Fig. 5

HCQ treatment decreases nuclear translocation and expression of NFAT. a Jurkat cells were pretreated without (W/O) or with 10× concentration of HCQ (1 × = 600 ng/ml) for 24 hours and then stimulated with ionomycin (1 μg/ml) for 2 hours or left unstimulated. Nuclear and cytoplasmic fraction protein of cells was extracted. Western blot analysis was performed using nuclear protein and antibodies specific for NFATc2 and lamin (upper panel). Western blot analysis using cytoplasmic protein and antibodies specific for NFATc2 and tubulin was also performed (lower panel). All data shown are representative of three independent experiments with similar results. b CD4⁺ T cells were left unstimulated or stimulated with ionomycin (1 μg/ml) and PMA (50 ng/ml) for 5 or 20 min in the presence or absence of the preceding 10× concentration HCQ, as indicated. NFATc2 localization was analyzed using immunocytochemistry and confocal microscopy (original magnification × 1000). All images shown are representative of four independent experiments with similar results. c Jurkat cells were pretreated with or without 10× concentration HCQ for 24 hours and were then activated by ionomycin (5 μg/ml) for 2 hours. NFATc1 expression was measured by flow cytometry. (n = 11). d Purified CD4⁺ T cells from SLE patients were treated without or with 10× concentration HCQ for 24 hours and activated without or with ionomycin (5 μg/ml) or ionomycin (1 μg/ml)/PMA (50 ng/ml) for 2 hours. Flow cytometry was used to measure NFATc1 expression (n = 8). HCQ hydroxychloroquine, NFAT nuclear factor of activated T cells, PMA phorbol 12-myrisate 13-acetate, NEG no HCQ treatment