FIGURE 6.

Effect of RNAi of Tbβ3 on calcium homeostasis and acidic calcium stores in PCF trypanosomes. A and B, acidic Ca²⁺ stores, assessed by the sequential addition of 1 μm ionomycin (Ion) and 1 μm nigericin (Nig) in Fura 2-loaded PCF trypanosomes. Tet(+) traces, cells grown 6 days in the presence of tetracycline; Tet(−) traces, control cells grown in the absence of tetracycline. Acidic stores were considerably reduced in tetracycline-induced cells as compared with controls grown in the absence of tetracycline. C, steady state intracellular Ca²⁺ concentration ([Ca²⁺]_i) in Fura 2-loaded PCF. Cells were incubated with gentle agitation at room temperature for 20 min in the presence of 1 mm extracellular EGTA or calcium (CaCl₂), as indicated, and [Ca²⁺]_i was subsequently determined. There was no statistically significant difference in [Ca²⁺]_i, indicating that intracellular Ca²⁺ homeostasis is maintained at normal levels. Values are mean ± S.E. (p < 0.05, Student's t test, n = 3). D, graphical representation of tracings from A and B, where the bars represent the difference in [Ca²⁺]_i between the base-line [Ca²⁺]_i and the [Ca²⁺]_i after the addition of ionomycin plus nigericin. The values are mean ± S.E. (error bars). The difference in the [Ca²⁺]_i was statistically significant (p < 0.05, Student's t test, n = 3).