Possible Points of Canonical Wnt Inhibition.

<p>This study has highlighted six possible points of canonical Wnt inhibition occurring in HSCs. (1) Predominantly non-canonical ligand expression. Non canonical signalling is inhibitory to canonical signalling (2) Decreased Fzd receptor expression, reducing response to canonical ligands secreted by other cells (3) Increased sFRP expression (4) Increased expression of other morphogens such as Notch, able to directly inhibit βCatenin. In the nucleus, HSCs express higher levels of the repressive TCF3 in comparision to the other TCF/LEF family members associated with transcriptional activation(5) (6) Transdifferentiation is associated with upregulation of SOX9, direct inhibitor of βCatenin activity.</p

HSCs upregulated non-canonical effectors upon Wnt stimulus.

<p>(A) Western blot for total (T) and phosphorylated (P) forms of JNK and CamKii in two preparations of rat aHSCs treated for 24 hours with either control (-), Wnt5a or Wnt10b conditioned media. (B) Western blot for Dvl2 in two preparations of rat HSCs at 0,4,6,8 and 10 days in culture. (C) Western Blot for Dvl2 in LX-2 cells overexpressing Wnt3a, Wnt5a or Wnt10b. (D) Diagram illustrating points in Wnt pathway for which inhibitors were chosen: Wnt secretion (IWP2) and Dvl-PDZ domain function (Dvl-PDZ). (E) Western Blot for Dvl2 in rat HSCs treated with 1–20 μM of IWP2 (F) qRT-PCR for profibrotic markers in vehicle control (DMSO) and 20μM IWP2 treated rat aHSCs, (n = 4). (G)qRT-PCR for NFAT3, Sox9 and Axin2 in vehicle control (DMSO) and 5μM PDZ-Inhibitor treated rat aHSCs, (n = 3) (H) qRT-PCR for profibrotic markers in vehicle control and 5μM PDZ-Inhibitor treated rat aHSCs (n = 3). qRT-PCR results expressed as fold change normalised to control ± SEM *p<0.05 (Student’s <i>t</i>-test).</p

Activated HSCs express Wnt4, Wnt5a and Wnt6.

<p>(A) Products of RT-PCR for Wnt4, Wnt5a, Wnt6, Wnt7b, Wnt9b and Wnt3a in day 7 rat aHSCs, liver and brain (+ve) visualised on agarose gels using UV. Water only (-ve) serves as negative control. β-Actin serves as a loading control (B) qRT-PCR for Wnt4 and Wnt5a in culture activated rat HSCs (quiescent (qHSC) or day 7 activated HSC (aHSC) (n = 5). (C) qRT-PCR for Wnt4 and Wnt5a in rat HSCs after 0,4,6,8 and 10 days in culture, (n = 3) (D) Western Blot demonstrates increased Wnt5a expression in rat HSCs activated after 7 days in culture. Wnt3a protein expression is absent in both quiescent (qHSC) and activated (aHSC) cells. αSMA and TGFβ 1 serve as markers of HSC activation. Huh7 protein lysate serves as a positive control (E) Western Blot for Wnt5a protein in whole cell rat aHSC (Day 7) lysate and concentrated conditioned media (F) qRT-PCR for Wnt4 and Wnt5a in HSCs isolated from mouse livers injured by carbon tetrachloride injection (CCl₄) or Bile Duct Ligation (BDL) (n = 3) (G) Products of RT-PCR for Wnt3a, Wnt10b and Wnt5a in 3 preparations of human HSCs visualised on agarose gels using UV. Wnt overexpressing LX-2 cells were used as a positive control (+ve). qRT-PCR results expressed as fold change normalised to control ± SEM. *p<0.05 (Student’s T-test).</p

Expression of TCF/LEF transcription factors appears reduced in LX-2 cells.

<p>(A) Western Blot for TCF1, TCF3, TCF4 and LEF1in separate samples of LX-2 and HEK293 cells (B) TOPFLASH assay in HEK293 cells after 24hours incubation with conditioned medium from either HEK293 or LX-2 cells, (n = 4). (C) TOPFLASH assay in LX-2 cells overexpressing LEF1 and Ser37-βcatenin (n = 3) (D) TOPFLASH assay in HEK293 cells overexpressing Ser37-βCatenin or Ser37-βCatenin and sFRP4, (n = 3). Luciferase results represented as fold change of Firefly to Renilla *p<0.05 (Student’s <i>t</i>-test).</p

Wnt receptor expression alters upon HSC transdifferention.

<p>(A) Products of RT-PCR for Fzd Receptors and non canonical receptors in rat qHSCs and aHSCs visualised on agarose gels using UV. (B) qRT-PCR for Fzd receptors in rat qHSC and aHSC. (n = 5) (C) qRT-PCR for non-canonical receptors in rat qHSC and aHSC (n = 3), results expressed as fold change normalised to control ± SEM. *p<0.05, ***p<0.001 (Student’s T-test).</p

Wnt5a stimulus influences HSC survival and expression of profibrotic markers in Kuppfer cells.

