Down-regulation of ATF3 by RNA interference impairs proliferation and compromises viability of Hodgkin cells. (A) L428 and L540Cy Hodgkin cells were transfected with control and ATF3-directed pSUPER siRNA expression plasmids, purified, and harvested 48 hours after electroporation. Whole-cell lysates were subjected to Western blot analysis using the indicated antibodies. (B) L540Cy HRS cells were transfected with the indicated pSUPER siRNA constructs, purified, and pulsed with [³H]-thymidine for 24 hours before harvesting and assessing incorporated radioactivity. Data are presented as cpm of [³H]-thymidine incorporation (mean ± SD of 5 measurements). (C) L428 and L540Cy HRS cells were transfected with control and ATF3-directed pRepH1 siRNA expression constructs and selected for siRNAexpressing cells by addition of puromycin 24 hours after electroporation. The percentage of viable and apoptotic cells was determined by annexin V-FITC/propidium iodide (PI) staining and flow cytometry. Viable L428 (left panel) and L540Cy (right panel) cells after 14 days in culture. The fraction of viable cells, negative for both annexin V-FITC and PI, is shown as percentage of total cells. Measurements were performed in triplicate. Error bars indicate SD.