Fig. 2.

Reducing Schlafen 3 by siRNA inhibited the stimulation of dipeptidyl dipeptidase (DPPIV) activity and villin expression by strain. Schlafen 3 protein abundance was substantially reduced 72 h after transfection with siRNA to Schlafen 3. A: typical immunoblot showing Schlafen 3 protein level reduction in cells transiently transfected with siRNA targeted to Schlafen 3 compared with cells transfected with a nontargeting sequence (NT1) (A; 1 of 3 similar experiments). B: cells were plated on collagen I and transiently transfected with siRNA targeted to Schlafen 3 or with nontargeting NT1 sequences before lysis and DPPIV-specific activity assay. Strain increased DPPIV activity to 1.63 ± 0.09 (B; n = 6, *P < 0.05). Reducing Schlafen 3 prevented any stimulation of DPPIV activity by strain compared with unstretched cells, while basal activity lowered to 0.35 ± 0.05 (B; n = 6, *P < 0.05), strongly suggesting that Schlafen 3 mediates the strain effect on DPPIV stimulation. C: to determine the role of Schlafen 3 in villin expression following strain, we transiently transfected cells plated on collagen I with siRNA targeted to Schlafen 3 or with nontargeting NT1 sequences for 72 h before strain and assessed villin expression by Western blot. Schlafen 3 siRNA moderately reduced villin levels and eliminated the strain effect (n = 4, *P < 0.05).