HeLa cells were plated at 2.5 × 104 cells per well in serum-free media and left overnight to adhere. Five wells per condition were then incubated in serum-free media with no treatment or containing WT or mutant TNF (10 ng/ml). Cells were placed into the incubator and fixed at either 24 or 48 hours after treatment, followed by DAPI (4′,6-diamidino-2-phenylindole) staining and imaging on an EVOS2-FL fluorescent microscope (Thermo Fisher Scientific, UK). Tile scans of every well were reconstructed in Fiji, and nuclear counts per well were calculated for each time point.

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