HFE2 is partially cleaved at the N-terminus in iron-deficient animals.

<p>(A) Partial cleavage of full length HFE2 (arrow) to an approximately 5 kDa shorter protein (double arrow) under iron deficiency conditions. Weaned rats and mice were kept on an iron deficient diet for three or four weeks respectively (ID), control animals (C) received standard laboratory diet. Loading controls are similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148540#pone.0148540.g002" target="_blank">Fig 2A</a>. (B) Comparison of the effects of iron-deficient diet (ID) or EPO treatment (E) on TMPRSS6 protein content and HFE2 cleavage in rats. Both treatments increase TMPRSS6 protein content; cleaved HFE2 band (double arrow) is apparent only in iron-deficient animals. (C) Cleaved HFE2 protein has the same number of N-linked oligosaccharides as the full length protein. Samples from control (C) or iron-deficient (ID) rats and mice were incubated with PNGase F (PNG +) or water (-). PNGase F treatment reduced the size of full length HFE2 protein (arrow) by about 10 kDa; the same size reduction of approximately 10 kDa was observed for the cleaved HFE2 protein (double arrow) present in iron-deficient samples. (D) Schematic representation of full length mouse HFE2 protein. The 361 amino acid chain has Q33 as the N-terninal amino acid (UniProt entry Q7TQ32), and is bound with a GPI anchor to the plasma membrane (PM). Arrow denotes the approximate position of the observed cleavage site, which is located N-terminally of the three N-linked oligosaccharide (NGlc) chains.</p

Erythropoietin treatment increases TMPRSS6 protein content in <i>Bmp6</i>-mutant mice.

<p>(A) Increase of TMPRSS6 protein content in <i>Bmp6</i>-mutant mice mice treated with saline (C) or EPO (E) at 50 I.U./mouse for four days. Arrow denotes TMPRSS6-specific band. ATP1A is used as loading control. (B) TMPRSS6 band density quantification. (C and D) Effect of EPO treatment on <i>Tmprss6</i> and <i>Hamp</i> mRNA content. <i>Tmprss6</i> and <i>Hamp</i> mRNA content is determined relative to <i>Actb</i> mRNA.</p

Immunoblot of TMPRSS6, HFE2 and ATP1A in the 3000 g fraction.

<p>Detection of TMPRSS6, HFE2 and ATP1A is enhanced in the 3000g fraction of mouse liver homogenates (3000g) in comparison with whole liver homogenates prepared in 1% NP40 (NP40) or microsomes (Micros). Samples were prepared from C57BL/6 mice (C), <i>mask</i> mice (<i>m</i>), <i>Hfe2</i>+/+ mice (WT) and <i>Hfe2</i>-/- mice (KO). Arrows denote the TMPRSS6-, HFE2- and ATP1A-specific bands. In contrast to the NP40 whole liver homogenate or liver microsomes, HFE2 is present in the 3000 g fraction solely as a full-length protein.</p

High dose of iron dextran decreases TMPRSS6 protein content.

<p>(A) Effect of iron on TMPRSS6 and HFE2 protein content. Mice were treated with 200, 350 and 750 mg/kg of iron dextran one week before sacrifice, control mice (C) received physiological saline. ATP1A is used as loading control. Arrow denotes the TMPRSS6-specific protein band, arrowhead denotes a nonspecific band. Graph represents TMPRSS6 band density quantification, asterisk denotes statistical significance (p<0.05, n = 3). (B) Effect of a 1000 mg/kg of iron dextran administered to mice 24 h prior to sacrifice. Graph represents TMPRSS6 band density quantification, asterisk denotes statistical significance (p<0.05, n = 4).</p

TMPRSS6 protein content and neogenin protein content is decreased in <i>Hfe2-/-</i> mice.

<p>(A) Immunoblot of TMPRSS6 protein in the liver 3000 g fraction of <i>Hfe2</i>+/+, (<i>Hfe2</i> WT), <i>Hfe2</i>-/+ (Het), <i>Hfe2</i>-/- (KO), C57BL/6 (WT) and <i>mask</i> (<i>m</i>) mice. Arrow denotes the TMPRSS6-specific band; arrowhead indicates a nonspecific band. <i>Mask</i> mice samples represent results from two experiments with two pairs of mice. (B) and (C) Immunoblots of HFE2, neogenin (NEO1) and ATP1A (loading control) in the same samples. Arrows denote HFE2- and NEO1-specific bands.</p

Erythropoietin treatment increases TMPRSS6 protein content.

<p>(A) Increase of TMPRSS6 protein content in rats and mice treated with saline (C) or EPO (E) at 500 or 50 IU/animal for four days. Arrows denote TMPRSS6-specific bands, arrowhead denotes a nonspecific band present in rat liver samples. ATP1A is used as loading control. (B) TMPRSS6 band density quantification. Asterisk denotes statistical significance (p<0.05), n = 5 for rats and n = 6 for mice. (C) Lack of effect of EPO treatment on <i>Tmprss6</i> mRNA. <i>Tmprss6</i> mRNA content is determined relative to <i>Actb</i> mRNA.</p

Caída libre de los cuerpos (Simulación)

En este artículo se muestra la simulación por computador y sus resultados tanto gráficos como numéricos de la posición, velocidad y aceleración de uno o dos cuerpos que pueden ser lanzados hacia arriba o hacia abajo en forma simultánea o con tiempos diferentes, en el mismo planeta o en planetas diferentes, desde la misma posición o posiciones diferentes y en el mismo sentido o sentidos opuestosAbstract : In this article is shown the simulation by computer and their results graphic as well as numerical of the position, velocity and aceleration when an object or two objects are launched to up or to down simultaneous or times diferents in the same planet or diferents planets, from the same position or diferents positions and same sense or opposite senses

Knockdown of <i>Cep164</i> causes S-phase delay.

