Fig.6

BTK inhibitors promoted CYLD-dependent apoptosis of rituximab-resistant non-GCB-DLBCL cells. a Construction of rituximab-resistant cell line by the concentration gradient method. Rituximab-resistant OCI-Ly10 cells (OCI-Ly10R) and rituximab-sensitive OCI-Ly10 cells (OCI-Ly10S) were treated with DMSO or rituximab and cell viability was assayed by CellTiter-Glo assay. b OCI-Ly10R and OCI-Ly10S cells were labeled of 51Cr, lysis was collected individually and γ emission was measured. c OCI-Ly10R and OCI-Ly10S cells were treated with CCK-8 reagent, the OD value at 450 nm was measured and the inhibition rate was calculated. d Parental OCI-Ly10 cells were exposed to sequentially increasing concentrations of rituximab for 48 h. Cells then stained with anti-CD20-FITC antibody and phenotyping of OCI-Ly10S and OCI-Ly10R cells were performed on flow cytometer. e, f OCI-Ly10S (e) and OCI-Ly10R (f) cells were treated with DMSO, 50 μg/ml rituximab, 10 μM PCI-32765 or combination of 50 μg/ml rituximab and 10 μM PCI-32765 for 48 h. Lysates were blotted with the indicated antibodies. g OCI-Ly10S and OCI-Ly10R cells were treated with DMSO, 50 μg/ml rituximab, 10 μM PCI-32765 or combination of 50 μg/ml rituximab and 10 μM PCI-32765 for 72 h. Cell viability was assayed by CellTiter-Glo assay. h OCI-Ly10R cells were transduced with lentiviruses encoding a non-targeting or CYLD-targeting shRNA. Lysates were blotted with the indicated antibodies. i Control or CYLD-deficient OCI-Ly10R cells were treated with DMSO or 50 μg/ml rituximab or 10 μM PCI-32765 or combination of 50 μg/ml rituximab and 10 μM PCI-32765 for 72 h. Cell viability was assayed by CellTiter-Glo assay. j Control or CYLD-deficient OCI-Ly10R cells were treated with DMSO or 10 μM PCI-32765 for 48 h. Lysates were blotted with the indicated antibodies. The ATP levels for each cell line treated with DMSO were set at 100%. The bars represent the mean ± SD from three independent experiments. **P < 0.01