ATF4 deficiency in P30 T17M retinas results in upregulation of autophagosome associated genes.

<p><b>(A)</b> Level of autophagy–related LC3-II (lipidated form) was diminished in T17M retinas as compared to C57BL6, ATF4^+/- and T17M ATF4^+/- retinas (<i>P</i> = 0.005, <i>P</i><0.01 and <i>P</i><0.05 respectively). Beclin-1 protein significantly up-regulated in T17M ATF4^+/- retinas as compared to C57BL6 and T17M retinas (<i>P</i><0.05 and <i>P</i><0.01 respectively). The T17M ATF4^+/-retinas showed increased peIF4E protein levels as compared to T17M retinas (P<0.05). The data are presented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a> for details.</p

Proposed mechanisms of retinal degeneration in T17M and T17M ATF4^+/- mice.

<p>In T17M retinas, we observed up-regulation of pPERK, ATF4, CHOP, p53 proteins resulting in activation of apoptosis. In T17M retinas deficient in ATF4 we detected up-regulation of peIF4E, NRF2, GADD34 and autophagy genes resulting in delay of retinal degeneration. Increased and decreased levels of proteins in the retina are present as small and large fonts, correspondingly. Increased and decreased effects in solid and dashed lines, respectively are shown as well. Proposed in the literature links between signaling molecules are showed by numbers corresponding to their citations in the References section.</p

ATF4 knockdown in P30 T17M photoreceptors has a positive influence on the RHO expression machinery (N = 4 for all groups).

<p>(<b>A</b>) A significant decrease in the mouse RHO mRNA, analyzed by one way ANOVA, was found in the T17M retina as compared to C57BL6 and ATF4^+/- mice <i>(P</i><0.001 for both). However, deficiency of ATF4 in these retinas led to a dramatic increase (16-fold) of mouse and human RHO mRNA expression as compared to T17M mice (<i>P</i><0.01 and <i>P</i><0.0001, respectively). (<b>B</b>) Increase in RHO mRNA led to elevated production of the RHO monomer and dimer in T17M ATF4^+/- retinas, whereas T17M mice experienced a dramatic (99% and 98%) loss of RHO production in photoreceptors, as measured by detection of dimer and monomer bands (<i>P</i><0.01 for both bands as compared to C57BL6). (<b>C</b>) Representative images of western blots treated with antibodies against RHO. (<b>D</b>) Immunohistological analyses of P30 retinas with anti-RHO antibody (1D4, in green) revealed normal localization of rhodopsin in the T17M ATF4^+/- OS, compared with T17M mice with partially mislocalized RHO, suggesting improved clearance of RHO in the ADRP ATF4 deficient retina. ONL, outer nuclear layer; IS, Inner segments; OS, outer segments. Scale bar indicates 50 mm. The data are presented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a> for details.</p

ATF4 knockdown protects T17M mice from loss of retinal integrity and photoreceptors.

<p>(<b>A)</b> We found dramatic increases in the average ONL thicknesses of both hemispheres of 1, 2, and 3-month-old T17M ATF4^+/- retinas compared to T17M mice (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a>). Representative spidergrams of the distribution of ONL thicknesses across the retina at 1, 2, and 3 months of age are shown. The data were analyzed by two-way ANOVA. The T17M ATF4^+/- retinas (N = 8) demonstrated an increase in the superior and inferior ONL thickness at 1, 2, and 3 months of age as compared to T17M mice (N = 11). All regions from both ADRP retinas were significantly different from C57BL6 (N = 7) and ATF4 (N = 7) mice (<i>P</i><0.0001 for both strains and all regions). The data are presented as mean ± SEM. (<b>B</b>) The ATF4 deficiency in T17M mice protected their retinas from photoreceptor cell loss, resulting in an increase in the number of photoreceptors relative to T17M as measured by two way ANOVA (<i>P</i><0.0001). The number of rows of photoreceptor nuclei between T17M ATF4^+/- (N = 4) and C57BL6 (N = 4) retinas was similar at P30, whereas the T17M mice lost 43% of their photoreceptor cells (<i>P</i><0.0001 as compared to all groups). However, by 3 months, both the T17M ATF4^+/- and the T17M mice experienced a loss of photoreceptor cells as compared to C57BL6 mice (<i>P</i><0.0001). The data are presented as mean ± SEM. <b>(C)</b> Representative images of H&E stained retinal sections from all four groups. Images of one-month-old (upper) and 3-month-old (bottom) control and experimental retinas. Scale bar indicates 50 μm.</p

