Cell viability and LC3 expression following CsA treatment.

<p>GH3 cells were incubated in DMEM with and without 10% fetal bovine serum in the presence or absence of CsA (0 to 10 µM) for 10 h. Cell survival was determined using Cell Counting Kit-8 (A) and LC3 expression was determined by Western blotting (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108981#s4" target="_blank">Materials and Methods</a>. Immunofluorescence staining of LC3 and Lamp2 (C, D), DAPI staining (E), and DNA fragmentation (F) were captured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108981#s4" target="_blank">Materials and Methods</a>. E: a, with FBS; b, without FBS; c, 1.0 µM CsA; d, 2.5 µM CsA; e, 5.0 µM CsA; f, 10 µM CsA. Scale bars: C, 100 µm; D, 25 µm; E, 100 µm. ***<i>p</i><0.001 vs. serum treatment. ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001 vs. serum treatment.</p

Effect of CsA-mediated autophagic and apoptotic death on Cu/Zn- and Mn-SOD levels.

<p>GH3 cells were incubated in DMEM with or without 10% fetal bovine serum in the presence or absence of CsA (0 to 10 µM) for 10 h. Cu/Zn- and Mn-SOD levels for autophagy (A) and apoptosis (D) were determined by Western blotting and the relative amount for autophagy (B, C) and apoptosis (E, F) was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108981#s4" target="_blank">Materials and Methods</a>. *<i>p</i><0.05, ***<i>p</i><0.001 vs. serum treatment. ^###<i>p</i><0.001 vs. no serum treatment.</p

Effect of CsA-mediated autophagic and apoptotic cell death on Bax and Bcl-2 levels.

<p>GH3 cells were incubated in DMEM with or without 10% fetal bovine serum in the presence or absence of CsA (0 to 10 µM) for 10 h. Levels of Bax and Bcl-2 for autophagy (A) and apoptosis (E) were determined by Western blotting and the relative amount of Bax and Bcl-2 for autophagy (B, C) and apoptosis (F, G) was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108981#s4" target="_blank">Materials and Methods</a>. Bcl-2 was imaged on an OLYMPUS DP controller and manager using an inverted microscope (D). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 vs. serum treatment. ^#<i>p</i><0.05, ^###<i>p</i><0.001 vs. no serum treatment. Scale bar is 100 µm.</p

Effect of CsA-mediated autophagic and apoptotic death on calbindin-D9k levels.

<p>GH3 cells were incubated in DMEM with or without 10% fetal bovine serum in the presence or absence of CsA (0 to 10 µM) for 10 h. Calbindin-D9k levels for autophagy (A) and apoptosis (C) were determined by Western blotting and the relative amount of calbindin-D9k for autophagy (B) and apoptosis (D) was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108981#s4" target="_blank">Materials and Methods</a>. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 vs. serum treatment. ^##<i>p</i><0.01, ^###<i>p</i><0.001 vs. no serum treatment.</p

Regulation of glucose parameters in hypoxia induced hyperglycmic model and CaBP-9K KO mice.

<p>The mice were divided into four groups (n = 8 per each group, I: normoxia-wildtype, II: hypoxia-wildtype, III: normoxia-CaBP-9k KO, IV: hypoxia-CaBP-9k KO). (A) OGG1 expression for confirmation of hypoxia induction. (B) Measurement of blood glucose level. (C) Measurement of plasma insulin level. (D) Calculated insulin resistance index; HOMA-IR. (E) IPGTT and AUC. AUC was calculated by trapezoidal method with graphpad prism. (F) IPITT and AUC. (G) Immunofluorescence assay with CaBP-9k, insulin and DAPI. The pancreas was immunostained with insulin and CaBP-9K antibodies. The tissue sections were also stained with DAPI for counter staining. * indicates <i>P</i><0.05 compared with normal oxygen conditioned group; ^# indicates <i>P</i><0.05 compared between Wildtype-CaBP-9k KO.</p

Regulation of glucose parameters in hypoxia induced hyperglycmic model and CaBP-9K KO mice.

<p>The mice were divided into four groups (n = 8 per each group, I: normoxia-wildtype, II: hypoxia-wildtype, III: normoxia-CaBP-9k KO, IV: hypoxia-CaBP-9k KO). (A) OGG1 expression for confirmation of hypoxia induction. (B) Measurement of blood glucose level. (C) Measurement of plasma insulin level. (D) Calculated insulin resistance index; HOMA-IR. (E) IPGTT and AUC. AUC was calculated by trapezoidal method with graphpad prism. (F) IPITT and AUC. (G) Immunofluorescence assay with CaBP-9k, insulin and DAPI. The pancreas was immunostained with insulin and CaBP-9K antibodies. The tissue sections were also stained with DAPI for counter staining. * indicates <i>P</i><0.05 compared with normal oxygen conditioned group; ^# indicates <i>P</i><0.05 compared between Wildtype-CaBP-9k KO.</p

Expression of insulin secretion related K_ATP channel in pancreas.

<p>(A)–Sulfonylurea receptor1 (SUR1) (B)–ATP-sensitive K⁺ channel, an inward-rectifier potassium ion channel 6.2 (K_ir6.2) mRNA expression were examined with realtime PCR. Results were normalized relative to β-actin. (* indicates <i>P</i><0.05 compared with normal oxygen conditioned group, ^# indicates <i>P</i><0.05 compared between Wildtype-CaBP-9k KO).</p

Expression of ER-stress maker genes in hypoxia induced hyperglycemic model and CaBP-9K KO mice.

<p>WT and CaBP-9K KO mice were exposed to normoxic or hypoxic condition for 10 days (n = 8 per each group). The mRNA was extracted from pancreas tissues and the transcriptional levels of CHOP (A), BiP (B), ERO1 (C), and PDI (D) genes were analyzed by real-time PCR. (E) Western blot analysis of BiP, PDI, ERO-1a and Calnexin was performed for evaluating protein levels of ER stress marker genes. Results were normalized relative to β-actin. * indicates <i>P</i><0.05 compared with normal oxygen conditioned group; ^# indicates <i>P</i><0.05 compared between Wildtype-CaBP-9k KO.</p

Expression of calcium modulating genes in hypoxia induced hyperglycemic model and CaBP-9K KO mice.

<p>WT and CaBP-9K KO mice were exposed to normoxic or hypoxic condition for 10 days (n = 8 per each group). The mRNA was extracted from pancreas tissues and the transcriptional levels of CaBP-9k (A), PMCA1 (B), Cav1.2 (C), CALR (D) and CANX (E) genes were analyzed by real-time PCR. Results were normalized relative to β-actin. * indicates <i>P</i><0.05 compared with normal oxygen conditioned group; ^# indicates <i>P</i><0.05 compared between Wildtype-CaBP-9k KO.</p

Developmental potentials of bi and uniparental porcine embryos^*.

<p>*The number of replicates was 5.</p><p>†Those zygotes having two pronuclei (IVF and AG zygotes) or one large pronucleus or two pronuclei (PG zygotes) were selected after staining with Hoechst 33342.</p><p>‡The cells of blastocysts were counted on Day 7.</p>a–c<p>Values with different letters within each column are significantly different, <i>p</i><0.05.</p