpopulation among NKYS-EV and NKYS-FLAG-TOX2 cells at 2-day interval up to day 8. Human IL-2 was removed from culture medium. This experiment was repeated 3 times. F TOX2 increases colony formation of NKTL cells. Representative images of colony formation captured from NKYS-EV and NKYS-FLAG-TOX2 cells (upper panel). The numbers of colony in 10 random field was illustrated in mean ± SD (lower panel). These data were from three independent experiments. *p = 0.021