Role of GSK3β-mediated NFATc1 activation and PD-L1 induction in DLBCL cells. (A) Cytoplasmic extracts (CE) and nuclear extracts (NE) were purified from representative non-GCB and GCB DLBCL cell lines and subjected to western blot analysis for PD-L1, actin (loading control cytoplasmic extracts), NFATc1, pSTAT3, and lamin B (loading control NE). (B) LP (DLBCL) cells were treated with increasing doses of enzastaurin for 24 hours, and protein expression levels of pGSK3β, GSK3β, and actin were analyzed by western blotting using cytoplasmic extracts. Nuclear extracts were used for western blot analysis for NFATc1 and lamin B protein expression. (C) LP (DLBCL) cells were transfected with a control vector or GSK3β plasmid (mutant [mut] or wild-type [wt]). Protein expression levels of GSK3β, pGSK3β, actin, and NFATc1 (nuclear extraction) were determined by western blotting. (D) LP (DLBCL) cells transfected with wt GSK3β were treated with the GSK3β inhibitor enzastaurin (ENZ) or BTK inhibitor ibrutinib (IBN) for 24 hours, and protein expression levels of pGSK3β, GSK3β, and actin were analyzed by western blotting. (E) HBL-1 non-GCB DLBCL cells were stimulated with IgM, and, in some cases, IgM-stimulated cells were treated with ENZ or IBN for 24 hours. Nuclear extracts were examined for NFATc1 and pSTAT3 expression, whereas cytoplasmic extracts were analyzed for PD-L1 expression. Actin was used as a loading control. (F) OCI-LY10 cells were treated with different doses of ibrutinib, APC-196, or BGB-3111 for 24 hours. Purified nuclear extracts were used to examine pSTAT3, STAT3, NFATc1, and lamin B (loading control) protein expression levels by western blotting. Cytoplasmic extracts were used to examine PD-L1 and actin (loading control). (G) HBL-1 and LP cells were treated with ibrutinib, APC-196, or BGB-3111 for different times (24 or 48 hours), and cytoplasmic and nuclear extracts were purified and examined for expression levels of NFATc1, pSTAT3, lamin B, PD-L1, and actin (loading control).