Axl and CDCP1 are over expressed in nilotinib resistant CML cells. (A) Total mRNA (1 μg) from Ks and K-rn cells were reverse transcribed into cDNA and used in Q-PCR to quantify the mRNA expression of Axl (black box) and CDCP-1 (white box) between Ks and K-rn cells. In parallel, the level of protein expression was quantified by densitometry analysis of 5 separate Western-blots. Results are expressed as a fold increase expression for the mRNA by the ΔΔ Ct method and the protein by the ratio of K-rn/Ks. (B) Seven CML patients (2 males, 5 females) were investigated as previously described.⁸  All patients were analyzed at the beginning or before nilotinib treatment and under treatment at the moment of nilotinib failure in chronic phase (number 6 and 7) or in accelerated phase (number 1 to 5). Quantitative Real-time PCR to amplify Axl (black box) and CDCP-1 (white box) transcripts was carried out on the cDNA. Analysis was done by comparison of the 2 time points (before nilotinib and under treatment at the moment of failure) by the comparative ΔΔCt method giving the fold increase of the amount of target normalized to the endogenous reference as 2^{- ΔΔCt}. (C) Purified CD34 positive cells from blood samples of CML patients (8 and 9 responding to nilotinib, 10 resistant to nilotinib) were analyzed by flow cytometry as described in “Methods” for Syk phosphorylation (phosphospecific antibody p-Syk 525/526) and Axl expression. Control using non immune IgG (gray histogram) and specific antibody (white histogram) are shown. (D) Bcr-Abl, Axl, CDCP-1 and Lyn were detected by Western-blotting using protein samples from CML 9 and 10 patients. (E) CD34 positive cells from nilotinib-responding (11: S) or resistant (12 and 13: R) CML patients were isolated and solubilized in Laemli buffer. Proteins were separated by SDS-PAGE and specific proteins were detected by Western-blotting as indicated. Actin was used as a loading control.