ATF4 knockdown launches the antioxidant cellular defense mechanism in P30 T17M and protects the ADRP retina against oxidative stress (N = 4).

<p><b>(A)</b><i>Nrf2</i> mRNA expressions were elevated in T17M retinas (<i>P</i><0.05 as compared to all groups). But, <i>Ho-1</i> mRNA was only upregulated in T17M ATF4^+/- retinas (<i>P</i><0.05; <i>P</i><0.01 and <i>P</i><0.001 when compared to T17M, ATF4^+/- and C57BL6 respectively). <b>(B)</b> Results of western blotting, analyzed by one way ANOVA, demonstrated that the antioxidant NRF2 and HO-1 expression was significantly up-regulated in T17M ATF4^+/- retinas as compared to C57BL6 retinas (<i>P</i><0.01 and <i>P</i><0.05, respectively) and T17M retinas (<i>P</i><0.01 for NRF2). Expression of HO-1 protein in T17M ATF4^+/- had a tendency for up-regulation. In addition, p62 was significantly lower in T17M retinas as compared to all other groups (P<0.05). <b>(C)</b> Representative images of western blots treated with antibodies against p62, NFR2, HO-1, and β-actin proteins. <b>(D)</b> Oxidative stress was significantly diminished in the T17M ATF4^+/- retinas compared to T17M retinas with ongoing oxidative stress. Representative images of retinal cryostat sections stained with H2DCFDA. ROS positive cells are evident in the ONL and INL of the T17M retina and are indicated with arrows. The data are presented as mean ± SEM. See also for details <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a>.</p

Overexpression of ATF4 accelerates and induces retinal degeneration in T17M mice and C57BL6 mice, respectively.

<p><b>(A</b>) ERG amplitudes were registered as described in the methods and were analyzed by one-way ANOVA. Data analysis demonstrated that subretinal injections of AAV2/5- ATF4 led to a loss of photoreceptor function in the right ATF4-injected T17M retina compared to the left AAV2/5- GFP-injected eye (N = 6, <i>P</i><0.05 for both a and b-wave amplitudes). The C57BL6 retina overexpressing ATF4 also experienced a decline in a- and b- wave ERG amplitudes (N = 4, for both <i>P</i><0.01) as compared to GFP-injected controls or uninjected animals (N = 4). Results of the scotopic ERG amplitudes registered at 25 cd*s/m2 are shown. <b>(B)</b> Overexpression of ATF4 in the injected (right) retinas was measured by western blotting. A 2.3-fold (N = 6, <i>t</i>-test p = 0.017) increase in ATF4 was found in the right eye when compared to the left eye. This resulted in upregulation of CHOP protein (N = 6, <i>t</i>-test p = 0.046) in ATF4 overexpressing eyes. Representative images of western blots probed with antibody against ATF4, CHOP, and β-actin are shown on the side. <b>(C)</b> Overexpression of ATF4 and CHOP proteins in T17M and C57BL6 retinas was associated with a loss of photoreceptor cells measured by counting the nuclei rows in H&E stained retinal sections (N = 4, <i>t</i>-test <i>P</i><0.001 and N = 3, <i>t</i>-test <i>P</i><0.001). <b>(D)</b> Representative images of the H&E stained T17M and C57BL6 retinas injected with AAV2/5-GFP and AAV2/5-ATF4. <b>(E)</b> Photoreceptor cell death in T17M retinas overexpressing ATF4 was associated with highly activated caspase-3/7. The activation of apoptotic cell death markers was also detected in the wild type retinas over-expressing ATF4 (N = 4, <i>t-</i>test <i>P</i><0.001 and <i>P</i><0.0001, respectively). The data are presented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a>.</p

IL-17 has opposing effects on the infiltration of CD11b+ macrophages and CD8+ T cells in DMBA/TPA treated skin.

