Figure 4

Spautin-1 reverses cytosolic calcium overload induced by cerulein or L-arginine in vitro and in vivo. (A, B) AR42J cells were treated with the indicated concentration of cerulein (A) or L-arginine (B) for 24 h and then cells were stained with Fluo-4-AM according to manufacturer’s instructions. The fluorescence images were analyzed by Image J. The graphs provided are quantified using Image J as means ± SD of three independent sets of experiments. Student t test was used. (C, D) AR42J cells were treated with normal saline, 200 nmol/L cerulein (C) or 10 mmol/L L-arginine (D) for 21 h and were then in the presence or absence of 2 µmol/L BAPTA-AM for an additional three hours. The trypsin activity was next analyzed by the fluorescence intensity. Data from three independent experiments were tested with Student t test and shown as means ± SD. (E, F) AR42J cells were treated with 200 nmol/L cerulein (E) or 10 mmol/L L-arginine (F) for 24 h in the presence or absence of 10 µmol/L spautin-1. After that cells were stained with Fluo-4-AM according to manufacturer’s instructions, the fluorescence images were analyzed by image J. The graphs provided are quantified using Image J as means ± SD of three independent sets of experiments. Student t test was used. Lysates from AR42J cells (G, H) or mice pancreas (I, J) with indicated treatment were analyzed by Western blotting with anti-CaMKII, anti-phos-CaMKII and antitubulin antibodies.