shRNA-induced knocking down of TG2 delays the differentiation process of NB4 cells. NB4 cells were cultured in standard medium in presence of 1μM ATRA for 0, 24, 48, 72, and 96 hours. (A) NBT reduction assays were performed (5 × 104 cells) at the indicated time points in triplicates in a 96-well plate by the addition of 1 mg/mL NBT and 2 μL (100 μg/mL) phorbol myristate acetate for 30 minutes. At least 400 cells were counted in each sample and according to their intracellular blue formazan deposit contents percentage of NBT positivity was determined. Percentage of NBT positivity is expressed as mean % ± SD for n = 3 parallel experiments. Light-microscopic image inserts show typical NBT positivity of the ATRA-treated control, virus control, and TG2 knockdown NB4 at the second-day stage of the differentiation (Zeiss Axiovert 135 inverted microscope, equipped with Achroplan 10×/0.25 Ph1; Carl Zeiss). (B) Induction of CD11c differentiation marker in ATRA-treated control, virus control, and TG2 knockdown NB4 cells were determined at the given time points of differentiation by real-time Q-PCR. Measurements were conducted in triplicates, values are expressed as mean % ± SD of the mean. (C) Differences in surface expression of CD11c between control, virus control, and TG2 knockdown NB4 cells were analyzed by flow cytometry at the indicated time points. Cells (8 × 105) were labeled with mouse antihuman CD11c IgG. Graph shows the average of the mean values of fluorescence measured in FL2 channel with ± SD. Typical flow cytometric profiles of CD11c surface expression in each cell types at the third day are shown by the inserted histograms.
