FIGURE 4.

Orai1 and STIM1 protein expression levels, store-operated Ca²⁺ entry (SOCE)-mediated mouse small intestinal longitudinal contraction and intestinal transit time alterations in diabetic mice and the effect of Orai1 and STIM1 siRNA transfections. Representative immunoblotting images (A) and summarized data (B,C) showing the expression levels of Orai1 (A,B) and STIM1 (A,C) proteins in small intestinal smooth muscle cells of age-matched control (CON) and diabetic (DM) mice. Alpha-actin was used as a loading control. Values are shown as the mean ± SEM (n = 4–5); *P < 0.05, age-matched control vs. diabetic mice. Mouse small intestine segments were precontracted by the application of 100 μM carbachol in Ca²⁺-free Krebs solution to evoke SOCE. Then, re-addition of 2 mM Ca²⁺ induced longitudinal contractions of small intestine segments. Representative traces (D) and summarized data (E,F) showing SOCE-mediated small intestinal longitudinal contraction in age-matched control (CON) and diabetic (DM) mice (D,E). Representative immunoblotting images and summarized data showing Orai1 and STIM1 expression levels in diabetic mice small intestinal smooth muscle cells transfected with Orai1 siRNA or STIM1 siRNA or scrambled control siRNA. Alpha-actin was used as a loading control (F,G). Values are shown as the mean ± SEM (n = 4); *P < 0.05, scrambled siRNA vs. Orai1 or STIM1 siRNA transfection. SOCE-mediated small intestinal longitudinal contraction in diabetic mice transfected with scrambled, Orai1 or STIM1 siRNA (H). Values are shown as the mean ± SEM (n = 6–16); *P < 0.05, age-matched control vs. diabetic mice; ^#P < 0.05, scrambled siRNA vs. Orai1 or STIM1 siRNA. Representative images (I,K) and summarized data (J,L) showing intestinal transit distance in age-matched control (CON) and diabetic (DM) mice (I,K), or diabetic mice transfected with scrambled, Orai1, or STIM1 siRNA (J,L). The dark charcoal marker appeared as indicated (red arrows). Values are shown as the mean ± SEM (n = 4–11); *P < 0.05, age-matched control vs. diabetic mice; ^#P < 0.05, scrambled siRNA vs. Orai1 or STIM1 siRNA.