Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 1. Model for RNA interference. dsRNA is processed to 21- to 23- nt siRNA duplexes by Dicer RNase III and presumably other factors. The siRNA duplexes are incorporated into RISC, which targets ho- mologous mRNAs for degradation. Ago2 and yet to be characterized proteins participate in RISC formation.

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques:

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 2. Selection of siRNA duplexes. (A) The design of siRNA duplexes for target mRNAs that contain the sequence AA(N19)UU. (B) The design of siRNA duplexes in the absence of AA(N19)UU target sequences. As long as one adenosine is present in the targeted region, siRNA duplexes with 30- TT overhangs can be used without effect on specificity of target recognition or RNAi efficiency.

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Selection, Sequencing

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 3. Analysis of siRNA duplex formation. Single-stranded (ss) sense RNA (s), antisense RNA (as), and annealed siRNA duplexes (ds) were separated on 4% NuSieve agarose gels. A 25-bp DNA ladder (M) was loaded as marker. The bands were visualized by UV light after ethi- dium bromide staining. The siRNA duplex was used for targeting la- min A/C mRNA [1].

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Marker, Staining

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 4. Detection of RNAi in mammalian cells by cotransfection of reporter constructs. (A) Reporter constructs. The firefly (Pp-luc) and sea pansy (Rr-luc) luciferase reporter-gene regions from plasmids pGL2-Control, pGL3-Control, and pRL-TK are illustrated. The promoter, enhancer and poly (A) region are indicated. The sequences of GL2 and GL3 luciferase are 95% identical but completely unrelated to that of RL. The region targeted by the siRNA duplexes is indicated as a black bar below the coding region. (B) siRNA duplexes targeting GL2, GL3, and RL luciferase. GL2 and GL3 differ by only three single-nucleotide substitutions. As nonspecific control, a duplex with the inverted GL2 (invGL2) sequence was included. The 2-nt 30 overhang of 20-deoxythymidine is indicated as TT. (C–G) RNA interference measurements. Ratios of target to control luciferase were normalized to that of a buffer control (bu, black bars); patterned bars indicate ratios of firefly (Pp-luc) GL2 or GL3 luciferase to sea pansy (Rr- luc) RL luciferase (left axis); white bars indicate RL to GL2 or GL3 ratios (right axis). The cell line used for the interference experiment is indicated at the top of each plot. Cotransfection of plasmids was performed with 0.21 lg siRNA duplex/well for HeLa cells (C, D) and with 0.84 lg for the other cell lines tested (E–G); see also protocol 4.

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Cotransfection, Construct, Luciferase, Control, Sequencing

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 5. Variation of the length of siRNA duplexes with preserved 2-nt 30 overhangs. The siRNA duplexes were extended to the 30 side of the sense siRNA (A) or the 50 side of the sense siRNA (B). The siRNA duplex sequences and the respective interference ratios are indicated. For HeLa SS6 cells, siRNA duplexes (0.84 lg) targeting GL2 luciferase were transfected together with pGL2-Control and pRL-TK plasmids (protocol 4). For comparison, the in vitro RNAi activities of siRNA duplexes tested in D. melanogaster lysate are indicated [23].

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Luciferase, Transfection, Control, Comparison, In Vitro

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 7. Recovery of lamin A/C knockdown cells. HeLa SS6 cells were transfected with the lamin A/C siRNA duplex (protocol 5) and fixed and stained with lamin A/C-specific antibody at the indicated time points (protocol 6). (D) Cells transfected with a control GL2 luciferase siRNA duplex. Cells were split every second or third day to maintain exponential growth. Although the majority of cells has recovered from the knockdown 9 days posttransfection, a few cells still display reduced lamin A/C levels (C).

