RDA isolation of homozygous genomic deletion at the Smpd3 locus in F4328 mouse osteosarcoma cells. (A) Schematic representation of the homozygous deletion of the mouse chromosome 8 locus in F4328. A total of 4 nonredundant RDA clones (-1, -5, -14, and -22 [arrow])—were found in the intergenic region between Zfp90 and Smpd3. Genomic PCR analysis of the first exon of Zfp90 (1), intergenic region (2), the first (3) and the second (3) exons, and the second intron (4 and 5) of Smpd3 in the F4328 and unrelated control F3733 and F4711 cell lines are shown. (B) Mouse genomic DNAs from F4328 and control F3081, F3678, and F3587 cell lines were digested by BamHI and subjected to Southern blot analysis using RDA clone-1 or -14 as probes. A DNA fragment that was isolated by RDA due to a BglII polymorphism served as a probe for the loading control. (C) Expression of Smpd3 and Zfp90 genes in the F4328 cell line was analyzed by RT-PCR analysis. Primers were chosen in exons 6 and 10 for Smpd3, which are outside the genomic deletion. Gapdh was used as an internal loading control. (D,E) Reconstitution of F4328 cell lines with SMPD3. SMPD3 (□) or empty vector control (■) constructs were retrovirally transduced into F4328 cells. (D) The resulting cells were plated in 96-well format for measurement of growth rate. Cell numbers were measured by the absorbance at 600 nm after staining with DNA staining dye CYTO60 and plotted as fold increase compared with day 1. Error bar indicates standard error of mean. Protein expression of SMPD3, detected by anti-Flag antibody, is shown in the inset. (E) Cells in 96-well plate format were treated with the indicated concentration of TNFα for 4 days, and cell viability was measured by MTS assay. Percentage of viability is plotted compared with the mock-treated cells. Statistical significance of each matching data point was calculated (*P < .001; **P > .05).