ALS Model: FUS/TLS Stess Granules Assay Cell Line
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that is characterized by premature degeneration of motor neurons, resulting in a progressive, fatal paralysis. Basis of this in vitro model is that ALS-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. A stable cell line expressing fluorescent FUS/TLS has been developed and validated to form stress granules under under conditions of oxidative stress.
Innoprot offers this cell line as a stable cell line but it is also offered as vials of division-arrested cells (DA cells) in order to perform a small number of assays. Each vial of these DA cells contains 2 million cells, enough to perform a complete experiment in a 96 well-plate.
Assay Details: U2O2 cells stably express human FUS/TLS were induced with IPTG 5mM during 48h to produce the hFUS-tGFP protein. Subsequently, cells were incubated with the compounds at 10 uM during 24 hours and then, cells were treated with 300 uM sodium arsenite during 120 min to induce the stress granules formation. The FUS/TLS-tGFP fluorescent granules were quantified using the BD Pathway HCS Reader and Attovision Compartmentalization Software.
Results indicate that the detecting dynamic range is dependent on the inhibitor biophysics and biochemical characteristics and the treatment time. This stress granules assay was validated with an average of Z= 0.62+/- 0.01 for High Content Screening with a 24 hours treatment.
Fig 1: Cellular fluorescence redistribution after an oxidative insult. Panels A-B show the distribution of FUS/TLS-tGFP protein mostly localized in the nucleus of the cell. After a treatment with Sodium Arsenite 300 M during 2 hours the protein is localized in Stress granules in the cytosol. A pre-treatment of 24 h with Arimoclomol 10 M (E) reduces approximately a 12% the effect of Sodium Arsenite. A pre-treatment of 24 h with Riluzole 10 M (F) reduces approximately a 15% the effect of Sodium Arsenite.
Fig 2: Protective effect of Arimoclomol and Riluzole against oxidative stress. Arimoclomol pre-treatment reduces Arsenite's toxicity a 12% and Riluzole a 15%. Z for this experiment was 0.62 +/- 0.01 and n=12.