Figure 3. Response of primary CD34⁺CML-CP cells to imipramine blue (IB) in combination with nilotinib.

(A) Dot plots showing apoptotic response of CML391 to IB (1 μM), nilotinib (1 μM), or combination treatment for 72 h. (B, C) After 72 h treatment with drugs or diluent control DMSO /No Drug Control (NDC), primary CML-CP cells were harvested and counted (CML388 (B) and CML391 (C)). The cells were also stained with Annexin V and/or DAPI for viability. The dot plots shown are from wells treated with the drug combination illustrating that some cells counted under phase contrast microscopy and scored as viable owing to Trypan Blue exclusion are in fact committing to apoptosis and/or dying. (D) To assess their function after drug treatment, surviving cells were plated into semi-solid culture medium for 12 days after which time colony formation was scored for total number. (E) Fluorescence in situ hybridisation (FISH) for BCR-ABL1 was performed on plucked colonies. Multiple yellow fusions were seen with K562 cells (positive control for FISH, ×100 magnification); 2 red and 2 green signals were evident in the normal donor (negative control for FISH, ×40 magnification); 2 red, 2 green, 1 yellow (fusion) signal as indicated by the arrow were seen in colonies plucked from CFC set up with CML-CP cells from an untreated well (CML388, ×100 magnification; this is an unusual but nonetheless positive fusion pattern). CFC from wells treated with 2 μM IB were leukemic i.e. BCR-ABL1 positive by FISH (×40 magnification).