Journal: Cells

Article Title: Altered Ca 2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

doi: 10.3390/cells11010049

Figure Lengend Snippet: PV RBCs show increased Gárdos channel activity. ( A ) CCCP method analysis of PV and CT RBC membrane potential changes upon 100 µM NS309 addition. At the end of the experiment, cells were lysed with 3M NaCl 1% Triton X lysis solution to obtain the zero membrane potential (pHi = pHo) for absolute calculation of membrane potential. CT (mean—black line; SD—grey) and PV (mean—red line; SD—pink) RBCs. Data are displayed as mean with 95% confidence interval; CT ( n = 6) and PV ( n = 8). ( B ) Cell volume assay on Gárdos activity; 0.05% RBCs suspension was prepared in PBS with a final concertation of 0.2% BSA, 1 mM CaCl 2 , and 100 µM NS309. RBC size was measured using CASY before and 2.5, 5, 7.5, and 10 min after 100 μM NS309 addition. Mean with SD, n = 6, Mann–Whitney test. ( C – E ) Patch-clamp analysis. Upon cell catch external solution was added to the wells followed by 10 µM NS3623, 10 µM NS309, 5 µM TRAM-34, and 30 µM GdCl 3 . Currents were measured at room temperature applying −100 to +80 mV ramp voltage protocol for 300 ms, at a holding potential of −30 mV. The cell response was measured in pA at +80 mV. Statistical analysis of the currents at +80 mV in NS309 and TRAM-34 responding cells in ( C ) CT and PV RBCs ( n = 17; n = 48, respectively), ( D ) BaF3 EpoR JAK2 WT ( n = 20) and BaF3 EpoR JAK2 V617F ( n = 48), and ( E ) HEL ( n = 61) and HEL cells treated with 0.3 µM ruxolitinib for 24 h ( n = 63). Cell was considered responsive if it displayed at least a 20% current change. The left panel represents the cell current upon the addition of NS3623 (baseline), NS309, and TRAM-34. The central panel displays NS309-induced current increase, while the right panels represent TRAM-34-induced current decrease. The data are presented as median and box plots (25–75%) with whiskers (10–90%). Mann–Whitney test or Wilcoxon test, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns—not significant.

Article Snippet: Three minutes after RBC addition, 100 µM of NS309 was added (a compound which shifts Ca 2+ sensitivity of Gárdos of one order of magnitude [ ] (Alomone labs, Jerusalem, Israel) followed by RBC lysis 8 min after RBC addition using 3M NaCl 1% Triton solution (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activity Assay, Lysis, MANN-WHITNEY, Patch Clamp