High content screening (HCS) microscopy is a very powerful tool for the identification of therapeutics to target cancer. One attractive group of target proteins are transcription factors, as these often exist in a steady-state in the cytoplasm, but when activated they translocate from the cytoplasm to the nucleus – this change in subcellular localization is very profound, and can, therefore, serve as the basis for a high content cell-based essay.

The identification of chemical compounds that can induce this redistribution of transcription factors from the cytoplasm to the nucleus would be of great benefit to cancer therapy strategies. Using the information that you have gained from the lectures and workshops in this module, in addition to the references provided, answer the following three questions:

1. Write a short commentary describing what an image analysis pipeline might look like if you were to have a cancer cell line stably expressing a GFP-tagged transcription factor. Give consideration to any other cellular markers (and therefore fluorescent channels) that you might need in your essay.

Specifically, detail what image analysis steps you would need to use to measure its change in redistribution from the cytoplasm to the nucleus on treatment with a panel of potential therapeutics. Be specific in terms of the function of each step in the pipeline, giving reasoning for why each step is important. You should also include a diagram to illustrate your ideas for the pipeline. Your answer should be a maximum of 500 words.

2. Your HCS assay (as described above) is ready to be employed on a panel of 100,000 novel chemical compounds. Write a short commentary detailing

• what type of multi-well plate format might be most appropriate considering the size of the library;
• what might be the appropriate controls, and their position and frequency in the plates; and
• what would be an appropriate number of different concentrations and replicates to test.

Your answer should be a maximum of 300 words.

3. Further information from any HCS assay can be gained by my ‘multiplexing’, namely adding in more fluorescent reporters to provide additional information about the state of the cells. The assay described above could be multiplexed by adding in another marker/channel showing the mitochondria, and specifically their ‘health’.

Detail what markers might be appropriate to add to your experiment to address mitochondrial health, and the additional image analysis steps that might be needed to measure any changes in the mitochondria in your essay. Your answer should be a maximum of 300 words.