shRNA directed against RPL35A mRNA causes decreased proliferation and apoptosis. (A) The efficacy of expression knockdown was assessed by real-time quantitative RT-PCR 4 days after transduction of UT-7/Epo or TF-1 cells with the lentiviral siRNA construct. Bar graph represents expression of RPL35A transcripts after normalization to GAPDH in each of 4 shRNA constructs compared with cells infected with a control shRNA-lentiviral construct targeting firefly luciferase. Bars represent the aggregated mean expression level from 5 independent experiments, 3 in TF-1 and 2 in UT-7/Epo. Error bars indicate SD. (B-E) UT-7/epo cell proliferation was assessed by quantitation of fluorescent dye DNA binding. GFP-positive UT-7/epo cells were sorted 3 days after lentiviral infection, subsequently plated (day 0) in 96-well plates in 5 replicates at 10³ cells/well, and assayed at the indicated times after sorting. Plating variation was corrected by adjusting the LOG2-transformed intensity at each day by the LOG2-transformed intensity on the initial day of plating. Curves represent the average of 4 platings from 2 independent experiments. Error bars indicate SDs. Differences between shLuc control-infected cells and RPL35A sh-2 were not significant. *sh-1, P < .05, compared with shLuc control cells. **sh-3 and 4, P < .01, compared with shLuc control cells. (F) Apoptosis was quantified by flow cytometric analysis after annexin V staining. GFP-positive UT-7/epo cells were sorted 3 days after infection and returned to culture for 24 hours. Cells were subsequently assessed on day 1 (top panel) and day 5 (bottom panel) after sorting. The histogram plots show cell counts versus annexin V intensity in shLuc (gray shading) and sh-3 (solid line) with apoptotic cells from each group enumerated above the gating threshold (M1). After sorting, no significant increase in annexin V–positive cells was discernable between control and sh-3–infected cells (K-S, P > .1). After adjustment for the proportion of annexin V–positive cells on day 1 (treatment group day 5 − treatment group day 1), RPL35A knockdown by day 5 resulted in a nearly 2.5-fold increase in annexin V–positive cells compared with controls (4.7% vs 11.7%; K-S, P < .001). Of note, a significant shift in the overall intensity of annexin V staining of the entire population of cells was also observed, suggesting that the overall effect on apoptosis was greater. Similar results were observed in sh-1– and sh-4–infected cells (not shown). Sh-Luc indicates Luciferase-control transduced cells; sh-1, -2, -3, and -4, respective RPL35A shRNAs.