Induction of PKCϵ and its effects on TRAIL-induced apoptosis in human erythroblasts. (A-B) Western blot detection of total PKCϵ protein expression in CD34 cells cultured in serum-free medium with the indicated cytokines. β-Actin was monitored for protein loading. (C) TRAIL-induced apoptosis in cells pretreated for 48 hours with 250 μg/mL PKCϵ inhibitor (+) or control peptide (–). Cell death is expressed as percentage of controls. (D) Semiquantitative RT-PCR analysis of residual PKCϵ mRNA expression after PKCϵ-siRNA transfection. RNA was recovered 48 hours after siRNA transfection. Lanes 1 to 5: PCRs of 10^–1 to 10^–5 dilutions of total cDNA obtained from 1 μg reverse-transcribed total RNA. (E) Western blot analysis of residual PKCϵ protein expression after PKCϵ-siRNA transfection. PKC-δ was used as control for PKCϵ-siRNA specificity. (F) PKCϵ-siRNA transfection increases cell sensitivity to TRAIL. Residual cell viability of erythroid cell lines HeL and K562 treated with PKCϵ-siRNA and challenged with TRAIL is reported as percentage of controls (some samples, in absence of TRAIL). Mean of 3 independent experiments is reported. *P < .05. (G) PKCϵ overexpression reduces TRAIL sensitivity of CD34-derived erythroblast. CD34 cells, derived from 3 unrelated donors and cultured with IL-3, SCF, and EPO for 24 hours, were transfected with GFP-PKCϵ or GFP-PKCϵm. Forty-eight hours later, cells were treated with 50 ng/mL TRAIL. Apoptosis was monitored 48 hours after TRAIL treatment, staining cells with annexin V–FITC and PI. TRAIL-induced cell death is reported as percentage of controls (mock-transfected CD34 cells from the same patients).