Figure 3

hLsg1 is an essential protein. (A) Rapid disappearance of hLsg1 in cells transfected with hLsg1 specific siRNA. HeLa cells were transfected with an hLsg1-specific siRNA (+) or with a scrambled siRNA as a negative control (-), and were harvested at 0, 12 and 24 h post-transfection. Extracts were prepared and 30 μg of each sample were separated on an 8% polyacrylamide gel and analyzed for hLsg1 by western blotting. Untreated cell lysate (30 μg) from confluent HeLa cells was run as a control (Co). To assess the specificity of the siRNAs, 30 μg of each extract was run on a 12% SDS-PAGE gel and analyzed for actin content by western blotting. (B) Cell count. HeLa cells plated on coverslips and transfected with no siRNA (black box), hLsg1-specific siRNA (white box), or a scrambled siRNA (grey box) were fixed at 0, 12, 24 and 36 h post-transfection. Cells were labeled with Dapi and anti-hLsg1 antibodies and the cell number was counted. The graph is the sum of three independent experiments (C) YRG family member lethality. Literature survey and database searches indicating that YRG family members are essential.