Effect of JNK silencing on Akts phosphorylation.

<p>Cells were transfected with <i>Jnks</i> (si1, si2, si3) or GFP (siGFP) siRNAs for 2 days. Transfected cells were (<b>A,C</b>) treated with cytokines (4 hrs) or (<b>B,D</b>) overnight starved then treated with insulin (30′). Protein extracts were used for western blotting with anti-phospho-Akt2 (p-Akt2⁴⁷⁴) and anti-Akts antibodies (<b>A,B</b>), or anti-phospho-Akt1 (p-Akt1⁴⁷³) and anti-Akts antibodies (<b>C, D</b>). Equal protein loading was assessed by blotting membranes with an antibody against tubulin. (<b>E</b>) Graphical presentations summarizing the effects of the different <i>Jnk</i> siRNAs <i>vs</i> siGFP in stimulated conditions; control values are further set to 1. The data are the means±SD of three independent experiments. Significant differences were obtained for p-Akt2/p-Akt1 in si1 and si2 <i>vs</i> p-Akt2/p-Akt1 (siGFP) in cytokines (*P<0.05) or starved-insulin treated cells (*P<0.05 or **p<0.01).</p

Effect of JNK silencing on downstream substrates of insulin signaling.

<p>Cells were transfected with <i>Jnks</i> (si1, si2, si3) or GFP (siGFP) siRNAs for 2 days and then exposed to cytokines (4 hrs). Western blot analysis was performed to determine (<b>A</b>) phospho-GSK3β or (<b>B</b>) phospho-FoxO3A and FoxO3A. Equal protein loading was assessed by blotting membranes with an antibody against tubulin. (<b>C</b>) Graphical presentations summarizing the effects of the different <i>Jnk</i> siRNAs <i>vs</i> siGFP in cytokine-treated cells; control values are set to 1. The data are the means±SD of three independent experiments. (**P<0.01) for cyto-Mix-siGFP <i>vs</i> cyto-Mix-si1, and cyto-Mix-si2. (***p<0.001) for cyto-Mix-siGFP <i>vs</i> cyto-Mix-si3. Significant differences were obtained for phospho-GSK3β in si1, si2, and si3 <i>vs</i> siGFP (***p<0.001).</p

Expression of different regulators of the insulin-signaling pathway.

<p>Protein expression levels of the phosphatases (<b>A</b>) PTEN, PHLPP2, and (<b>B</b>) PHLPP1 were studied in conditions similar to those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035997#pone-0035997-g001" target="_blank">Figs.1</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035997#pone-0035997-g002" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035997#pone-0035997-g003" target="_blank">3</a>. No relevant alterations of the protein expression levels in any condition could be demonstrated.</p

Effect of the islet isolation procedure on activation of MAPKs.

<p><i>(</i><b><i>a/b</i></b><i>)</i> For measuring JNK activation by JNK assays, protein extracts (50 µg) were mixed with the GST-Jun fusion protein (GST-c-Jun) and used as described in <i>“</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099796#s2" target="_blank"><i>Materials and Methods</i></a><i>”</i> Photographs represent ^γ33P-phosphorylation of the substrate (p-GST-c-Jun) following SDS-PAGE. The levels of <i>(</i><b><i>c/d</i></b><i>)</i> p38 and <i>(</i><b><i>e/f</i></b><i>)</i> ERK_1/2 protein phosphorylation and expression were determined by Western blot using antibodies against: phospho (p)-p38, p38, p-ERK_1/2 and ERK_1/2. The band density values were calculated as a ratio of <i>(</i><b><i>a/b</i></b><i>)</i> p-GST-c-Jun normalized to GST-c-Jun, <i>(</i><b><i>c/d</i></b><i>)</i> p-p38s normalized to p38s and <i>(</i><b><i>e/f</i></b><i>)</i> p-ERK_1/2 normalized to ERK_1/2. Results are means ± SEM of five to thirteen separate experiments (n = 5–13) and are presented as graphics: white bars; pancreas (>98% exocrine tissue), black bars; purified <i>D₀</i> islets (90% endocrine tissue) and grey bars; cultured <i>D₇</i> islets (>90% endocrine tissue). <i>(</i><b><i>a/b</i></b><i>)</i> JNK activation: Statistically significant differences are assessed by ANOVA or Student's <i>t</i>-test with <i>^*P<0.05</i> or <i>^**P<0.01</i> for all groups prior <i>vs.</i> after SW (n = 5) and for <i>D₀ vs. D₇</i> islets (n = 13). No significant differences are found among all groups from <i>T₀</i> to <i>T₁₅</i>. <i>(</i><b><i>c/d</i></b><i>)</i> p38 activation: Differences are significant for groups <i>T₀–₁₅</i> compared to <i>T_sw-₄₀</i> during pancreas digestion (n = 5, <i>^*P<0.05</i>) and for <i>D₀ vs. D₇</i> islets (n = 13, <i>^*P<0.05</i>). <i>(</i><b><i>e/f</i></b><i>)</i> No significant differences are found among all groups.</p

