Fig. 2.

RWPE-2 cells are less sensitive than RWPE-1 cells to pore opening, which is enhanced by ERK inhibition. (A) PTP opening of RWPE-1/2 cells treated with arachidonic acid (AA) (1 μM) or with EM20-25 (EM, 50 μM) is measured with the whole-cell CRC assay. In B, cells were also pretreated with the ERK inhibitor PD98059 (PD, 40 μM). Calcium Green-5N fluorescence is reported as arbitrary units on the y axis. Because the probe does not permeate mitochondria, Ca²⁺ uptake into the organelles is displayed by a rapid decrease of the fluorescence spike after administration to the cells of subsequent Ca²⁺ pulses (5 μM each). CsA (red) increases the number of spikes before the permeability transition, recorded as a marked fluorescence increase, occurs. This establishes the PTP dependence of Ca²⁺ release. In the histograms, both the Ca²⁺ concentration required to open the pore (Left) and the ratio between the CRC detected in the presence (CRC) and absence (CRC₀) of the agonist (Right) are reported.