Response of BTK mutant REC-1 cells to the different covalent and noncovalent BTK inhibitors. (A) VAF of the different BTK mutations acquired in the REC-1 cells are indicated. Each of the cell lines (WT or BTK mutant) were treated in triplicates with increasing concentrations of the indicated covalent and noncovalent BTK inhibitors. Cell viability was measured after 96 hours using CellTiter Glo (Promega). Heatmap on the left shows the fold change in the IC50s of the respective cell lines compared to the WT cells for each of the BTK inhibitors. The cell viability heatmap is a summary of 3 independent experiments. Intracellular calcium flux was determined after exposing the different REC-1 cell lines (WT or BTK mutant) to 1 μM of the covalent or noncovalent BTK inhibitors for 1 hour, followed by stimulation with 10μg/mL of anti-IgM. The heatmap on the right shows percentage inhibition of the maximum calcium flux obtained upon BCR stimulation. The values for calculating the percentage inhibition were taken from representative experiments shown in supplemetal Figure 7A. Calcium flux measurement for each independent REC-1 line treated with 8 different BTK inhibitors and dimethyl sulfoxide control was performed at least twice. (B) COS-7 cells were transfected as indicated with 100 ng/well of vector encoding PLCγ2 and 400 ng/well of empty vector (∅) or increasing amounts (100, 200, and 400 ng/well) of vector encoding WT, C481S, C481F, G409R, L528W, L528S, G480R, D539H or K430R mutant BTK. Twenty-four hours after transfection, the cells were incubated for 18 hours with myo-[2-³H] inositol, and inositol phosphate formation was then determined. Results are one representative from 3 independent experiments and illustrated as mean ± SD of 3 technical replicates (C) Representative western blot showing IGF1R expression in BTK mutant REC-1 cells in comparison to WT. (D) Expression of IGF1R in the BTK mutant REC-1 cells normalized to that of WT. IGF1R expression was quantified using ImageJ from 2 repetitions of the western blots and normalized to the corresponding GAPDH controls. (E) The BTK mutant and WT REC-1 cells were treated with increasing concentrations of the IGF1R inhibitor linsitinib for 96 hours and cell viability was measured using CellTiter Glo (Promega). The data shown is a summary of 3 independent experiments performed in triplicates.