Fig. 3: Effects of ethanolamine on the growth, viability, mitochondrial activity, differentiation, and autophagy of NB4 cells.

Three independent cultures of retinoid-sensitive NB4 cells were pre-treated with vehicle (DMSO), 20 and 50 μM ethanolamine (ET) for 12 h. Subsequently cells were treated with vehicle or ATRA (1 μM) for another 48 h. The concentrations of ET used (20 μM; 50 μM) are indicated by the numbers shown under the diagram. A Upper diagram: The number of cells was evaluated with the use of a Cell Viable Analyzer. Each value is the Mean + SD of three replicate and independent cultures. **Significantly different relative to the vehicle pretreated and vehicle treated cells (white column; p < 0.01, unpaired Student’s t test). Lower diagram: the viability of cells was evaluated with the use of a Cell Viable Analyzer and the results are expressed as the percentage of viable cells. Each value is the Mean + SD of three replicate and independent cultures. The diagrams shown are representative of two independent experiments producing similar results. B At the end of the treatment, cells were co-stained with Mito-Tracker green and Mito-Tracker red before subjecting them to FACS analysis. The left diagrams show the levels of Mito-Tracker green fluorescence. The right diagrams show the values of MitoTracker-red/green fluorescence (red fluorescence following normalization for the green fluorescence value). Each value is the Mean + SD of the three independent cultures. *Significantly different (p < 0.05, unpaired Student’s t test). The diagrams shown are representative of two independent experiments producing similar results. C Mitochondria were isolated from NB4 cells exposed to vehicle, ET, ATRA or the combination of the two compounds as indicated. Mitochondrial Complex I, Complex III, and Complex IV enzymatic activities were measured on four replicates and the results are expressed as the Mean + SD. The data are representative of two independent experiments providing similar results. **Significantly lower relative to the corresponding control sample pre-treated and exposed to vehicle alone (white column; p < 0.01, unpaired Student’s t test). *Significantly lower relative to the corresponding control sample pre-treated and exposed to vehicle alone (white column; p < 0.05, unpaired Student’s t test). D The panel shows the expression levels of the myeloid markers, NBT-reductase, CD11b, CD11c, and CD33 as indicated. NBT-reductase activity was determined with a spectrophotometric assay. The surface expression of CD11b, CD11c and CD33 was determined by FACS analysis. In the case of CD11b, CD11c and CD33, the leftmost diagrams show representative FACS curves. The rightmost column diagrams illustrate the quantitative data obtained on the three independent samples analyzed. The results are expressed as the Mean + S.D. (N = 3) of the values determined. **Significantly lower relative to the corresponding control sample treated with ATRA alone (black column) (p < 0.01, unpaired Student’s t test). The diagrams shown are representative of two independent experiments producing similar results. E and F The panels illustrate the results of Western blot experiments performed on pooled cellular extracts obtained from three separate cultures of NB4 cells exposed to vehicle, ET, ATRA or the combination of the two compounds, as indicated. The antibodies used target the differentiation markers, PU.1 and IRF1 (E), as well as the autophagic markers ATG5 and LC3I/LC3II (F). The same amounts of proteins were loaded in each lane, as demonstrated by the Western blot signals obtained with the anti-β₂actin antibodies. In the case of PU.1, IRF1 and LC3I-II, the Western blots shown are representative of two independent experiments producing similar results. The numbers above the PU.1 LC3I-II and IRF1 lanes show the densitometric data of the Western blot. The values represent the PU.1/Actin, IRF1/Actin or LC3I-II/Actin ratios and are normalized for the results obtained after treatment with ATRA (lane 2 from the left). The PU.1/Actin or IRF1/Actin values are taken as 1.0. In the case of ATG5, the Western blots shown are representative of three experiments. The diagrams above the ATG5 lanes show the densitometric data of the Western blots performed in the three independent experiments. The values (mean ± SD, N = 3) represent the ATG5/Actin ratio and are normalized for the results obtained after treatment with vehicle alone (leftmost lane) whose ATG5/Actin value is taken as 1.0. R.I. = Relative Intensity.