<p>(A) Rat aHSCs were treated with control or Wnt5a conditioned medium and visualised by bright field microscopy. (B) Proliferation was assessed by MTT assay (C) Acridine Orange staining was used to quantify apoptotic response upon serum withdrawal (D) Migratory potential was assessed by scratch wound assay (E) Apoptotic response in LX-2 cells overexpressing Wnt 5a orWnt10b was also assessed by acridine orange staining in standard culture conditions (10% FCS) or serum free conditions. Data presented as number of apoptotic cells as percentage of total cell number. (F) qRT-PCR for profibrotic markers in control or Wnt5a conditioned medium treated rat Kuppfer cells, (n = 3) (G) Western Blot for TGFB1 expression in control or Wnt5a conditioned medium treated Kuppfer cells. qRT-PCR results are expressed as fold change normalised to control ± SEM *p<0.05, **p<0.01, ***p<0.001 (Student’s <i>t</i>-test).</p

Variant Histone H2afv reprograms DNA methylation during early zebrafish development

<p>The DNA methylome is re-patterned during discrete phases of vertebrate development. In zebrafish, there are 2 waves of global DNA demethylation and re-methylation: the first occurs before gastrulation when the parental methylome is changed to the zygotic pattern and the second occurs after formation of the embryonic body axis, during organ specification. The occupancy of the histone variant H2A.Z and regions of DNA methylation are generally anti-correlated, and it has been proposed that H2A.Z restricts the boundaries of highly methylated regions. While many studies have described the dynamics of methylome changes during early zebrafish development, the factors involved in establishing the DNA methylation landscape in zebrafish embryos have not been identified. We test the hypothesis that the zebrafish ortholog of H2A.Z (H2afv) restricts DNA methylation during development. We find that, in control embryos, bulk genome methylation decreases after gastrulation, with a nadir at the bud stage, and peaks during mid-somitogenesis; by 24 hours post -fertilization, total DNA methylation levels return to those detected in gastrula. Early zebrafish embryos depleted of H2afv have significantly more bulk DNA methylation during somitogenesis, suggesting that H2afv limits methylation during this stage of development. H2afv deficient embryos are small, with multisystemic abnormalities. Genetic interaction experiments demonstrate that these phenotypes are suppressed by depletion of DNA methyltransferase 1 (Dnmt1). This work demonstrates that H2afv is essential for global DNA methylation reprogramming during early vertebrate development and that embryonic development requires crosstalk between H2afv and Dnmt1.</p

Interaction of p50 and p65 with MMP3 gene promoter.

<p>Chromatin proteins and DNA in THP1 cells were cross-linked in 1% formaldehyde for 10 minutes. Cells were then lysed, and the lysate sonicated to reduce DNA length to 200–1000 bp. The sonicated chromatin was subjected to immunoprecipitation using a p50 or p65 antibody, with rabbit IgG as a control, followed by PCR amplification of 406 bp and 407 bp DNA fragments surrounding the <i>MMP3</i> gene 5A/6A site and then agarose gel electrophoresis of the PCR products. Intensity of bands on the agarose gels were quantified with the computer software ImageJ. The fold enrichment of DNA precipitated with p50 antibody or p65 antibody, relative to the IgG control, was calculated by comparing the intensity of the band of the amplicon from the p50 or p65 antibody precipitate to the intensity of the band of the amplicon from the IgG control. The graph shows fold enrichments of DNA precipitated with p50 antibody or p65 antibody, relative to the IgG control. Data shown are mean (± standard error of mean) from four independent experiments. * denotes p<0.05 comparing DNA precipitated with p50 antibody or p65 antibody to the IgG control.</p

The MMP3 gene 5A allele more readily interacts with p50 and p65, than the 6A allele.

<p>Chromatin proteins and DNA in THP1 cells were cross-linked in1% formaldehyde for 10 minutes. Cells were then lysed, and the lysate sonicated to reduce DNA length to 200–1000 bp. The sonicated chromatin was subjected to immunoprecipitation using a p50 or p65 antibody, or incubated with a rabbit IgG, followed by PCR amplification of 179 bp and 180 bp DNA fragments surrounding the <i>MMP3</i> gene 5A/6A site using fluorescence labelled primers. The PCR amplicons were analyzed using an ABI 3730xl analyzer and Genemapper software. Three independent experiments were performed. (A). Representative output from the Genemapper programme. The panels from top to bottom represent input chromatin DNA, chromatin immunoprecipitated with the p50 antibody, and chromatin immunoprecipitated with the p65 antibody. (B). Chart shows mean (± standard error of mean) of the relative peak heights of the 5A and 6A alleles in the p50 and p65 antibody precipitates, standardized against the relative peak heights of the 5A and 6A alleles in the input DNA, in three independent experiments. * denotes p<0.05 comparing 5A versus 6A alleles.</p

NFκB has a greater upregulatory effect on the 5A allele than the 6A allele.

<p>RAW264 cells were transfected with an <i>MMP3</i> 5A-firefly luciferase reporter plasmid or an <i>MMP3</i> 6A-firefly luciferase reporter plasmid, and the pRL-TK plasmid (expressing <i>renilla</i> luciferase) to serve as a reference for transfection efficiency, together with a plasmid expressing p50 and/or a plasmid expressing p65. Luciferase reporter assay was performed using a dual-luciferase reporter assay system. Data shown are mean (± standard error of mean) of the ratio of firefly luciferase activity over <i>renilla</i> luciferase activity from four independent experiments. In panel (A), * indicates p = 0.07 comparing 5A and 5A plus p50. In panel (B), ** indicates p<0.01 comparing 5A and 5A plus p65. In panel (C), * denotes p<0.05 comparing 5A with 5A plus p50 and p65.</p