Semi-quantitative rt-PCR analysis showing total ET-1 mRNA expression in the retinae of non-transgenic (NTg), heterozygous (He) and homozygous (Hm) TET-1 mice.

<p>A: Ethidium bromide-stained agarose gels of rt-PCR products of ET-1, showing increased ET-1 mRNA expression in transgenic mouse retina. The molecular size marker (M) was 1 kb plus marker. B: Histograms showing the levels of ET-1 mRNA expression normalized to that of GAPDH. n = 4 for each experimental group. *: <i>p</i><0.05 (Kruskal-Wallis Test).</p

Immunohistochemical staining of aquaporin-4 (AQP4).

<p>Up-regulation of AQP4 signal was found in the Müller cell processes in IPL (arrows) and Müller cell bodies in INL (arrowheads) in the ipsilateral retina of Hm TET-1 mice compared with the corresponding contralateral retina, while similar intensity of AQP4 signal was present in the contralateral and ipsilateral retinae of NTg mice. n = 5 for each experimental group. Scale bar = 50 µm.</p

Severity of the presence of pyknotic nuclei in cells in ganglion cell layer (GCL) after transient inner retinal ischemia.

<p>Severity of the presence of pyknotic nuclei in cells in ganglion cell layer (GCL) after transient inner retinal ischemia.</p

Thicknesses of inner nuclear layer (INL), inner plexiform layer (IPL) to inner limiting membrane (ILM) and the whole inner retina (µm).

<p>Thicknesses of inner nuclear layer (INL), inner plexiform layer (IPL) to inner limiting membrane (ILM) and the whole inner retina (µm).</p

Immunohistochemical staining of glial fibrillary acidic protein (GFAP).

<p>Compared with the contralateral retinae of NTg and Hm TET-1 mice, GFAP signal was up-regulated in the ipsilateral retinae in the astrocytes around capillaries in the inner limiting membrane (arrowheads) and the Müller cell processes in IPL (arrows). n = 5 for each experimental group. Scale bar = 50 µm.</p

Chromatin changes at the AR gene in response to hypertonic stress.

<p>(A-C) NIH-3T3 cells were subjected to hypertonic stress, and ChIP was conducted with antibodies against (A) histone H3, (B) histone H2B, and (C) histone H3. DNA was analyzed by real-time PCR using AR_INTER (a), AR_ORE (b), AR_PP (c), and AR_EX2 (d) primer pair respectively as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008435#pone-0008435-g001" target="_blank">Figure 1</a>. *, p<0.01 and **, p<0.001 by one-way ANOVA. (C) Cells were either left untreated (isotonic), or treated with hypertonic medium for 30 min followed by incubation in isotonic medium for 30 min, 6 and 16 h respectively (isotonic recovery). ChIP was conducted with antibody against histone H3. DNA was analyzed by real-time PCR using AR_ORE primer pair. The results are presented as the ratio of immunoprecipitated DNA to total input DNA and normalized to the value of cells maintained under isotonicity. *, p<0.001 by one-way ANOVA. (D) Cells were subjected to hypertonic stress, and ChIP was conducted with antibodies against OREBP. DNA was analyzed by real-time PCR using AR_ORE. The results were normalized to the ratio of immunoprecipitated DNA to total input DNA at time  = 0. (E and F) Cells were treated with hypertonicity for 0 and 8 hr, respectively. ChIP was conducted with antibodies against OREBP (E) or histone H3 (F). DNA was analyzed by real-time PCR using a primer pair specific for the TonEA of the SMIT gene. The results were normalized to the ratio of immunoprecipitated DNA to total input DNA at time  = 0. *, p<0.05 by Student's t-test. For A-G, data are the mean ± SEM (n = 3).</p

Association between OREBP, histone acetylation and histone eviction.

<p>(A) Role of OREBP in histone H4 acetylation. WT and OREBP^−/− MEFs were maintained under isotonicity or treated with hypertonic medium for 1 h. ChIP was conducted with antibody against acetylated histone H4 and histone H4, respectively. DNA was analyzed by real-time PCR using AR_ORE primer pair. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. The value for acetylation histone H4 level was divided by H4 occupancy value. The value of WT MEFs under isotonic conditions was set to be 1.0. *, p<0.001 by one-way ANOVA. I, isotonic medium; H, hypertonic medium. (B-E) Effect of TSA treatment on histone eviction at ORE and AR expression. WT MEFs were treated with DMSO or TSA (1 µg/ml) for 2 hr. ChIP was conducted with antibody against (B) acetylated histone H4, (C) histone H3, and (D) OREBP. In (B), DNA was analyzed by real-time PCR using AR_ORE and AR_INTER primer pairs. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. Values of DMSO treatment were set to be 1.0. *, p<0.05 by one-way ANOVA. In (C) and (D), DNA was analyzed by real-time PCR using AR_ORE primer pair. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. The results were normalized to the ratio of immunoprecipitated DNA to total input DNA of DMSO treatment. *, p<0.01 by one-way ANOVA. (E) Quantitative analysis of AR transcriptions by RT-PCR. Cells were treated with DMSO or TSA (1 µg/ml) for 8 h. Total RNA was prepared and relative AR mRNA level was determined. The result was expressed as fold change relative to the expression level of WT MEFs treated with DMSO. Data are the mean ± SEM (n = 3). *, p<0.001 by one-way ANOVA.</p

Evaluation of histone acetylation status at the OREs of AR in response to hypertonicity.

