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First published online 25 January 2005
doi: 10.1242/jcs.01652

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RNAi of FACE1 protease results in growth inhibition of human cells expressing lamin A: implications for Hutchinson-Gilford progeria syndrome

Jens Gruber^*, Tina Lampe^*, Mary Osborn and Klaus Weber

Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Am Fassberg 11, 37077 Göttingen, Germany

View larger version (58K): [in a new window]   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.

View larger version (72K): [in a new window]   Fig. 2. Silencing of FACE1 causes abnormalities in nuclear morphology. (A) Control HeLa cells (a) show a normal nuclear morphology whereas FACE1-silenced HeLa cells display a range of abnormal nuclear morphologies (b-h) when stained with Hoechst dye. The abnormalities included minor effects such as herniations in the nuclear periphery (b) and folds (c-f), or more severe effects such as lobules (f) and fragments (g) as well as formation of micronuclei (h). The number of cells with severe abnormalities increased significantly at later times after transfection. (B) Comparative statistics. Cells were divided into four classes. `Normal' nuclei have a morphology as in a; `folds and herniations' as in c-f, fragmentation as in g; and micronuclei as in h. The percentage of normal nuclei decreased from 60% at 24 hours to 20% at 72 hours. Folds and herniations reached a maximum (>40%) 48 hours after transfection and then decreased slowly. The number of cells with micronuclei increased throughout the observation time and reached a maximum (>30%) 120 hours post transfection. (C) Cells treated with FACE1 siRNAs in-phase superimposed with prelamin A stain (red) and Hoechst DANN stain (blue) to show micronuclei. Bars, 15 µm.

View larger version (74K): [in a new window]   Fig. 3. Aberrant mitosis and apoptosis are consequences of FACE1 knockdown. (A) RNAi mediated knockdown of FACE1 led to mitotic arrest of HeLa SS6 cells within 24 hours of transfection. Arrested cells (top) displayed abnormal mitotic spindle architecture and disturbed chromosome congression. Control cells (bottom) transfected with GL2 siRNA showed normal mitosis. Cells were stained with tubulin antibody and with Hoechst dye. (B) TUNEL assay on FACE1-silenced (top) and control cells (bottom) 72 hours after siRNA transfection shows that the mitotically arrested cells have entered apoptosis. (C) Quantification of mitotic arrest after FACE1 silencing. The fraction of cells showing mitotic arrest increased with time after transfection, reaching a maximum of 35% of the total cell population at 48 hours. Cells transfected with either GL2 or with lamin A siRNA did not arrest in mitosis. (D) Quantification of apoptosis after FACE1 silencing. The percentage of apoptotic cells reached a maximum of 25% 72 hours after transfection and then decreased. Apoptosis was observed only in FACE1-silenced cells, but not in control (GL2 siRNA) or in lamin A siRNA-transfected cells. (E) Cell growth measured with the CellScreen apparatus was reduced dramatically after silencing with FACE1 siRNA. Bar, 15 µm.

View larger version (59K): [in a new window]   Fig. 4. Double RNAi suggests prelamin A is responsible for phenotypic changes after FACE1 silencing. (A) Silencing of lamin A prior to FACE1 prevented phenotypic changes. Human HeLa SS6 cells were transfected with the siRNA specific for the first target and after 24 hours with the second siRNA. Cells were stained for tubulin and for DNA 24 hours after the second transfection. (a,b) Knockdown of lamin A before FACE1 did not cause abnormal mitosis, apoptosis or changes in the nuclear morphology. (c,d) Silencing of FACE1 prior to silencing of lamin A led to aberrant mitotic arrest. (B) Successful transfection in the double knockdown experiments was confirmed by the branched DNA assay. Data represent the relative mRNA levels 24 hours after the second transfection for the LMNA-FACE1 combination () and for the FACE1-LMNA combination. Double siGL2 transfection was used for normalisation (GL2). That the second transfection was effective was further confirmed by transfecting either FACE1 or lamin A siRNA 24 hours after siGL2 delivery (GL2-FACE1 and GL2-LMNA). Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 5. Immunofluorescence detection of FACE1 in the endoplasmic reticulum and the nuclear periphery. (A) HeLa cells were stained with dihexyloxacarbocyanine iodide to label the endoplasmic reticulum and then with the rabbit FACE1 antibody against residues 296-313. FACE1 is seen in the endoplasmic reticulum identified by DiOC6 staining and is also seen in spots at the nuclear periphery. (B) Immunofluorescence with the FACE1-specific antibody 48 hours after transfection of FACE1 or GL2 siRNA demonstrates silencing of FACE1 in cells transfected with FACE1 siRNA, but not in cells transfected with GL2 siRNA. Bar, 10 µm.