Figure 6
Figure 6. Patients’ B cells were resistant to PMA-induced cell death. (A) and (C) EBV-transformed B cells or PBMCs were cultured with PMA (100 ng/mL) or APO-1-3/protein A (1 μg/mK each). After 24 hours (for EBV B cells) or 48 hours (for PBMCs), cells were stained with DiOC6 (40 nM) for 15 minutes and live cells were counted by flow cytometry by constant time acquisition. (B) EBV-transformed B cells were stimulated with or without PMA or APO-1-3 for 6 hours, stained with propidium iodide (PI; 50 ng/mL) for 15 minutes, and analyzed by flow cytometry. (D) EBV-transformed B cells were stimulated with or without PMA for 3 hours, and cells were stained with FITC-activated caspase-3 (BD Pharmingen) according to the manufacturer’s instructions. (E) and (F) The patient’s EBV-transformed were transduced with pLVX-IRES-ZsGreen control vector or pLVX-IRES-ZsGreen-RRKCD using lentivirus system. Overexpression of PKCδ was analyzed by western blots (WB; E). Cells were stimulated with PMA (100 ng/mL) for 24 hours, live cells were counted by flow cytometry by constant time acquisition (F). Analysis was performed by gating on live ZsGreen expressed cells. Data are represented as means ± SE of 3 separate experiments. *P < .05 by Student t test for comparison with untreated cells.

Patients’ B cells were resistant to PMA-induced cell death. (A) and (C) EBV-transformed B cells or PBMCs were cultured with PMA (100 ng/mL) or APO-1-3/protein A (1 μg/mK each). After 24 hours (for EBV B cells) or 48 hours (for PBMCs), cells were stained with DiOC6 (40 nM) for 15 minutes and live cells were counted by flow cytometry by constant time acquisition. (B) EBV-transformed B cells were stimulated with or without PMA or APO-1-3 for 6 hours, stained with propidium iodide (PI; 50 ng/mL) for 15 minutes, and analyzed by flow cytometry. (D) EBV-transformed B cells were stimulated with or without PMA for 3 hours, and cells were stained with FITC-activated caspase-3 (BD Pharmingen) according to the manufacturer’s instructions. (E) and (F) The patient’s EBV-transformed were transduced with pLVX-IRES-ZsGreen control vector or pLVX-IRES-ZsGreen-RRKCD using lentivirus system. Overexpression of PKCδ was analyzed by western blots (WB; E). Cells were stimulated with PMA (100 ng/mL) for 24 hours, live cells were counted by flow cytometry by constant time acquisition (F). Analysis was performed by gating on live ZsGreen expressed cells. Data are represented as means ± SE of 3 separate experiments. *P < .05 by Student t test for comparison with untreated cells.

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