<p>(A) Schematic illustration of the mouse AR genomic DNA showing exon 1 and 2, promoter and upstream region. The region amplified by primer sets AR_INTER, AR_ORE, AR_PP and AR_EX2 are indicated by arrows. Transcriptional start site is indicated by a bold arrow. The DNA probe for southern blotting analysis in the MNase digestion assay (ORE_MNase) is indicated by a grey bar. (B) ChIP assays of AR promoter. ChIP assays were performed with antibodies against acetylated histone H3 or acetylated histone H4 using NIH-3T3 cells that were induced with hypertonicity for 0, 2, 6 and 10 h, respectively. DNA was analyzed by real-time PCR using AR_ORE primer pair. The results are normalized to the ratio of immunoprecipitated DNA to total input DNA at time 0. Data are the mean ± SEM (n = 3). *, p<0.05 by one-way ANOVA test when compared to the value at time 0. (C) Quantitative analysis of AR transcriptions by RT-PCR. NIH-3T3 cells were incubated in hypertonic medium for 0, 3, 6, 8 and 15 h. Total RNA was prepared. Relative expression level of AR was determined. The expression level of β-actin was used to normalize the AR expression. Results were expressed as fold change relative to the expression level at time 0. Data are the mean ± SEM (n = 3).</p

Histone acetylations and histone occupancy at the AR gene in response to hypertonicity.

<p>(A) Histone H4 hyperacetylation at the AR gene in response to hypertonic stress. Cells were induced with hypertonic stress, and ChIP was conducted with antibodies against acetylated histone H4 and histone H4. DNA was analyzed by real-time PCR using AR_INTER (a), AR_ORE (b), AR_PP (c), and AR_EX2 (d) primer pair respectively. The ratio of immunoprecipitated DNA to total input DNA for each of the antibodies was determined. The value for the level of acetylation of histone H4 was divided by H4 occupancy value. Values at time 0 were set to be 1.0. *, p<0.01 by one-way ANOVA. (B) Histone occupancy at the ORE region of WT and OREBP^−/− MEFs. Cells were maintained under isotonicity or treated with hypertonicity for 1 hr. ChIP was conducted with antibody against histone H3. DNA was analyzed by real-time PCR using AR_ORE primer pair. The results are presented as the ratio of immunoprecipitated DNA to total input DNA and normalized to the value of WT MEFs maintained under isotonicity. *, p<0.01 and **, p<0.001 by one-way ANOVA. I, isotonic medium; H, hypertonic medium. Insert, Western blotting analysis of MEFs using anti-OREPB antibodies. (C) MNase assay. (Upper) Nuclei were prepared from WT and OREBP^−/− cells that were induced with hypertonic stress for 0 and 60 min, respectively, and chromatin was subjected to limited digestion with MNase for increasing times. The nucleosome ladder was resolved in agarose gel and visualized by ethidium bromide staining; (Lower). The DNA was subjected to southern blot analysis using a ³²P-labeled probe spanning the ORE (ORE_MNase, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008435#pone-0008435-g001" target="_blank">Fig. 1A</a>). The triangles denote increasing times of MNase digestion. a, mononucleosome; b, dinucleosomes; c, trinucleosomes. (D) Quantitative analysis of AR transcriptions by RT-PCR. WT and OREBP-/- fibroblasts were incubated in isotonic or hypertonic medium for 16 h. Total RNA was prepared. Relative expression level of AR was determined. The expression level of β-actin was used to normalize the AR expression. Results were expressed as fold change relative to the expression level at time 0. Data are the mean ± SEM (n = 3).</p

Effects of Huanglian-Jie-Du-Tang and Its Modified Formula on the Modulation of Amyloid-β Precursor Protein Processing in Alzheimer's Disease Models

<div><p>Huanglian-Jie-Du-Tang (HLJDT) is a famous traditional Chinese herbal formula that has been widely used clinically to treat cerebral ischemia. Recently, we found that berberine, a major alkaloid compound in HLJDT, reduced amyloid-β (Aβ) accumulation in an Alzheimer’s disease (AD) mouse model. In this study, we compared the effects of HLJDT, four single component herbs of HLJDT (Rhizoma coptidis (RC), Radix scutellariae (RS), Cortex phellodendri (CP) and Fructus gardenia (FG)) and the modified formula of HLJDT (HLJDT-M, which is free of RS) on the regulatory processing of amyloid-β precursor protein (APP) in an <i>in vitro</i> model of AD. Here we show that treatment with HLJDT-M and its components RC, CP, and the main compound berberine on N2a mouse neuroblastoma cells stably expressing human APP with the Swedish mutation (N2a-SwedAPP) significantly decreased the levels of full-length APP, phosphorylated APP at threonine 668, C-terminal fragments of APP, soluble APP (sAPP)-α and sAPPβ-Swedish and reduced the generation of Aβ peptide in the cell lysates of N2a-SwedAPP. HLJDT-M showed more significant APP- and Aβ- reducing effects than berberine, RC or CP treatment alone. In contrast, HLJDT, its component RS and the main active compound of RS, baicalein, strongly increased the levels of all the metabolic products of APP in the cell lysates. The extract from FG, however, did not influence APP modulation. Interestingly, regular treatment of TgCRND8 APP transgenic mice with baicalein exacerbated the amyloid plaque burden, APP metabolism and Aβ production. Taken together, these data provide convincing evidence that HLJDT and baicalein treatment can increase the amyloidogenic metabolism of APP which is at least partly responsible for the baicalein-mediated Aβ plaque increase in the brains of TgCRND8 mice. On the other hand, HLJDT-M significantly decreased all the APP metabolic products including Aβ. Further study of HLJDT-M for therapeutic use in treating AD is warranted.</p></div