FIG. 6.

siRNA-mediated depletion of endogenous paxillin (Pax) significantly attenuates glucose-stimulated focal adhesion remodeling and insulin secretion in primary rat β-cells. β-Cells were transfected with either scrambled (Scr) siRNA or a cocktail of three siRNAs specific for rat paxillin and studied 72 h later. A: Paxillin knockdown was verified by Western blot with equal loading confirmed with an antiactin antibody. A representative blot from three independent experiments is depicted (left panel), and paxillin-to-actin ratios were quantified by densitometry (right panel). B: Cell death (apoptosis plus necrosis) measured using the transferase-mediated dUTP nick-end labeling assay. C: siRNA-transfected β-cells were incubated for 2 h with 2.8 mmol/L glucose and stimulated for 20 min with 16.7 mmol/L glucose. Lysates were analyzed by Western blot with the indicated antibodies, and the relative intensities of the phosphorylated and total protein bands were quantified by densitometry and expressed as a ratio. Ratios were normalized to scrambled siRNA-transfected cells. D and E: siRNA-transfected β-cells were incubated as described above. Cells were then fixed and stained with either phospho-paxillin and actin or Evans blue. Both phospho-paxillin–containing focal adhesions and cell surface areas were quantified and normalized to scrambled siRNA-transfected cells. F: siRNA-transfected primary β-cells were incubated with low (2.8 mmol/L) followed by high (16.7 mmol/L) glucose for 1 h, and insulin release was monitored and expressed as a percentage of total cell content. Data are means ± SEM from three independent experiments. *P < 0.05 and **P < 0.001 vs. scrambled siRNA-transfected cells.