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. 2020 Nov 20;11:580557. doi: 10.3389/fimmu.2020.580557

Figure 3.

Figure 3

Galectin-9 increases the intracellular activity of granzyme B and upregulates pro-apoptotic processes in Jurkat T cells in a VISTA- and Tim-3-dependent manner. VISTA and Tim-3 expression on the surface of resting Jurkat T cells and those treated for 24 h with 100 nM PMA was measured using anon-cell Western (A). VISTA surface expression was confirmed using immunofluorescence microscopy (B). Total cellular levels of Tim-3 and VISTA were measured in permeabilised Jurkat T cells using flow cytometry (C). Following pre-treatment of Jurkat T cells for 24 h with 100 nM PMA, they were exposed for 24 h to 2.5 µg/ml galectin-9 in the absence or presence of VISTA or/and Tim-3 neutralizing antibodies. Granzyme B activity was then measured in these cells (D). To assess pro-apoptotic processes Jurkat T cells were pre-treated for 24 h with 100 nM PMA and then exposed for 24 h to 2.5 µg/ml galectin-9. Caspase-3 activity and PARP cleavage were then measured by colorimetric assay and Western blot, respectively (E).