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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Cell Death Differ. 2010 Mar 19;17(9):1381–1391. doi: 10.1038/cdd.2010.25

Figure 7.

Figure 7

Aberrant Lck expression correlates with resistance to dexamethasone in CLL. (a) Representative single cell calcium traces in unstimulated PBLs from CLL2 showing that these cells undergo ligand-independent signaling. Ligand-independent calcium oscillations were detected in three of three CLL patient samples. (b) PBLs from CLL14 (2 × 106 cells per ml) were treated with vehicle (0.1% ethanol) or 10−6 M dexamethasone for 24 – 96 h. (c) MEC1 cells (2 × 105 cells per ml) were treated with vehicle or 10−6 M dexamethasone for 24 h. Cell viability was measured in triplicate by trypan blue dye exclusion. Similar results were obtained by MTS assay. (d) Lck mRNA was measured by real-time PCR in 10 freshly isolated primary CLL samples and in PBLs from a patient with circulating marginal zone lymphoma. Lck was also measured in the prolymphocytoid CLL line MEC1, primary thymocytes, WEHI7.2, CEMC7, and Jurkat cells. All values were measured in triplicate relative to normal B cells. β-Actin was used as an endogenous control for each PCR reaction. (e) Fyn and Lyn were measured as in d. Values represent the mean change in mRNA expression relative to normal B cells. Significance was determined by Student’s t-test. (f) Lck protein was measured by western blotting in six freshly isolated primary CLL samples. β-Actin was used as a loading control (lysate from CLL2 was not loaded in lane three due to insufficient sample volume). (g) A scatter-plot showing a significant negative correlation between Lck mRNA level and response to dexamethasone. Lck mRNA was measured as in d (x axis) and response to dexamethasone (y axis) was determined by calculating fold change in cell killing of CLL cells (10−6 M dex, 24 h). The scatter-plot shows the Spearman’s rank order assigned for each variable pair (N = 9). The correlation coefficient is − 0.700 (P<0.05). Data are representative of multiple independent experiments. Error bars indicate the difference between maximum and mean values from triplicate measurements or the S.E.M from real-time PCR and viability data, respectively.