Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-content imaging and RNAi screens for investigating kinase network plasticity

doi: 10.1007/978-1-4939-7154-1_10

Figure Lengend Snippet: Pipetting scheme for reverse transfection of adherent tissue culture cells with arrayed siRNA oligonucleotides. Steps either side of the dashed line represent preparation of parallel copies of the library siRNA transfections combined with either non-targeting (NT) siRNA or a specific additional modifier siRNA (siMOD). This siRNA screen can either look for epistatic modifiers of the siMOD target gene or, vice versa, to test a candidate siMOD target for epistatic impact on the various targets in the library. Values in bold and in parentheses indicate the current volume per well of liquid in the plates upon completion of the indicated steps. (A) A plate of an siRNA library arrayed in a 96 well format is diluted in siRNA buffer to yield a 200 nM copy on a new plate containing 70 ul final volume. The siRNA dilution buffer is supplemented with either 55.5 nM NT or siMOD siRNA, or non-supplemented if the screen does not involve a modifier (see Note 1). (B) Lipid-siRNA complexes are generated by adding diluted lipid mix to the plate(s). (C) The resulting complexes are then sub-divided into three copies on new plates, each with opaque walls and a transparent base, to which the cells are subsequently added. Final oligonucleotide concentrations once the cells have been added are 20 nM for the library siRNA and 5 nM for the modifier siRNA.

Article Snippet: HiPerfect siRNA transfection lipid (Qiagen, catalogue number 301704).

Techniques: Transfection, Generated

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-content imaging and RNAi screens for investigating kinase network plasticity

doi: 10.1007/978-1-4939-7154-1_10

Figure Lengend Snippet: Example scatter plots of data produced using this analysis strategy. The R-script in Fig. 15 has been used in conjunction with an output Nuclei.csv file from a siRNA screen. The x-axes display GFP-ratio values calculated from the GFP-CDK2 reporter and the y-axes the mean nuclear fluorescence intensity of CyclinA antibody staining in arbitrary units. Positions of the assay thresholds (bold white lines) are set based on observations of a relevant test set of wells present on each plate of the screen and numbers in each of the quadrants represent the percent cells of the plotted population contained therein. (A) Individual cell assay values from a test set of siRNA representing NT oligonucleotide, siRNA targeting Cyclin A and a siRNA mixture targeting the G1 phase cyclin-dependent kinases CDK6 and CDK4. The Cyclin A knockdown data was used to set an arbitrary threshold for Cyclin A expression positivity/negativity (horizontal white line) whereas the rightwards trend of the double CDK4 and CDK6 knockdown data, resulting in enrichment of CDK2 negative G1 cells, was used to estimate a suitable value for the vertical threshold of the GFP-CDK2 reporter. (B) Example screen data where a candidate epistatic modifier (siMOD) is shown cancelling loss of CDK2 activity and Cyclin A in cells, otherwise driven by loss of CDK6. Non-targeting siRNA control and CDK6 siRNA data values in the absence (upper panels) and presence (lower panels) of a candidate epistatic siMOD siRNA are shown, respectively. NT siRNA substitutes for the absent siMOD siRNA in the upper panel data such that the total siRNA concentration is constant across all transfection conditions in the screen.

Article Snippet: HiPerfect siRNA transfection lipid (Qiagen, catalogue number 301704).

Techniques: Produced, Fluorescence, Staining, Expressing, Activity Assay, Concentration Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Tumor Suppressor Protein Pdcd4 Inhibits Translation of p53 mRNA *

doi: 10.1074/jbc.M111.269456

Figure Lengend Snippet: siRNA-mediated knockdown of Pdcd4 up-regulates p53 expression. A, Western blot analysis of HeLa cells (WT) and two stable Pdcd4 knockdown clones (K11 and K12). The Western blots were developed with antisera against Pdcd4, p53, and β-actin as indicated. Cells were grown in normal growth medium or in medium supplemented with 10 μm proteasome inhibitor MG132 for 4 h before harvesting. B, HeLa cells were transiently transfected with control siRNA or with two different Pdcd4-specific siRNAs. Cell extracts were analyzed by Western blotting for the expression of Pdcd4, p53, and β-actin. C, HepG2 cells were transiently transfected with control siRNA or with two different Pdcd4-specific siRNAs. Cells were treated with or without the proteasome inhibitor MG132 as indicated, and cell extracts were analyzed by Western blotting for the expression of Pdcd4, p53, and β-actin. D, total RNA isolated from siRNA-transfected HepG2 cells was analyzed by quantitative real-time PCR for the expression of p53 (black bars) and Pdcd4 (white bars) mRNAs. E, the nuclear-cytoplasmic distribution of p53 mRNA in HepG2 cells transfected with control or Pdcd4-specific siRNAs was analyzed by real-time PCR.

Article Snippet: Approximately 2.5 × 10 5 cells were plated the day before transfection in 6-cm plates and received 1.5 ml of fresh growth medium prior to transfection. siRNA was transfected using Metafectene TM Pro (Biontex Laboratories GmbH) or HiPerFect (Qiagen), according to the manufacturers' protocols.

Techniques: Knockdown, Expressing, Western Blot, Clone Assay, Transfection, Control, Isolation, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Tumor Suppressor Protein Pdcd4 Inhibits Translation of p53 mRNA *

doi: 10.1074/jbc.M111.269456

Figure Lengend Snippet: Knockdown of Pdcd4 increases the polysomal association of p53 mRNA. Cytoplasmic extracts of HepG2 cells transiently transfected with control or Pdcd4-specific siRNA were fractionated by sedimentation through 10–50% sucrose density gradients. Aliquots of the gradient fractions were analyzed by agarose gel electrophoresis to visualize the distribution of 18 S and 28 S ribosomal RNAs (upper). Subsequently, fractions 1–3, 4–7, and 8–11 were pooled as nonribosomal, monoribosomal, and polyribosomal fractions as indicated. Total RNA isolated from these pools was then analyzed by quantitative real-time PCR for p53 (black bars) and Mdm2 (white bars) mRNAs. The bar height reflects the amounts of p53 and Mdm2 RNAs normalized to the amount of the corresponding RNA in the nonribosomal pool.

Article Snippet: Approximately 2.5 × 10 5 cells were plated the day before transfection in 6-cm plates and received 1.5 ml of fresh growth medium prior to transfection. siRNA was transfected using Metafectene TM Pro (Biontex Laboratories GmbH) or HiPerFect (Qiagen), according to the manufacturers' protocols.

Techniques: Knockdown, Transfection, Control, Sedimentation, Agarose Gel Electrophoresis, Isolation, Real-time Polymerase Chain Reaction