Fig. 2. CSF2RB knockdown impairs FLT3-ITD-induced cell growth, sensitizes cells to FLT3 inhibition, and decreases phosphorylation of STAT5.

A–J Ba/F3 cells transduced with FLT3-ITD, FLT3-ITD-positive AML cell lines MOLM-13 and MV4–11, as well as FLT3 expressing cell line THP-I, were transduced with constructs bearing inducible CSF2RB shRNA or control shRNA, respectively, and cultured in the presence of doxycycline for at least 48 h prior to and during experiments. A–D Proliferation of control shRNA vs. CSF2RB shRNA-expressing cells was determined by counting viable cells after trypan blue staining in Neubauer counting chamber at indicated time points. Cells were seeded in triplicates in a density of 1 × 10⁴cells/ml at day 0. Fresh medium was added regularly to maintain optimal cell densities over time. Cell lysates were subjected to SDS–PAGE and western blot analysis using indicated antibodies to confirm knockdown. E–G Cell viability was determined using MTS assay. Cells were seeded in a density of 6000 cells per well in 96-well plates and cultured in the presence of midostaurin at the indicated concentrations. Proliferation was measured in triplicates as formazan absorption after 72 h at 490 nM. H–J Cells were seeded in a density of 6000 cells per well in 96-well plates and cultured in the presence of midostaurin at the indicated concentrations. Cell viability was measured daily by formazan absorption at 490 nm. K, L Cells were serum-deprived for 8 h, with or without the addition of 25 nM midostaurin. Whole-cell lysates were subjected to SDS–PAGE and western blot analysis using the indicated antibodies. Quantification of pSTAT5 relative to total STAT5 was calculated from three biologically independent replicates.