TOX protein levels are increased in SS CD4⁺ T cells and in CTCL cell lines. (A) Western blot analysis showed TOX overexpression in 4 CTCL cell lines (Hut78, Hut102, HH, and SZ4) and in CD4⁺ T cells from the peripheral blood of a patient with SS (SS-5), compared to peripheral CD4⁺ T cells from an HP (Ctr 1) or a patient with BID (Ctr 2). For each sample, cell lysate was extracted as described in the “Methods and materials” section and probed with antibodies specific for TOX and actin proteins. (B) FACS analysis showed TOX elevation in the peripheral CD4⁺ T cells from a patient with SS (SS-12) and in the CTCL cell lines Hut78, HH, and SZ4, whereas normal CD4⁺ T cells were negative for TOX staining. A fluorescence-minus-one control was included as a negative control. (C) Immunofluorescence staining showed strong nuclear TOX staining in the CTCL cells lines Hut78, HH, and SZ4 but not in normal CD4⁺ T cells. For each cell type, 1 million cells were mounted onto the slide and subjected to staining as described in the “Methods and materials” section. Bars represent 25 μm. DAPI, 4′,6 diamidino-2-phenylindole.