Knocking down of TG2 in NB4 cells enhances the cell proliferation potency and reduces the immunological functions during ATRA-induced differentiation. NB4 cells (105 cells/mL) and its sublines (virus control and TG2-KD NB4 cells) were cultured in standard medium in presence of 1μM ATRA for 48 and 72 hours. (A) Cell numbers were determined by the trypan blue dye exclusion method at the 24, 48, and 72 hours states of the differentiation. Figure shows growth curves of control, virus control, and TG2-KD NB4 cells conducted in 3 parallel experiments. The increase in TG2-KD NB4 cell number was statistically significant (P ≤ .05) at 48 and 72 hours. (B) Cell-cycle of NB4 cells and its sublines (minimum 20.000 events collected from each) were analyzed by flow cytometry. Figure shows mean of S-phase ratios of cell cycles from 3 independent experiments at 0, 24, 48, and 72 hours of differentiating cells. The error bars represent SD of means. Increase number of TG2-KD NB4 cells in S-phase at 48 hours was calculated to be statistically significant (P ≤ .05). (C) After 48 and 72 hours of initiation of differentiation, 105 cells in 1 mL were allowed to adhere to plastic surface of 24-well plate for 30 minutes. After removal of nonadherent cells, the number of adherent cells were determined by randomly selected 10 fields of view seen through the eyepieces of the microscope performed in 3 independent experiments. Decrease in adherence in case of TG2-KD NB4 cells was statistically significant (P ≤ .05). (D) Cells (2.5 × 105) from each cell line were placed into the upper chamber of Matrigel Invasion Chamber after 48 hours and 72 hours of initiation of differentiation. Migration was elicited by 200nM fMLP or 10 ng/mL IL-8 containing medium in lower chamber. Numbers of cells that migrated through the chambers were determined as described previously in panel A. The migration time was 14 hours. The decrease in chemotactic activity was calculated to be significant at 48 and 72 hours (P ≤ .05). (E) ATRA differentiated control, virus control, and TG2-KD NB4 cells were fed with 2 types of FITC-labeled bacteria (L monocytogenes and S aureus) with the ratio of 1:50 cell/bacterium for 2 hours in the presence of serum at 72-hour stage of differentiation. Phagocytic capacity was evaluated by flow cytometric analysis. The bars represent mean and SEM of 3 independent experiments (P ≤ .05). Typical flow cytometric profiles of L monocytogenes (top panel) and S aureus (bottom panel) phagocyting 72 hours differentiated cells are shown. (F) Intracellular NADPH-oxidase activity of 48 and 72 hours ATRA differentiated control, virus control, and TG2-KD NB4 cells (106 cells/mL) was induced by 50nM phorbol myristate acetate in 1-mL reaction volume containing 5 μL of L-012 (100μM). Results are the mean ± SD of 3 experiments. Chemiluminescence was detected by MOONLIGHT 2010 luminometer at intervals of 10 seconds. The decrease in ROS production in TG2 knockdown NB4 was calculated to be statistically significant (P ≤ .05).
