Figure 3.

Catalytically active RNASET2 complements mutant 2B1 cells. (a) CHO cells were transiently transfected with empty vector (EV), wild type (WT), or catalytically inactive mutant (CI) murine RNASET2 sequences. Cells were analyzed by western blotting for expression of murine RNASET2 (antibody does not recognize hamster species) and hsp90 as a loading control, and by in-gel zymography (zym) for ribonuclease activity at pH 5 and pH 7. For zymography panels, bands represent areas of enzymatic activity in RNA impregnated gel. Images are representative of three independent experiments. (b–d) Mutant 2B1 cells were transfected with EV or plasmids encoding WT or CI murine RNASET2. Independent stable clonal cell lines were isolated for each construct and analyzed following growth in normal media (untreated, UT, black bars) or following supplementation with 500 μM palm for 48 h (hatched bars). RNA was isolated and analyzed by qRT-PCR for RNASET2 expression relative to 36B4 using primers specific for the murine RNASET2 transcript (b). RNASET2 protein levels were analyzed by western blotting of total cell lysates. Graph shows mean densitometry of RNASET2 species normalized to hsp90 (c). Cell death was assayed by PI staining and flow cytometric analysis on 10⁴ cells/sample (d). Data are expressed as mean+S.E. for a minimum of n=3 samples per condition/cell type. *P<0.05 for comparisons indicated