Figure 5.

Calcium blockers affect TG2 expression and NF-κB activity in CD45⁺CD19⁻ MCL-ICs. (A) Stem like MCL cells (CD45⁺CD19⁻ MCL-ICs) expressed TG2 protein. Western blot analysis of TG2 expression was performed using cell lysates from 5 different primary CD45⁺CD19⁻ MCL-ICs. The breast cancer cell line MDA-MB-231 was used as a positive control, and normal B cells were used as negative controls. β-actin was used as a loading control. Pt indicates patient. (B) Stem-like MCL cells expressed TG2 enzymatic activities comparable to positive controls. TG2 enzymatic activity was measured using the TG2-CovTest TG2-specific colorimetric assay kit (Novus Biologicals) with cell extracts from 5 different primary CD45⁺CD19⁻ MCL-ICs. Recombinant human TG2 was used as a positive control, and normal B cells were used as negative controls. (C) Treatment with BAPTA/AM or POH suppressed TG2 enzymatic activity in CD45⁺CD19⁻ MCL-ICs. Five different CD45⁺CD19⁻ MCL-ICs were treated with BAPTA/AM (60μM) or POH (1mM) for 16 hours. TG2 enzymatic activity was measured using a TG2-specific colorimetric assay kit with cell extracts from untreated and treated CD45⁺CD19⁻ MCL-ICs. Changes in TG2 enzymatic levels before and after treatment with BAPTA/AM or POH were measured. The colorimetric values in untreated samples were subtracted from the values measured in treated samples. Results are shown as the means ± SD. (D) Calcium blockers inhibited NF-κB DNA-binding activities in primary CD45⁺CD19⁻ MCL-ICs. Nuclear extracts from CD45⁺CD19⁻ MCL-ICs that were untreated or treated with BAPTA/AM (60μM for 16 hours) or POH (1mM for 16 hours) were analyzed using ELISA assays to evaluate p50 and p65 DNA-binding activities. The relative ratio values of NF-κB DNA-binding activities after drug treatment are shown as the means ± SD. *P < .05 by unpaired t test.