Antisense oligonucleotide-inhibition of HCP expression enhances EPO-induced SKT6 cell hemoglobinization. (A) SKT6 cells were transfected with HCP antisense (AS) or control (S) oligonucleotides at 20 μmol/L and 40 μmol/L concentrations using DMRIE-C as a liposomal agent (see the Materials and Methods). At 48 hours posttransfection, frequencies of hemoglobinized cells in EPO-exposed versus control cultures were assayed by staining with DAF. Values are means ± standard deviations for three replicate cultures and are expressed as the percentage of maximal response to EPO. (B) To further illustrate effects of the treatment of SKT6 cells with HCP antisense oligonucleotides, data from above (A; 40 μmol/L oligonucleotide exposure) are expressed directly as frequencies of hemoglobinized cells, and SKT6 cells treated with oligonucleotides are compared with cells treated in parallel with DMRIE-c alone (right histograms). (C) In experiments in which SKT6 cells were transfected with antisense (AS) versus control (S) oligonucleotides at 40 μmol/L, the specific inhibition of HCP expression was assessed. After stimulation with EPO (+10 U/mL for 48 hours) cell lysates were prepared, and levels of HCP expression were assayed by immunoprecipitation and Western blotting. Based on densitometry, an estimated twofold inhibition of HCP expression was achieved by antisense oligonucleotide treatment.