HMGB1 and osteosarcoma: GFP-tagged mutant HMGB1 transgenes were transiently expressed in human osteosarcoma (U2OS) cells, and the nucleolar dynamics were quantified as the ratio of mobile versus immobile fractions of GFP–HMGB1 in nucleoli using fluorescence recovery after photobleaching (FRAP) (Fig. 1d).