Article Title: Tonic B-cell receptor signaling in diffuse large B-cell lymphoma
Figure Lengend Snippet: Effect of BCR surface density on tonic BCR signaling. (A) BCR surface density, measured by anti-IgH isotype antibody staining and FCM, increases with doxycycline (Dox) concentration in cells of OCI-Ly19 and SUDHL-4 GCB-DLBCL lines engineered with both KO of the endogenous IgH locus and expression of its same-isotype IgH by a Dox-inducible expression vector. Red and blue arrows on the y-axis mark the similarly determined BCR surface density of unmodified (WT) cells of the lines. For each line, BCR surface density is based on flow cytometric measurement of fluorescence intensity with an antibody specific for its IgH isotype; therefore, values are not directly comparable between isotypes or to BCR surface density determined with a different antibody (eg, to IgL) or by CD79A-GFP fluorescence. (B) Absolute growth curves for GCB-DLBCL lines with inducible IgH expression, relative to the starting point at the indicated Dox concentration (3 days for OCI-Ly19, 5 days for SUDHL-4), showing that growth rates increase with Dox concentration. (C) Correlation between BCR surface density and relative proliferation, defined here as the ratio of slopes of absolute growth curves at different Dox concentrations to that of the curve for maximal Dox concentration (100 ng/mL), for GCB-DLBCL lines at different levels of induced IgH expression. (D) Correlation between BCR surface density and cell size, estimated by the forward scatter value, for GCB-DLBCL lines at different levels of induced IgH expression. (E) Correlation between BCR surface density and AKT activity, measured by FRET efficiency of an AKT activity reporter, for OCI-Ly19 cells with different levels of induced IgH expression, achieved by different durations (up to 7 days) of Dox withdrawal. Data from 2 independent experiments are displayed.
Article Snippet: The FRET-based AKT activity reporter AktAR2 (Addgene) was modified by N-terminal addition of the 10 N-terminal amino acids (AA) from Lyn kinase.
Techniques: Staining, Concentration Assay, Expressing, Plasmid Preparation, Flow Cytometry, Fluorescence, Activity Assay