An unexpected link between immunogenic cell death and inhibition of gene transcription

Figures & data

Figure 1. Principle of the measurement of transcription inhibition.

A. Following the treatment with an experimental compound, transcription can be analyzed by quantifying the incorporation of click-it chemistry-detectable 5-ethynyl uridine (EU). Incorporated EU molecules can be microscopically visualized with fluorescently labeled azide upon fixation.B. Alternatively, cells can be fixed after treatment and stained with antibodies specific to nucleolin (NCL) and fibrillarin (FBL) which facilitate RNA synthesis in the fibrillar center of the nucleus. Thus, the absence of colocalization of these two proteins is indicative for impaired transcription.C. The emitted pool of immunogenic cell death (ICD)-related danger associated molecular patterns includes endoplasmic reticulum (ER)-resident calreticulin (CALR), whose exposure at the cell surface depends on eIF2α phosphorylation (peIF2α), ATP that is released in an autophagy-dependent fashion, the production of type I interferon (IFN) as well as the exodus of high mobility group box 1 (HMGB1) from the nucleus (Nuc) and annexin A1 (ANXA1) from the cytoplasm (Cyto) during cell death. For the purpose of drug discovery, the hallmarks of ICD, including the inhibition of DNA-to-RNA transcription, can be measured by click-it chemistry such as 5-ethynyl uridine (EU)-containing RNA or biosensor cell lines expressing LC3 (present in the membranes of autophagosomes), CALR or HMGB1 fused to a fluorescent protein. Other hallmarks such as the release of ATP can be detected with the chemical dye quinacrine, ANXA1 released in the supernatant can be measured by ELISA and the production of type I IFN can be monitored on the mRNA level.