Figure 4.
Lynup-Bslightly enhances ex vivo viability of murine CLL cells, but Lynup-B-induced BCR responsiveness is masked in the Eµ-TCL1 transgenic background. (A) Flow cytometry analysis of viable (Annexin V−) B cells upon treatment with the Lyn/Src inhibitors dasatinib and bosutinib at stated concentrations for 24 hours. (B) Flow cytometry analysis of viable (Annexin V−), IgM-stimulated B cells upon treatment with the Lyn/Src inhibitors dasatinib and bosutinib at stated concentrations for 24 hours. (C) Flow cytometry analysis of viable (7-AAD− Annexin V-) B cells upon treatment with BCR inhibitors dasatinib, entospletinib, ibrutinib and idelalisib at 5 µM for 24 hours. (D) Flow cytometry analysis of viable (7-AAD− Annexin V−), IgM-stimulated B cells upon treatment with BCR inhibitors dasatinib, entospletinib, ibrutinib, and idelalisib at 5 µM for 24 hours. (E) Western blot analysis of purified B cells isolated from spleens of TCL1tg/wt and TCL1tg/wt Lynup-B mice. Cells were kept untreated or stimulated with 20 mg/mL IgM for 10 minutes before lysis. (F) Tyrosine phosphorylation profile of purified B cells from TCL1tg/wt (n = 3) and TCL1tg/wt Lynup-B (n = 3) mice upon IgM stimulation. Kinases in B-cell lysates actively phosphorylated substrates on the PamChip. Tyrosine phosphorylation was detected by a FITC-conjugated PY20 antibody to quantify the phosphorylation signal. LFC values between untreated and IgM-stimulated samples were calculated. Each column of the heatmap represents the mean LFC of 3 mice per genotype. A red row indicates higher phosphorylation of the peptides upon IgM treatment, and a blue row implies a lower phosphorylation after IgM treatment. Supplemental Table 4 provides the LFC values for each phosphorylated peptide on the PamChip. (G) Dephosphorylation profile of purified B cells from TCL1tg/wt (n = 2) and TCL1tg/wt Lynup-B (n = 2) mice upon IgM stimulation. Phosphatases in the B-cell lysates dephosphorylated proprietary nitrophosphotyrosine residues on a chip. Unphosphorylated nitrotyrosine residues were detected by an anti-nitrotyrosine antibody. LFC values between untreated and IgM-stimulated samples. Each column of the heatmap represents the mean LFC of 2 mice per genotype. A red row indicates higher dephosphorylation of the peptides after IgM treatment, and a blue row implies a lower dephosphorylation after IgM treatment. Supplemental Table 5 provides the LFC values for each dephosphorylated peptide on the phosphatase chip.

Lynup-Bslightly enhances ex vivo viability of murine CLL cells, but Lynup-B-induced BCR responsiveness is masked in the Eµ-TCL1 transgenic background. (A) Flow cytometry analysis of viable (Annexin V−) B cells upon treatment with the Lyn/Src inhibitors dasatinib and bosutinib at stated concentrations for 24 hours. (B) Flow cytometry analysis of viable (Annexin V−), IgM-stimulated B cells upon treatment with the Lyn/Src inhibitors dasatinib and bosutinib at stated concentrations for 24 hours. (C) Flow cytometry analysis of viable (7-AAD− Annexin V-) B cells upon treatment with BCR inhibitors dasatinib, entospletinib, ibrutinib and idelalisib at 5 µM for 24 hours. (D) Flow cytometry analysis of viable (7-AAD− Annexin V−), IgM-stimulated B cells upon treatment with BCR inhibitors dasatinib, entospletinib, ibrutinib, and idelalisib at 5 µM for 24 hours. (E) Western blot analysis of purified B cells isolated from spleens of TCL1tg/wt and TCL1tg/wt Lynup-B mice. Cells were kept untreated or stimulated with 20 mg/mL IgM for 10 minutes before lysis. (F) Tyrosine phosphorylation profile of purified B cells from TCL1tg/wt (n = 3) and TCL1tg/wt Lynup-B (n = 3) mice upon IgM stimulation. Kinases in B-cell lysates actively phosphorylated substrates on the PamChip. Tyrosine phosphorylation was detected by a FITC-conjugated PY20 antibody to quantify the phosphorylation signal. LFC values between untreated and IgM-stimulated samples were calculated. Each column of the heatmap represents the mean LFC of 3 mice per genotype. A red row indicates higher phosphorylation of the peptides upon IgM treatment, and a blue row implies a lower phosphorylation after IgM treatment. Supplemental Table 4 provides the LFC values for each phosphorylated peptide on the PamChip. (G) Dephosphorylation profile of purified B cells from TCL1tg/wt (n = 2) and TCL1tg/wt Lynup-B (n = 2) mice upon IgM stimulation. Phosphatases in the B-cell lysates dephosphorylated proprietary nitrophosphotyrosine residues on a chip. Unphosphorylated nitrotyrosine residues were detected by an anti-nitrotyrosine antibody. LFC values between untreated and IgM-stimulated samples. Each column of the heatmap represents the mean LFC of 2 mice per genotype. A red row indicates higher dephosphorylation of the peptides after IgM treatment, and a blue row implies a lower dephosphorylation after IgM treatment. Supplemental Table 5 provides the LFC values for each dephosphorylated peptide on the phosphatase chip.

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