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Knockdown, Transfection, Staining, Control, Luciferase

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 6. Knockdown of lamin A/C protein. (A) Lamin A/C siRNA duplex. The targeted region of lamin A/C mRNA is indicated with respect to the start codon (pos. 1). HeLa SS6 cells were transfected with either lamin A/C siRNA (B, D) or GL2 Pp-luc control siRNA duplex (C, E) according to protocol 5. Knockdown of lamin A/C was monitored as described in protocol 6 using lamin A/C antibody 636 (Santa Cruz Biotechnology, No. sc- 7292; or Novocastra, No. NCL-LAM-A/C) The bright cell located at the bottom left-hand corner of (B) was not transfected.

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Knockdown, Transfection, Control

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 8. Western blot analysis of HeLa SS6 cells transfected with various concentrations of lamin A/C siRNA duplex. Transfection of siRNA duplex was performed according to protocol 5 and the amount of duplex was varied from 0.84 lg to 8.4 pg, which corresponds to a final duplex con- centration in the medium of 100 nM–1 pM, respectively. Cells transfected with buffer or 100 nM GL2 Pp-luc siRNA duplex served as control. Western blotting was performed according to protocol 7.

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Western Blot, Transfection, Control

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 9. Double-stranded siRNAs are required for gene silencing. HeLa SS6 cells were transfected with either duplex siRNA (ds) or single-stranded sense (s) or antisense (as) siRNA according to protocol 5. For transfection, 8.4 ng (A) or 0.84 lg (B) of sense, antisense, or dsRNA were used, which corresponds to 1 or 100 nM final siRNA duplex concentration and 2 or 200 nM single-stranded siRNA concentration. As nonspecific control, GL2 Pp-luc siRNAs were used. Western blotting was performed according to protocol 7, harvesting cells 2 days posttransfection. Following lamin A/C detection by ECL, the blot was stripped and reprobed with vimentin antibody to control for loading (see bottom).

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Transfection, Concentration Assay, Control, Western Blot

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 10. Double knockdown of lamin A/C and NuMA in HeLa SS6 cells. (A) NuMA siRNA duplex. Cells transfected with 0.42 lg lamin A/C and 0.42 lg NuMA siRNA duplexes (B, D) and with 0.84 lg of GL2 luciferase siRNA duplex control (C, E) according to protocol 5. Two days after transfection, cells were examined with lamin A/C (B, C) or NuMA antibodies (D, E). Primary antibodies for lamin A/C (mouse monoclonal) and NuMA (affinity purified polyclonal [41]) were applied together, followed by simultaneous incubation of the secondary antibodies (Cy3 anti-mouse, Alexa 488 anti-rabbit) according to protocol 7. The majority of cells shows reduced expression of both target proteins; the single bright cell lighting up in (B) and (D) was nontransfected.

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Expressing

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 11. Knockdown of the abundant cytoskeletal intermediate filament protein vimentin and comparison of the efficiency of different siRNA du- plexes. (A) Sequences of vimentin siRNA duplex. (B) Immunofluorescence detection of vimentin 3 days posttransfection of HeLa SS6 cells. siRNA duplexes V1 and V4 are not efficient enough to suppress vimentin expression. 50-Phosphorylation of siRNA duplex V1 (V1-P) has no effect. As control, transfection with GL2 luciferase siRNA duplex (GL2) was performed. Vimentin protein was detected with vimentin V9 antibody (Novo- castra, No. NCL-VIM-V9) as described in protocol 6. (C) Western blot analysis of HeLa SS6 cells 3 days posttransfection with vimentin siRNA duplexes according to protocol 7. In agreement with the immunofluorescence results (B) V3 appears to be the most efficient siRNA duplex. Some of the residual vimentin has to be accounted for by incomplete transfection.

