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Fig. 4. Effect of type 3 RyR knockdown, RyR inhibition and Ins(1,4,5)P₃R inhibition on early local Ca²⁺ signaling upon activation of the cADPR/Ca²⁺-signaling system. T cells [control clone E2 (A) or type 3 RyR-knockdown clone 25 (B)] were loaded with Fura-2/AM and analyzed by single-cell confocal Ca²⁺ imaging as described in the Materials and Methods. Either cADPR was microinjected (pipette concentration 20 µM) or cIDPRE was added extracellularly (final concentration 500 µM). During a 15-second time interval, characteristic confocal ratio Ca²⁺ images acquired in the pacemaker phase are displayed (A,B; bar, 5 µm) and amplitudes of selected ROIs (see inset) are plotted. Ruthenium Red (RuRed; pipette concentration 10 µM) was microinjected directly before start of the measurement. Data acquisition rate was 0.1 ratios/second before cADPR microinjection or cIDPRE addition, and 2 ratios/second thereafter. Data in C and D are presented as mean±s.d. [n=9 ROIs for each subcellular region (edge, cytosol, nucleus) taken from three different individual cells]. Asterisks denote significant differences as indicated (P0.05, Student's t test).

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