<p>Mice were treated with DMBA and TPA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032126#pone-0032126-g001" target="_blank">figure 1</a> and skin tissues were harvested at the end of the experiment (26 weeks). A). Frozen tissue sections were stained with CD11b antibodies (brown) and counterstained with hematoxylin (blue, upper panels) or with anti-CD8 antibodies (green) and counterstained with a fluorescence dye DAPI (blue, lower panel). Photos were taken microscopically (20× objective). Numbers of positive cells were counted microscopically. Islets show the enlarged areas of circled squares with positive cells. Graphs show the mean number of positive cells per field +/− SEM, <i>n</i> = 10, * p<0.05, ** P<0.01. B). Gr-1 and CD11b double positive cells in spleens were stained with fluorescence labeled antibodies and positive cells were analyzed in a flow cytometer. The numbers in the right upper quadrant indicate the percent of positive cells in whole spleen cells. C). Statistical analysis of GR-1/CD11b+ (myeloid derived suppressor cells), CD19/B220+ (B cells), CD8+ and CD4+ T cells in spleens (mean +/− SEM, n = 8, ** P<0.01). Data are representative of two independent experiments.</p

Neutralization of IL-17 inhibits the progression of tumors in DMBA/TPA induced cutaneous carcinogenesis.

<p>Wild type C57Bl/6 mice were topically treated once with DMBA and then twice weekly with TPA. All mice developed cutaneous tumors at 12 weeks. The mice were divided (10 mice/group) and treated intravenously with adenovirus encoding GFP (Ad-GFP), IL-17R:Fc (Ad-IL-17R:Fc) or left untreated (Ctrl) at week 12 and 16. All mice were continuously treated with TPA twice a week throughout the experiments. The tumor growth was monitored weekly. Bar graph shows the mean tumor number per mouse +/− SEM at the end of experiments (* P<0.05). Data are representative of 2 independent experiments.</p

Effect of delphinidin treatment on markers of angiogenesis in tumors of athymic nude mice implanted with NSCLC cells.

<p>Athymic nude mice were implanted with [A] NCI-H441, and [B] SK-MES-1 cells. Mice were treated with delphinidin, tumor tissues were collected in 10% formalin and blocks were prepared in paraffin and immunofluorescence of CD31 and immunohistochemistry of VEGF were performed as described in “Materials and Methods”. Photomicrographs show representative pictures from three independent tumor samples. Bar = 20 µm.</p

The deficiency in IL-17 receptor inhibits TPA induced tumor promoting inflammation in the skin.

<p>Mice were treated topically with TPA every other day for a total of three applications. Skin tissues were harvested 24 hours after the last treatment. A). Paraffin embedded tissue sections were stained with hematoxylin. Skin samples from naïve untreated mice served as controls. Dashed lines indicate the border between the epidermis and dermis. The thickness of epidermis was measured microscopically (n = 10). B). Frozen tissue sections were stained immunohistochemcially with antibodies. CD11b+ (monocytes/macrophages) and Gr-1+ (granulocytes) cells were counted microscopically (n = 6). C) Concentrations of cytokines in the skin lysates were measured by ELISA (n = 4). D). S100A8 and S100A9 proteins in the skin lysates were detected by Western blots using specific antibodies. E). Concentrations of PGE2 in the skin lysates were measured by a PGE2 EIA kit (n = 3). F). Expression levels of mRNA for chemokines and cytokines in the skin lysates were quantified by real time RT-PCR (n = 3). Data are representative of 2–3 independent experiments (mean +/− SEM, * P<0.05: ** P<0.01).</p