Search for intermediates of Na+,K+-ATPase-mediated [Na+]i/[K+]i-independent death signaling triggered by cardiotonic steroids  Olga A. Akimova, Olga D. Lopina, Pavel Hamet, Sergei N. Orlov  Pathophysiology  Volume 12, Issue 2, Pages 125-135 (September 2005) DOI: 10.1016/j.pathophys.2005.03.003 Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 (A) Kinetics of modulation by ouabain of MTT staining (curve 1) and detachment (curve 2) of C7-MDCK cells. The cells were incubated in DMEM containing 3μM ouabain for up to 24h. The values of MTT staining in the absence of ouabain and the total protein content of attached and detached cells were taken as 100%. Means±S.E. from experiments performed in quadruplicates (cell attachment) and octaplicate (MTT staining) are shown. (B) Phase-contrast microscopy of control cells and cells treated with ouabain for 6 and 24h. Dead cells are shown by arrows. Pathophysiology 2005 12, 125-135DOI: (10.1016/j.pathophys.2005.03.003) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Kinetics of modulation of [Ca2+]i in C7-MDCK cells by 100μM ATP and 0.5μM thapsigargin (TG) in control medium (medium B) (a), in Ca2+-free medium (b), and in cells loaded with BAPTA and incubated in Ca2+-free medium (c). In Ca2+-free medium, CaCl2 was omitted, and 0.1mM EGTA was added. Cells were loaded with BAPTA as indicated in Section 2. Pathophysiology 2005 12, 125-135DOI: (10.1016/j.pathophys.2005.03.003) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Effect of sodium nitroprusside (SNP) and oxadiazoloquinoxalin (ODQ) on cGMP production (a), MTT staining (b) and attachment (c) of C7-MDCK cells. The cells were kept in DMEM±100μM ODQ for 20min. Then, they were treated with the indicated concentrations of SNP for 1h (a), 6h (b) and 24h (c), respectively. Where indicated, 3μM ouabain was added in 15min of treatment with SNP. The optical density of MTT-stained cells and the relative content of attached cells obtained in the absence of any additions (control) were taken as 100%. Means±S.E. values from experiment performed in quadruplicate (b) and triplicate (a and c) are shown. Pathophysiology 2005 12, 125-135DOI: (10.1016/j.pathophys.2005.03.003) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Dose-dependency of the action of N-ethylmaleimide (NEM) on MTT staining (a) and cell attachment (b) of control (1) and ouabain-treated (2) C7-MDCK cells. The cells were treated with NEM for 30min before 3μM ouabain was added. The optical density of MTT-stained cells and the relative content of attached cells obtained in the absence of any additions (control) were taken as 100%. Means±S.E. values from experiments performed in quadruplicate (MMT staining) and triplicate (cell attachment) are shown. Pathophysiology 2005 12, 125-135DOI: (10.1016/j.pathophys.2005.03.003) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 A representative record of lucigenin- (a and b) and luminol-dependent (c) chemiluminescence (ICL) in C7-MDCK cells. Cells in 35-mm flasks were washed and incubated in the presence of 1ml of medium B at room temperature. At the incubation time indicated by arrows, 20μM lucigenin (LG) or luminol (LU), 10μM ouabain (OU), 80μM digitonin (DG), 25μM NADP or NADPH and 10μg/ml of microperoxidase (MP) were added. Pathophysiology 2005 12, 125-135DOI: (10.1016/j.pathophys.2005.03.003) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 Effect of cytochalasin B (a and c) and vinblastin (b and d) on MTT staining (a and b) and cell attachment (c and d) in the absence (1) and presence (2) of ouabain. The cells were preincubated for 30min with or without of cytochalasin B and vinblastin, and then 3μM ouabain was added to part of the samples. The optical density of MTT-stained cells and the relative content of attached cells obtained in the absence of any additions (control) were taken as 100%. Means±S.E. values from experiments performed in quadruplicate (MTT staining) and triplicate (cell attachment) are shown. Pathophysiology 2005 12, 125-135DOI: (10.1016/j.pathophys.2005.03.003) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions