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siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a primary regulator of transferrin uptake

Thierry Galvez*, Mary N Teruel, Won Do Heo, Joshua T Jones, Man Lyang Kim, Jen Liou, Jason W Myers and Tobias Meyer*

Author Affiliations

Department of Chemical and Systems Biology and Bio-X Program, Stanford University School of Medicine, Stanford, CA 94305, USA

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Genome Biology 2007, 8:R142  doi:10.1186/gb-2007-8-7-r142

Published: 19 July 2007

Additional files

Additional data file 1:

Figure S1 illustrates the observed well-to-well variability in the transferrin uptake assay. HeLa cells were transfected with CTR d-siRNA (targeting yellow fluorescent protein, eYFP) or d-siRNA targeting the AP2M1 gene. Twelve wells within the same row of a 96-well plate were transfected with the same pool of d-siRNAs to measure the well-to-well variability of the intensity measurements. Means ± standard deviation of two experiments are represented. Figure S2 summarizes the strategy used to generate the diced-siRNA library. Specific primers for the selected genes were designed to amplify approximately 600 bp PCR fragments from a cDNA library. A second amplification was performed with a set of nested primers bearing a T7 promoter sequence extension. Nested PCR products were in vitro transcribed, resulting double-stranded RNAs were diced with recombinant enzyme Dicer and the 21 mers d-siRNAs were finally purified from incomplete digests. Figure S3 shows the relationship between duplicated values of F for the 1,920 screened d-siRNAs (red). The data are compared to a simulated, two-dimensional Normal distribution of the stochastic noise (blue). The estimated standard deviation of the noise distribution is based on the measured deviations between duplicated measurements for the same d-siRNA. Figure S4 shows the distribution of the CAsH scores from the primary screen. The population of genes with CAsH >0.95 are indicated in red. Figure S5 is an example of the images used for quantitative analysis. (a) Images of HeLa cells illustrating the effect of d-siRNAs targeting mTOR (diced-mTOR) or GL3 luciferase (CTR) on fluorescent transferrin (red) uptake, as well as the effect of 24 h treatment with rapamycin (RAPA) compared to DMSO (CTR). Scale bars: 20 μm. Insets show a magnified view of the indicated image subset. (b) Immunofluorescent staining (green) of transferrin receptor performed on permeabilized cells in order to estimate the total number of transferrin receptors. Examples of cells transfected with d-siRNAs targeting mTOR, TSC2 or GL3 luciferase (CTR). Scale bars, 10 μm. Insets show a magnified view of the indicated image subset.

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Additional data file 2:

Genes targeted by the human d-siRNA signaling library.

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Additional data file 3:

Hits from the primary screen.

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Additional data file 4:

The 21 identified 'high confidence' genes that increase or decrease transferrin uptake in HeLa cells.

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Additional data file 5:

Nucleotide sequences of the designed external (outer) and nested primers for creating Dicer siRNAs targeting 1,920 signaling proteins.

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Additional data file 6:

Nucleotide sequences of the designed primers for the two independent sequences used to target the 21 high confidence hits as well as the sequences of the expected amplicons.

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