Fig. 6: Functional studies in APL-derived NB4 cells stably silenced for the S100A3 gene.

NB4 cells were stably infected with lentiviruses containing shSCRAM, shS100A3a, and shS100A3c or an equimolar combination of the two lentiviral vectors shS100A3a and shS100A3c. Infected cells were selected in puromycin for 10 days obtaining the following cell populations: SCRAM/NB4, S100A3a/NB4, S100A3c/ NB4 and S100A3a+c/NB4. a The indicated NB4 derived cell populations were treated with vehicle (DMSO) or ATRA (10–6 M) for 24 h. Cell extracts were subjected to western blot analysis with anti-RARα, anti-S100A3, and anti-actin antibodies. Each line shows cropped lanes of the same gel, hence the results can be compared across the lanes, as they were obtained with the same exposure time. M.W. = molecular weights of the indicated proteins. b The indicated NB4 derived cell populations were treated with vehicle and ATRA (10–7 M) for the indicated amount of time. The growth of each cell population was evaluated by counting the number of viable cells. Each experimental point is the mean ± S.E. of three independent cell cultures. The cell number values of the indicated shRNA expressing cell populations exposed to vehicle or ATRA are significantly lower than the corresponding values determined for the VOVE/NB4 and SCRAM/NB4 counterparts (**p < 0.01 according to a two-way Student’s t-test). c The indicated NB4 derived cell populations were treated with ATRA (10–8 M) for three days. Left: cells were subjected to the NBT assay. Each experimental point is the mean ± S.E. of three independent cell cultures. **The NBT absorbance values of the S100A3a/NB4, S100A3c/NB4, and S100A3a+c/NB4 cell populations are significantly higher than the corresponding values determined for the VOVE/NB4 and SCRAM/NB4 counterparts (p < 0.01 according to a two-way Student’s t-test). Right: the indicated NB4 derived cell populations were treated with vehicle or ATRA (10–8 M) for 2 days. Cells were subjected to FACS analyses for the CD11b, CD11c, CD38, and CD33 surface markers, as indicated. The results are representative of two other independent experiments. d The indicated NB4 derived cell populations were treated with vehicle or ATRA (10–7 M) for 1 day. Cell extracts were subjected to western blot analysis for the indicated proteins using specific antibodies. Each line shows cropped lanes of the same gel, hence the results can be compared across the lanes, as they were obtained with the same exposure time. The results are representative of two other independent experiments. M.W. = molecular weights of the indicated proteins. e The indicated cell lines were treated with vehicle (DMSO) or ATRA (1 μM) for 24 h. Total RNA was extracted and subjected to RT-PCR with Taqman assays for the cEBPβ and paxillin. The results are the mean ± SD of three replicates. **Significantly higher relative to the corresponding vehicle-treated sample (Student’s test p < 0.01).