Journal: Haematologica

Article Title: Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia

doi: 10.3324/haematol.2017.178699

Figure Lengend Snippet: COBLL1 expression in chronic lymphocytic leukemia (CLL) and non-malignant B cells. (A) COBLL1 mRNA expression in 86 mutated CLL (M-CLL), 92 unmutated CLL (U-CLL), and healthy B cells isolated from 4 tonsils and 5 peripheral blood (PB) samples. Individual dots represent individual patients. Full lines indicate median. dCtN - dCt value normalized for three independent datasets (see Methods); *** P <0.0001, Mann-Whitney test. (B) COBLL1 expression histogram follows a bimodal distribution pattern in U-CLL. (C) COBLL1 protein levels correspond very well with COBLL1 mRNA both in M-CLL and U-CLL cells. COBLL1 protein levels in CLL cells were determined using western blotting and anti-COBLL1 antibody. Actin was used as a loading control. Patient samples are ordered according to their IGHV mutation status and COBLL1 mRNA expression [in the ascending order; numbers indicate patients’ relative COBLL1 expression determined by qRT-PCR (see A)]. (D) COBLL1 mRNA expression does not change with time or treatment. COBLL1 expression was analyzed in each patient at two time points (T1 and T2, connected by line) with (left; 6 M-CLL, 6 U-CLL) or without (right; 1 M-CLL, 13 U-CLL) therapy in the interim. Patients were administrated mainly fludarabine-cyclophosphamide-rituximab (FCR) regimen. ○ M-CLL, ● U-CLL: open circle; FCR; full circle: other therapy. Wilcoxon signed rank test. ns: not significant. (E) COBLL1 expression does not change with time or treatment. Protein expression was detected in 2 U-CLL COBLL1 -low patients (1 and 2) and 2 U-CLL COBLL1 -high patients (3 and 4) at two time points with or without therapy in the interim. Western blotting and anti-COBLL1 and anti-actin (as a loading control) antibody was used.

Article Snippet: Peripheral blood (PB) B cells from CLL patients or healthy volunteers and non-malignant tonsillar tissue were separated by non-B-cell depletion (RosetteSep CD3 + Cell Depletion Cocktail, RosetteSep B Cell Enrichment Cocktail, StemCell Technologies or magnetic B-cell isolation kit II, Miltenyi Biotec).

Techniques: Expressing, Isolation, MANN-WHITNEY, Western Blot, Control, Mutagenesis, Quantitative RT-PCR

Journal: Haematologica

Article Title: Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia

doi: 10.3324/haematol.2017.178699

Figure Lengend Snippet: U-CLL COBLL1 -high cells show higher response upon BCR stimulation. (A and B). Chronic lymphocytic leukemia (CLL) cells (4 mutated CLL (M-CLL), 8 U-CLL COBLL1 -low, 6 U-CLL COBLL1 -high) were stimulated for 4 minutes with anti-IgM and response to BCR stimulation was analyzed using phospho-specific antibodies targeted against pPLCγ2, pSYK and pBLNK. (A) Representative examples of M-CLL, U-CLL COBLL1 -low and U-CLL COBLL1 -high patients. Histograms show a negative control (unstimulated non-stained sample, dotted line), unstimulated sample (full line) and IgM-stimulated sample (full line, gray area). Percentage of positive cells is indicated (unstimulated sample → stimulated sample). (B) Quantification of changes in the pPLCγ2, pSYK and pBLNK. Phosphorylation increase (y-axis) was calculated as a ratio of positive cells in IgM-stimulated versus unstimulated samples. Box-and-Whisker plots show quartiles and median. Dashed line indicates phosphorylation increase in non-malignant peripheral blood (PB) B cells (mean), • outliers, * P ≤0.05, ** P ≤0.01, *** P ≤0.001. Mann-Whitney test. (C) Western blot analysis of representative U-CLL samples treated with anti-IgM and analyzed for activation of BCR components using phospho-specific antibodies - PLCγ2 (pY1217), pSYK (pY525/526), pAKT (pS473) and pERK1/2 (pT202/Y204). Loading control: β-actin (left), total PLCγ2 (right). (D) Correlation of the response at the level of individual kinases (Spearman correlation). Statistically significant P -values are highlighted in bold with gray background. See Online Supplementary Figure S7 for details and raw data.

Article Snippet: Peripheral blood (PB) B cells from CLL patients or healthy volunteers and non-malignant tonsillar tissue were separated by non-B-cell depletion (RosetteSep CD3 + Cell Depletion Cocktail, RosetteSep B Cell Enrichment Cocktail, StemCell Technologies or magnetic B-cell isolation kit II, Miltenyi Biotec).

Techniques: Negative Control, Staining, Phospho-proteomics, Whisker Assay, MANN-WHITNEY, Western Blot, Activation Assay, Control