Fig 1. Jurkat cell responses to exogenous gal-9 in the presence of Gal-Nab1 and Gal-Nab2 antibodies.

A. Jurkat cells were treated during 24h with increasing concentrations (3 to 300 nM) of human recombinant gal-9 (gal-9S or M) and percentages of apoptotic cell death were assessed by flow cytometry analysis of annexin-V⁺ PI⁺ cells (technical duplicates). B. Example of flow cytometry plots for Jurkat cells treated or not with gal-9 (gal-9S; 40 nM) alone or in combination with lactose (5 mM), control isotype mAbs (ctrl IgG1) or anti-gal-9 mAbs (Gal-Nab1 and Gal-Nab2) at 67 nM (i.e. 10 μg/mL) followed by annexin-V and PI staining after 24h. C-D. Dose-response curves for apoptotic cell death (annexin-V⁺ PI⁺) (C) or PS translocation (annexin-V⁺ PI^-) (D) in Jurkat cells treated for 24h with increasing concentrations of Gal-Nab1 and Gal-Nab2 (6 to 130 nM). Empty squares indicate the percentages obtained in conditions without gal-9. Black crosses indicate the percentages obtained with isotype control IgG1 mAbs used at maximal concentration (130 nM). Data are presented as means ± SEM of four biological replicates. E-F. Variations of [Ca²⁺]_cyt in Jurkat cells treated with gal-9 (40 nM, added at t = 0 s) alone (black line) or with gal-9 combined with Gal-Nab1 (E) or Gal-Nab2 (F) (red line). [Ca²⁺]_cyt was assessed by Indo-1 fluorimetry as described under “Materials and Methods”. Data presented here are representative of three similar independent experiments.