FIGURE 1.

LANCL2-expression modifications alter the ABA-induced increase of the [Ca²⁺]_i and of the [cAMP]_i in human granulocytes. After 24 h from transfection, control (electroporated in the absence of siRNA), negative control siRNA-transfected, or LANCL2 siRNA-transfected cells were treated as follows: A, subjected to total RNA extraction and real-time PCR were performed (n = 3); B, loaded with Fura2/AM and exposed to 10 μm ABA (to obtain maximal response (16)) at 20 °C. The [Ca²⁺]_i was measured as described in Ref. 16. Each microscopic field contained ≥30 cells. Representative traces from three experiments, yielding closely comparable results, are shown; C, challenged with 10 μm ABA and processed for measurement of [cAMP]_i levels (at 60 s). Results are expressed as percentage increase over basal values, measured at time = 0 (n = 3). D, 24 h after transfection, control granulocytes (electroporated in the presence of the empty vector pcDNA6.2), LANCL2-transfected granulocytes, and granulocytes transfected simultaneously with the empty plasmid and with the siRNA for LANCL2 (siRNA) were processed for [Ca²⁺]_i measurement and challenged with 10 μm ABA (added at time = 0). Representative traces from three experiments, yielding closely comparable results, are shown. Inset: Western blot analysis of cell lysates from control, LANCL2-transfected granulocytes or empty plasmid/siRNA-transfected granulocytes stained with a monoclonal antibody against the V5 epitope (see “Experimental Procedures”). E, 24 h after transfection, granulocytes transfected with Gα_q/i, control granulocytes (electroporated in the presence of the empty vector), granulocytes transfected with α_t, and granulocytes transfected with the empty vector and preincubated with PTX, 2 μg/ml, were processed for [Ca²⁺]_i measurement and challenged with 10 μm ABA (added at time = 0). Representative traces from three experiments, yielding closely comparable results, are shown. F, 24 h after transfection, control granulocytes (electroporated in the presence of the empty vector) and granulocytes transfected with Gα_q/i preincubated with 1 μm xestospongin or with 100 μm 8-Br-cADPR, were processed for [Ca²⁺]_i measurement and challenged with 10 μm ABA (added at time = 0). Representative traces from three experiments, yielding closely comparable results, are shown.