Fig 1.
Proliferation of Mino and Mino/FR cells in presence of fludarabine and other anti-lymphoma agents.
Cells were grown for 6–8 days in presence of increasing concentrations of (A) fludarabine, (B) cladribine, (C) cytarabine, (D) gemcitabine, (E) ibrutinib, (F) bortezomib, (G) doxorubicin, (H) cisplatin, (I) bendamustine and (J) methotrexate. Relative toxicity of the drugs was determined by the WST-8 cell proliferation assay, Dashed lines with open circles or triangles indicate cell proliferation in absence of an anti-lymphoma drug. Other curves represent the cells grown in increasing concentrations (indicated by the associated number) of the tested drug. Maximal absorbance (highest number of viable cells) of cells grown without an anti-lymphoma agent in each experiment was set as 100%. Standard deviations were < 5% for all measurements.
Fig 2.
Correlation of protein expression ratios in forward and reverse SILAC experiments.
Heavy/Light protein ratios (log values) from the forward experiment were plotted against log values of Light/Heavy protein ratios obtained from the reverse labeling experiment.
Table 1.
Differentially expressed proteins in Mino/FR cells identified by the proteomic analysis.
Differentially expressed proteins with fold-change >1.5 are listed. Gene names are shown, proteins with fold-change >3 fold are present in bold letters).
Fig 3.
Verification of differential expression of the key proteins identified by proteomics.
(A) Relative expression of four differentially expressed proteins—deoxycytidine kinase (dCK), phoshatase SHP-1 (alias PTPN6), Bcl-2 and phoshoserine aminotransferase (PSAT-1)–was determined by Western Blotting using specific antibodies in Mino and Mino/FR cells. GAPDH was used as a loading control. (B) Relative expression of two surface CD markers (CD20 and CD38) determined by flow cytometry using specific antibodies. Open histograms represent Mino/FR cells, full histograms show Mino cells. Histograms demonstrate approximately 2-fold decreased expression of CD20 and CD38 in Mino/FR cells as indicated by decreased median fluorescence intensity.
Fig 4.
Mino/FR cell are highly sensitive to ABT-199.
Proliferation of Mino and Mino/FR cells in presence of of 0.01–10 μM Bcl-2 inhibitor ABT199. Cells were grown for 6–8 days in presence ABT199. Relative toxicity of the drugs was determined by the WST-8 cell proliferation assay. Dashed curves and open circles or triangles indicate cell proliferation in absence of ABT199. Maximum absorbance (highest number of viable cells) of cells grown without ABT199 experiment was set as 100%. Other curves represent the cells grown in increasing concentrations (indicated by the associated number) of ABT199. Standard deviations were < 5% for all measurements.
Fig 5.
Decreased levels of total BTK and its activated p-BTKY233 form in Mino/FR cells.
Relative expression of total and phosphorylated (active) p-BTK233 was determined by Western blotting using specific antibodies in Mino and Mino/FR cells. GAPDH was used as the loading control.
Fig 6.
Schematic illustration of the processes associated with fludarabine resistance in Mino/FR cells summarizes the landmark observations in our analyses.