JNK3 expression analysis.

<p><i>(</i><b><i>a/b</i></b><i>)</i> Protein extracts (30 µg) were prepared from tissue of different species (human, pig and mouse): skeletal muscle (S. Muscle), pancreas, brain and islets generated from wild type (<i>WT</i>) and <i>Jnk3</i>-knockout (<i>KO</i>) mice. JNK3 expression levels were examined by Western blot in <i>D₀</i> and <i>D₇</i> using a JNK3 monoclonal antibody. JNK1 and tubulin monoclonal antibodies were used as loading controls. Protein bands were scanned and quantified by Odyssey scan software. The values were summarized in graphics. Data are means ± SEM of eighteen independent experiments (n = 18, <i>^***P<0.001</i> for JNK3 in <i>D₀</i> (black bars) <i>vs. D₇</i> (grey bars)).</p

JNK3 and c-fos strongly correlate to OCR.

<p>Correlation analysis using <i>the Spearman's</i> correlation coefficient <i>rho</i> was determined as described in <i>“Statistical analysis”</i> by plotting the values of delta <i>(d)</i>-OCR_D7-D0 together with <i>(</i><b><i>a</i></b><i>) d-JNK3</i> (n = 18, <i>^***P<0.001</i>, 0.7853) and with <i>(</i><b><i>b</i></b><i>) d-fos</i> (n = 6, 0.8857).</p

Islet culture improves islet viability by increasing ATP content and OCR.

<p><i>(</i><b><i>a</i></b><i>)</i> ATP was measured and normalized to DNA content (nmol/mg protein/mg-DNA) as described in <i>“</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099796#s2" target="_blank"><i>Materials and Methods</i></a><i>”</i>. Graphic represents ATP/DNA values. Results are means ± SEM of five separate experiments performed in triplicate (n = 5, <i>^**P<0.01</i> for <i>D₀</i> (black bars) <i>vs. D₇</i> (grey bars)). <i>(</i><b><i>b</i></b><i>)</i> Islet samples were used for OCR measurements normalized to DNA content (nmolO₂/min/mg-DNA) as described in <i>“</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099796#s2" target="_blank"><i>Materials and Methods</i></a><i>”</i>. Graphic represents OCR/DNA values. Data are means ± SEM of eighteen separate experiments performed in triplicate (n = 18, <i>^***P<0.001</i> for <i>D₀ vs. D₇</i>). <i>(</i><b><i>c/d</i></b><i>)</i> Protein extracts (30 µg) from islet samples with <i>(</i><b><i>c</i></b><i>)</i> high or <i>(</i><b><i>d</i></b><i>)</i> low JNK3 protein content in <i>D₀</i> and <i>D₇</i> were tested by Western blot. Polyclonal antibodies against: p-Akt1, p-Akt2, p-GSK3β, JNK3, MKK7, Akts and PTEN were used. Equal protein loading was assessed by blotting membranes with antibodies against tubulin and GAPDH. Data are representative of at least three separate experiments (n = 3–8).</p