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In-Cell Western Assay and RNAi

The In-Cell Western™ Assay can be used in a functional siRNA screen measuring the effects of knockdowns in cultured cells.

Hoffmann et al.1 used In-Cell Western screening to assess the effects of knockdowns on mTORC1-dependent phosphorylation of ribosomal protein S6 (rpS6).

Schematic of the workflow for the siRNA screen

Schematic of the workflow for the siRNA screen. (Adapted from Greg Hoffman, Harvard.)

  • HeLa cells were transfected with siRNA and screened for phosphorylation of prS6 at Ser235/236.
  • NHS ester cell labeling was used for cell number normalization.
  • The Dharmacon Human Druggable Genome siRNA library gave 7,317 genes from the human genome that are likely targets for pharmacological inhibition.
  • A pilot small molecule screen was performed with a library of ~2500 compounds.
  • Knockdown of components required for both the growth factor and amino acid branches of the mTORC1 signaling network caused reduction in phospho-rpS6.
  • Known genes involved in growth factor-mediated inputs leading to rpS6 phosphorylation (IRS2, IGF1R, PI3-kinase, PDK1, and S6K2) scored as hits in the screen, validating the approach.
  • In addition to known pathway components, a number of uncharacterized genes scored as strong hits.
  • In-Cell Western RNAi screening was found to be faster and less expensive than high content microscopy (IF), and gave similar or better statistical reproducibility.
Results of pilot small molecule screen

Figure 1. (A) Results of pilot small molecule screen performed with a library of ~2500 known bioactive compounds. Compounds with average Z-score < -2 are considered hits and are shown in red. Known inhibitors of mTORC1 signaling found in the library are shown in green. Star-shaped symbols represent compounds with > 4-fold reduction in cell number. (B) Plate to plate reproducibility is shown for a representative plate from the small molecule library.

rnai screens

Figure 2.

  • 1. Hoffmann et al. A functional siRNA screen for novel regulators of mTORC1 signaling. Poster presentation, ASCB Annual Meeting (2008)

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