Fig. 1. Silencing of human FACE1 results in accumulation of prelamin A. (A) Human HeLa SS6 cells were immunostained with the antibody SC06214 recognising an epitope in the C-terminal region of unprocessed lamin A. This antibody detects prelamin A, but not lamin A. Transfection with an unspecific GL2 siRNA did not affect the normal localisation of prelamin A in subnuclear foci (row 1). Transfection with FACE1 siRNA results in progressive accumulation of unprocessed prelamin A in the nuclear lamina (row 2). Transfection of cells with lamin A siRNA targeting lamin A/C mRNA resulted in loss of prelamin A (row 3). Hoechst stain of the same cells (row 4). (B) Accumulation of prelamin A was confirmed by western blots of FACE1-silenced cells (siFACE1) and control populations (siGL2). Prelamin A accumulated in FACE1-silenced cells 48 hours after transfection, but was not detectable in extracts from cells transfected with the GL2 siRNA. (C) Silencing of FACE1 and lamin A in single siRNA experiments was confirmed by luminometric measurement of absolute mRNA levels via the branched DNA assay. Note that after 24 hours the mRNA is very much reduced for both targets and that these low levels are maintained at least until 96 hours after transfection. Bar, 20 µm.