Neuronal living cells in hippocampus of wild type and CHOP−/− mice.

<p>Tunicamycin (0.1 mg/ml) were intracerebroventricular injected in the hippocampus of wild type C57BL/6J (W; A) and CHOP−/− (B) mouse for 5–7 days. Neuronal cells were identified by immunofluorescence staining with Neu-N (green fluorescence). Representative images of three independent experiments are shown. The star symbol indicates that the neuronal living cells are markedly decreased in hippocampus of CHOP−/− mice.</p

Cell apoptosis in the hippocampus or cortex regions were isolated from wild type and <i>chop</i>−/− mice.

<p>Tunicamycin (0.1 mg/ml) were injected in the hippocampus of wild type C57BL/6J and CHOP−/− mouse. Apoptotic cells were performed by the TUNEL staining 5 days after tunicamycin injection. The staining of neuron nuclei in normal control hippocampus was shown on top (A), and the TUNEL positive cells from hippocampus or cortex (B) of wild type C57BL/6J and <i>chop</i>−/− mouse were quantified (C). Data are presented as mean ± S.E.M. for each group (n = 4). **P<0.01 as Tun+wild type versus Tun+<i>chop−/−</i> mice. H: hippocampus; C: cortex.</p

Expressions of ER stress-related molecules GRP78 and CHOP in the mouse hippocampus with or without tunicamycin treatment.

<p>C57BL/6J mice were intracerebroventricular injected with tunicamycin (0.02–1 mg/ml) at various time course (6–72 h). In A, the levels of GRP78 and CHOP proteins in hippocampus were determined by Western blotting 24 h after tunicamycin injection in a dose-dependent manner. Representative images of three independent experiments are shown. In B, the levels of GRP78 and CHOP proteins from hippocampus were determined by Western blotting after tunicamycin (0.1 mg/ml) injection in a dose-dependent manner. In C, the levels of GRP78 and CHOP mRNAs from hippocampus were determined by real-time PCR after tunicamycin (0.1 mg/ml) injection in a time-dependent manner. In B and C, quantification of proteins and mRNA levels were shown. Data are presented as mean ± S.E.M. (n = 3). *P<0.05 as compared with sham-control.</p

Expressions of ER stress-related molecules GRP78, IRE-1, phospho-IRE-1, XBP-1, JNK, phospho-JNK, phospho-PERK, phospho-eIF2α, and ATF6 in the hippocampus isolated from wild type and <i>chop</i> knockout mice.

<p>The expressions of GRP78, IRE-1, phospho-IRE-1, XBP-1, JNK, phospho-JNK, phospho-PERK, phospho-eIF2α, and ATF6 in the hippocampus isolated from C57BL/6J and <i>chop</i>−/− mice 5 days after intracerebroventricular injection of tunicamycin were performed by the Western blotting (A). Quantification of protein expressions were shown in B. Data are presented as means ± S.E.M. for three independent experiments. Each data was performed in triplicate. *P<0.05 as Tun+Wild Type versus Tun+<i>chop−/−</i> mice.</p

<i>Chop</i> gene and protein expressions in the hippocampus isolated from wild type and <i>chop</i> knockout mice.

<p><i>Chop</i> genome typing and protein expression in the hippocampus isolated from C57BL/6J mice and <i>chop</i>−/− mice 1 day after intracerebroventricular injection of tunicamycin (0.1 and 1 mg/ml) were detected by the PCR (A) and Western blotting (B), respectively. Representative images of three independent experiments are shown.</p

Behavior analysis in wild type and CHOP−/− mice with or without tunicamycin treatment.

<p>(A). Passive avoidance was performed 1 day before and 5 days after administration of tunicamycin (0.1 mg/ml) in wild type and CHOP−/− mice. The avoidance latency is significantly decreased in <i>chop</i>−/− mice than in wild type mice 5 days after tunicamycin administration. (B). Water maze was performed 1 day before and 5 days after administration of tunicamycin (0.1 mg/ml) in wild type and <i>chop</i>−/− mice. The time spent in finding the target platform is significantly increased in <i>chop</i> −/− mice than in wild type mice 5 days after tunicamycin administration. (C). In water maze, the time spent in the target quadrant is significantly decreased in <i>chop</i>−/− mice than in wild type mice 5 days after tunicamycin (0.1 mg/ml) administration. Data are presented as mean ± S.E.M. for each group (n = 5). *P<0.05 as compared with wild type mice or <i>chop−/−</i> mice without tunicamycin.</p

Expressions of pro-caspases 9 and 12 in the hippocampus isolated from wild type and <i>chop</i>−/− mice.

<p>Tunicamycin (0.1 mg/ml) were intracerebroventricular injected in the hippocampus of C57BL/6J and CHOP−/− mouse for 3–5 days. Pro-caspase 12 and pro-caspase 9 expressions were presented by the Western blotting from day 3 to day 5 after tunicamycin injection. Representative images of three independent experiments are shown.</p

Inhibition of AMPK enhances I/R-induced apoptosis in LLC-PK1 cells.

<p>Cells, which were transfected with scrambled shRNA or shRNA for AMPKα (shAMPK) (A) or pre-treated with 15 μM Compound C (B), were treated with 50 μM antimycin A and 5 mM 2-deoxyglucose for 1.5 h to induce ischemia (I) injury followed by reperfusion (R) for 24 h. Percentages of cells with the hypodiploid DNA content (sub-G1 cells) were determined by low cytometry. Data are presented as the means ± SDs in three independent experiments. *<i>P</i> < 0.05 and **<i>P</i> < 0.01 as compared with vehicle control group.</p

Autophagy in LLC-PK1 cells during <i>in</i><i>vitro</i> I/R.

<p>Cells were treated with 50 μM antimycin A and 5 mM 2-deoxyglucose for 1.5 h to induce ischemia (I) injury followed by reperfusion (R) for 1-24 h. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079814#pone-0079814-g003" target="_blank">figure 3A</a>, autophagy was determined by Western blotting using anti-LC3 antibody. The β-actin was used to an internal control. Data are presented as the means ± SDs in three independent experiments. *<i>P</i> < 0.05 and **<i>P</i> < 0.01 as compared with vehicle control group. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079814#pone-0079814-g003" target="_blank">figure 3B</a>, the GFP-LC3 puncta formation in LLC-PK1 cells was determined by immunofluorescence. Cells were transiently transfected with GFP-LC3 for 4 h before I/R treatment. Arrow indicates GFP-LC3 puncta formation (green). Nuclei were stained by Hoechst33258 dye (blue). Scale bar = 10 μm. Results shown are representative of at least three independent experiments. </p

The changes in the phosphorylations of AMPK, mTOR and LC-3 cleavage in renal proximal tubule cells during <i>in</i><i>vitro</i> I/R .

<p>LLCPK-1 (A) and NRK-52E (B and C) cells were induced ischemia (I) injury followed by reperfusion (R) for 1 to 24 h. The phospho-AMPKα, phospho-mTOR, AMPKα1, LC-3, and mTOR were determined by Western blotting. The percentages of cells, which treated with <i>in </i><i>vitro</i> I/R and Compound C (5 μM) with the hypodiploid DNA content (sub-G1 cells) were determined by flow cytometry (C). The β-actin was used to an internal control. Data are presented as the means ± SDs in at least three independent experiments. *P < 0.05 and **P < 0.01 as compared with vehicle control group.</p