<p>(A–D) Endogenous <i>Cep164</i> knockdown leads to block in S-phase and is rescued by inducible human wild type <i>CEP164</i> but not by human mutant <i>CEP164</i>. Under thymidine synchronization, <i>Cep164</i> knockdown cells (black) were lagging in the transition from S to G₂/M phase in comparison to the control siRNA treated cells (white) in all IMCD3 <i>N-GFP-CEP164-WT</i> (A) IMCD3 <i>N-GFP-CEP164-Q525X</i> (B) and IMCD3 <i>N-GFP-CEP164-R93W</i> (C) cell lines. Upon doxycycline induction of wild type human CEP164 construct IMCD3 <i>N-GFP-CEP164-WT</i> (light grey) cells were rescued from arrest in S-phase (A). In contrast, overexpression of the cDNA clone that represents the human truncating mutant <i>N-GFP-CEP164-Q525X</i> cells did not rescue the S-phase lag. In addition, overexpression of <i>N-GFP-CEP164-Q525X</i> (dark grey) caused an increase of cells in S-phase, indicating a dominant negative effect of the human truncating mutant (B). Cells were not rescued by <i>N-GFP-CEP164-R93W</i> (C) from the S-phase lag upon doxycycline induction. P values were calculated using two-way ANOVA and Bonferroni multiple comparison test. (n = 3, *p<0.05). (D) Western blot showing expression of N-GFP CEP164 WT and R93W by CEP164 antibody and N-GFP CEP164 Q525X by GFP antibody upon doxycycline induction. β-actin is used as loading control. (E) Lysates of IMCD3 cells were made 1 hour after 10J UV exposure. Western blot was performed for Cep164 and loading control β-actin. Stabilization of Cep164 is visible after DNA damage induction (n = 3). (F) Lysates of RPE cells were made 56 hours after knockdown of <i>CEP164</i> with 20 nM siRNA. Upregulation of DNA damage marker γH2AX is shown on western blot with loading controls H2AX and β-actin (n = 3). (G) Lysates of thymidine synchronized IMCD3 cells were made 56 hours after knockdown of <i>Cep164</i> with 20 nM siRNA. Upregulation of DNA damage marker γH2AX is shown on western blot with loading control H2AX and β-actin 6 hours after release of the thymidine block (n = 3). (H) Urine derived renal cells stained for γH2AX (green) and CEP164 (magenta) of NPHP patient and unaffected control. γH2AX intensities were quantified by ZEN2011 software. T-test reveals statistical difference (***p<0.001) (n = 15). (I) S-phase marker PCNA expression is increased in RPE and IMCD3 cells transfected with siCep164/CEP164p and –i compared to control transfected cells. β-actin was used as loading control. APH exposure (18 hour, 400 nM) enhanced the PCNA expression levels in both cell lines.</p

Schematic overview of signaling cascade involved in development of PKD and NPHP.

<p>Mutations in ciliary genes cause ciliary defects. Ciliary defects result in proliferation defects, loss of planar cell polarity (PCP) and deregulated cellular signaling. This causes cystic kidney disease. When the gene mutated is also involved in the DNA damage response (DDR) or DNA replication, impairments of these processes cause apoptosis, epithelial-to-mesenchymal transition (EMT) and consequently fibrosis in the NPHP patients.</p

CEP164 regulates cell cycle progression and proliferation.

<p>(A) RPE-FUCCI cells and their daughter cells were tracked by live cell imaging for 72 hours (hr) after transfection. <i>CEP164</i> depleted cells have a quicker cell cycle (∼35 hr) compared to control cells (∼48 hr) (>25 cells and their daughter cells per position (n = 3) per experimental condition per experiment (n = 3), One-way ANOVA (Dunnett's post hoc) (*p<0.05). Error bars represent SEM. (B) Each cell cycle stage in siControl and si<i>CEP164</i> transfected cells was measured. S-phase took significantly longer in si<i>CEP164</i> transfected cells and their daughter cells (8.6 hour) compared to control (5.7 hour) (**p<0.01). G₁-phase is significantly shorter in si<i>CEP164</i> transfected cells (10.7 hour) compared to control (20 hr) (*p<0.05). G₂- and M-phase were almost significantly shorter in si<i>CEP164</i> transfected cells (7.9 and 6.8 hr respectively) compared to control (11.6 and 11.1 hour respectively; both p = 0.06) (>25 cells and their daughter cells per position (n = 3) per experimental condition per experiment (n = 3), ± represent SEM, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004594#pgen.1004594.s001" target="_blank">Figure S1E</a> for details. (C) Fluorescence images of RPE-FUCCI cells expressing mKO2-hCdt1 (30/120) (magenta) and mAG-hGem (1/110) (green) constructs were pulsed for 30 minutes with EdU (10 µM) and stained with Alexa anti-EdU-647 to visualize cells in early S-phase (white) and DAPI to visualize the nuclei (blue). Cells expressing both constructs also show EdU incorporation (white). Scale bar represents 10 µm. (D) Quantification of a time series of serum released RPE-FUCCI cells after 24 hour serum starvation. Cell cycle stage and ciliation were quantified and correlated. Both decreased ciliary frequency and increased cell cycle entry were observed in <i>CEP164</i> depleted cells. Error bars represent SEM. (E) Quantification of RPE and IMCD3 cell proliferation using the CyQUANT NF Cell Proliferation Assay Kit. Fluorescence intensities of quadruplicate samples after 72 hours after transfection were measured. A significant reduction in cell number is visible after respectively <i>CEP164</i> and <i>Cep164</i> knockdown (n = 3, **p = 0.007, ***p = 0.001). Error bars represent SEM.</p