FIGURE 4.

LANCL2 silencing impairs the ABA-induced increase of the [Ca²⁺]_i, of the [cAMP]_i, and of insulin release in the rat insulinoma cell lines INS-1 and RIN-m. After 48 h from transfection, control (electroporated in the absence of siRNA), negative control siRNA-transfected, or LANCL2 siRNA-transfected cells were treated as follows: A, RIN-m (white bars) or INS-1 cells (gray bars) were subjected to total RNA extraction and real-time PCR analysis of LANCL2. Expression of LANCL2 is normalized to that of the housekeeping genes GAPDH and β-actin (n = 3); B, INS-1 cells were loaded with Fura2/AM and exposed to 10 μm ABA at 20 °C. The [Ca²⁺]_i was measured as described before (25). Each microscopic field contained ≥25 cells. Representative traces from three experiments, yielding closely comparable results, are shown. Similar traces were obtained with RIN-m cells (not shown). C, RIN-m (white bars) or INS-1 cells (gray bars) cells were challenged with 10 μm ABA and extracted after 60 s for measurement of [cAMP]_i levels. Results are expressed as percentage increase over basal values, measured at time = 0 (n = 3). D, INS-1 cells were incubated for 30 min at 37 °C in LG- (white bars) or HG-KRH (black bars), in the absence or in the presence of 100 nm ABA. Results are expressed as insulin secretion over unstimulated samples and are the mean ± S.D. of three different experiments. Untreated cells were not electroporated. Similar results were obtained with RIN-m cells (not shown). Basal insulin secretion was 4.0 ± 0.8 and 33.3 ± 7.5 ng/10⁶ cells/30 min in RIN-m and INS-1 cells, respectively. *, p < 0.01 by t test, compared with the respective negative control (transfected with negative control siRNA).