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Knockdown, Comparison, Expressing, Phospho-proteomics, Control, Transfection, Luciferase, Western Blot

Journal: Methods

Article Title: Analysis of gene function in somatic mammalian cells using small interfering RNAs

doi: 10.1016/s1046-2023(02)00023-3

Figure Lengend Snippet: Fig. 12. Knockdown of the kinesin-related motor protein Eg5 causes mitotic defects. (A) Eg5 siRNA duplex. HeLa SS6 cells were transfected with Eg5 siRNA (B, E) or GL2 luciferase control siRNA duplexes (C, D, F, G). Two days posttransfection, spindle formation was analyzed by staining cells for a-tubulin with a-tubulin antibody (Sigma, No. T-9026) (B–D). Nuclear chromatin was stained with Hoechst dye (E–G). Eg5 siRNAs triggered aberrant mitotic arrest [24] with monoastral microtubular arrays (B). This phenotype was previously observed by alternative methods such as antibody microinjection [42] or application of the small molecule inhibitor monastrol [43]. In control experiments applying GL2 siRNAs, normal bipolar spindles were observed during mitosis (C, F) or normal microtubules during interphase (D, G).

Article Snippet: A new siRNA transfection reagent, TransIT-TKO (Mirus, No. MIR 2150, www.genetransfer.com) has recently become available.

Techniques: Knockdown, Transfection, Luciferase, Control, Staining, Microinjection

Journal: The Journal of Cell Biology

Article Title: Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor–κB activation in muramyl dipeptide recognition

doi: 10.1083/jcb.200502153

Figure Lengend Snippet: Expression and cellular localization of endogenous NOD2. (A) Western blot analysis using rabbit NOD2 antiserum HM2559 and anti–β-actin. 10 μg of total protein from different intestinal epithelial cells (IEC), COS7, and HEK293 cell lines were loaded onto 4–12% Tris-glycine gel. (B) NF-κB activation in HT29 and Caco-2 cells after stimulation with 1 μg/ml MDP-LD or MDP-LL using NF-κB luciferase reporter assay and normalization with renilla after 18 h of transfection. Error bars represent SEM of at least four separate experiments. *, P < 0.05. (C) Confocal microscopy examination of endogenous NOD2 in HT29 and Caco-2 cells using rabbit NOD2 antiserum HM2559 or preimmune serum and anti–rabbit Texas red as a secondary antibody. Cytosolic and surface membrane localization (arrows) of NOD2 in HT29 cells is shown. Bar, 20 μm.

Article Snippet: For NF-κB assay or protein expression, cells were transfected using LipofectAMINE 2000 (Invitrogen), and for immunostaining experiments, cells were transfected using TransIT Transfection Reagent Kit (Mirus Bio Corp.) according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Activation Assay, Luciferase, Reporter Assay, Transfection, Confocal Microscopy, Membrane

Journal: The Journal of Cell Biology

Article Title: Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor–κB activation in muramyl dipeptide recognition

doi: 10.1083/jcb.200502153

Figure Lengend Snippet: Ligand-induced NF-κB activation and IL-8 release are dependent on NOD2 membrane association. (A) Expression of NOD2 and mutants without tag that were transfected into HEK293 cells was determined by Western blot analysis using NOD2 antiserum HM2563. (B) HEK293 cells were transfected with 1 ng NOD2 expression vector or mutants together with 1 μg MDP-LD. NF-κB activity was determined by using an NF-κB luciferase reporter assay and was normalized with renilla after 18 h of transfection. Fold increase of NF-κB activation was determined by comparing untransfected and nonstimulated HEK293 with MDP. (C) IL-8 released in the supernatant of HEK293 cells that were transfected with mutants (M) of NOD2 and stimulated 18 h with 1 μg MDP-LD was measured by ELISA. Error bars represent SEM of at least four separate experiments. NT, nontransfected. *, P < 0.05.

Article Snippet: For NF-κB assay or protein expression, cells were transfected using LipofectAMINE 2000 (Invitrogen), and for immunostaining experiments, cells were transfected using TransIT Transfection Reagent Kit (Mirus Bio Corp.) according to the manufacturer's protocols.

Techniques: Activation Assay, Membrane, Expressing, Transfection, Western Blot, Plasmid Preparation, Activity Assay, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay