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Identification of essential genes in cultured mammalian cells using small interfering RNAs

Jens Harborth^1,*, Sayda M. Elbashir^2,*, Kim Bechert¹, Thomas Tuschl^2, and Klaus Weber^1,

¹ Department of Biochemistry and Cell Biology and
² Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
^* These authors contributed equally to this work

View larger version (77K): [in a new window]   Fig. 1. Silencing of SV40 large T antigen in a stably transformed rat fibroblast cell line (F5). Triple fluorescence staining of cells transfected with T-antigen siRNA duplex (A,C,E), and with GL2 luciferase siRNA duplex serving as control (B,D,F). (A,B) Staining with T-antigen specific antibody, (C,D) staining with NuMA specific antibody, (E,F) Hoechst staining of chromatin. The few cells cells in A that are stained for T antigen were probably nontransfected. (G) Western blot of cells transfected with T antigen siRNA (left) or luciferase siRNA (right) probed with T antigen-specific antibody. The blot was stripped and reprobed with vimentin antibody (bottom). Magnification 480x (A-F).

View larger version (66K): [in a new window]   Fig. 2. Silencing of lamin A/C in HeLa cells results in displacement of emerin. Cells transfected with lamin A/C siRNA (A) and with luciferase siRNA (B) were stained with emerin antibody. Cells with reduced lamin A/C expression (A) show enrichment of emerin in the cytoplasm. Magnification 480x.

View larger version (105K): [in a new window]   Fig. 3. Silencing of lamin B1 and lamin B2. HeLa cells were transfected with lamin B1 siRNA (A,C), lamin B2 siRNA (E) or with luciferase siRNA (B,D,F). Cells were either double stained with lamin B1-specific antibody (A,B) and NuMA specific antibody (C,D) or stained for lamin B2 (E,F). (G) Western blot of cells transfected with lamin B1 (left) or luciferase (right) siRNA duplexes using lamin B1 antibody (top). The blot was stripped and re-probed with vimentin antibody to check for equal loading (bottom). Magnification 480x (A-F).

View larger version (58K): [in a new window]   Fig. 4. Silencing of the nuclear pore complex protein Nup153. (A) Phase-contrast of HeLa cells 3 days after transfection with Nup153 siRNA. (B) Western blot of cells transfected with Nup153 siRNA (left) or with luciferase siRNA (right) probed with Nup153-specific antibody. Magnification 300x (A).

View larger version (109K): [in a new window]   Fig. 5. Silencing of cytoplasmic actins in HeLa cells. (A) Phase-contrast of cells transfected with ß-actin siRNA duplex. Transfected cells show blebbing. (B) Western blot of cells transfected with ß-actin (left) or luciferase (right) siRNA duplexes using ß-actin antibody. The blot was stripped and re-probed with vimentin antibody to check for equal loading. (C) Phase-contrast of cells transfected with -actin siRNA duplex. Transfected cells show a blebbing phenotype similar to cells with ß-actin knockdown. Magnification 480x (A,C).

View larger version (79K): [in a new window]   Fig. 6. Silencing of the cytoskeletal intermediate filament protein vimentin. Cells were transfected with vimentin duplex 2 (bp 1145-1167) (A-C) or with GL2 luciferase duplex (D). (A,B,D) Staining with vimentin V9 antibody; (C) Hoechst staining. Note the strong decrease of vimentin staining in cells transfected with vimentin duplex 2 (A) when compared with the control (D). Only at longer exposure times (B) can short filamentous structures (vimentin squiggles) (arrow) be seen. (E) Western blot using vimentin antibody (top panel), and ß-actin antibody as control (bottom panel). Magnification 460x (A-D).

View larger version (58K): [in a new window]   Fig. 7. Silencing of the kinesin-related motor protein Eg5 and of the microtubular motor CENP-E. HeLa cells were transfected with Eg5 siRNA (A,D,G-I) or with luciferase control siRNA (B,C,E,F). Cells are stained for -tubulin (A-C) with a corresponding Hoechst stain (D-F). Cells transfected with Eg5 siRNA are arrested in mitosis and show monoastral microtubular arrays (A,D). By contrast, control cells show normal bipolar spindles in mitosis (B,E) and microtubule networks in interphase (C,F). A higher magnification of cells transfected with the Eg5 siRNA clearly shows the monopolar spindle (G), which does not overlap with the chromatin staining (H); for overlay see I. HeLa cells were transfected with CENP-E siRNA (J-M). Cells were stained with -tubulin antibody (J,K) and Hoechst (L,M). Note the production of multipolar arrays. The higher magnification of a metaphase cell (K,M) indicates that the loss of CENP-E affects chromosome congression to the spindle equator (arrows). Magnification 480x (A-F,J-M); 750x (G-I).

View larger version (108K): [in a new window]   Fig. 8. Silencing of the cyclin-dependent kinase 1 (cdk1). HeLa cells were transfected with cdk1 siRNA (A,C,D) or with luciferase siRNA (B) and stained for lamin A/C (A-C). Cells transfected with cdk1 siRNA round up. Higher magnification shows that the lamina of these cells starts to depolymerize (C). (D) Phase-contrast of HeLa cells 2 days after transfection with cdk1 siRNA. Magnification 480x (A,B); 760x (C); 300x (D).

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