1. Volume 29, Issue 3, Pages 354-366 (March 2016)
Smyd3 Is a Transcriptional Potentiator of Multiple Cancer-Promoting Genes and Required for Liver and Colon Cancer Development 
Michalis E. Sarris, Panagiotis Moulos, Anna Haroniti, Antonis Giakountis, Iannis Talianidis 
Cancer Cell 
Volume 29, Issue 3, Pages (March 2016)
DOI: /j.ccell
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2. Cancer Cell 2016 29, 354-366DOI: (10.1016/j.ccell.2016.01.013)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Smyd3 Is Required for DEN-Induced Liver and DMH/DSS-Induced Colon Cancer Formation
(A) Macroscopic appearance of livers in 7- and 8.5-month-old DEN-treated and untreated wild-type (WT), Smyd3-KO, and Smyd3-Tg mice.
(B) Quantitation of tumor foci numbers and areas in DEN-treated livers. Average values and standard errors (±SEM) were from n = 12 mice.
(C) Representative H&E staining of liver sections of 8.5-month-old DEN-treated wild-type (WT), Smyd3-KO, and Smyd3-Tg mice.
(D) Macroscopic appearance of colons of wild-type (WT) and Smyd3-KO mice after DMH/DSS treatment.
(E) Quantitation of tumor foci numbers and areas in DMH/DSS-treated colons (n = 9).
(F) Representative H&E staining of colon sections of DMH/DSS-treated wild-type (WT) and Smyd3-KO mice.
See also Figure S1.
Cancer Cell  , DOI: ( /j.ccell )
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4. Figure 2
Smyd3 Is Required for Cell Proliferation in DEN-Treated Liver and DMH/DSS-Treated Colons
(A) Representative immunohistological staining of liver sections from 8.5-month-old untreated and DEN-treated wild-type (WT) and Smyd3-KO mice with Ki67 antibody.
(B) Quantitation of Ki67-positive cells in untreated and DEN-treated mice. Bars represent average percentages and SEM of cells positively staining for Ki67 over all cells (estimated by DAPI staining) examined in 10 high-power fields (HPF) of sections from three different animals. ∗∗p <
(C) RT-PCR analysis of Cyclin A2 (CcnA2), Cyclin D1 (CcnD1), and Cyclin E1 (CcnE1) mRNA levels in the livers of 8.5-month-old untreated and DEN-treated mice. Bars represent mean mRNA levels normalized to Gapdh mRNA and ±SEM from n = 5 individual mice. ∗p < 0.01; ∗∗p <
(D) Representative immunohistological staining of colon sections from untreated and DMH/DSS-treated wild-type (WT) and Smyd3-KO mice with Ki67 antibody.
(E) Quantitation of Ki67-positive cells in the colon sections of untreated and DMH/DSS-treated mice. Data are presented as in (B).
(F) RT-PCR analysis of Cyclin A2 (CcnA2), Cyclin D1 (CcnD1), and Cyclin E1 (CcnE1) mRNA levels in the colons of untreated and DMH/DSS-treated mice. Data are presented as in (C) (n = 5).
See also Figure S2.
Cancer Cell  , DOI: ( /j.ccell )
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5. Figure 3
Smyd3 Is Required for Normal Cell Proliferation and Oncogene Activation
(A) Representative immunohistological staining of liver sections from wild-type (WT) and Smyd3-KO mice 48 hr after partial hepatectomy (PH) with Ki67 antibody. Panel at right: quantitation of Ki67+ cell occurrence. Bars represent average number of cells and SEM staining positively for Ki67 in 10 HPFs of sections from four mice. ∗∗p < Control mice were sham operated.
(B) Cyclin D1 (CcnD1) and Cyclin E1 (CcnE1) mRNA levels in the livers 48 hr after partial hepatectomy (PH). Bars represent mean mRNA levels normalized to Gapdh mRNA and ±SEM from n=4 individual mice. ∗p < 0.01; ∗∗p <
(C) Myc and Ctnnb1 mRNA levels in the livers of 8.5-month-old untreated (Control) and DEN-treated mice. Data are presented as in (B) (n = 5).
(D) Activation of Stat3. Western blot analysis of liver extracts from 8.5-month-old untreated (Contr.) and DEN-treated mice with STAT3 (Total Stat3) and phospho-STAT3 (P-Stat3) antibodies.
(E) Il6, Jak1, and Jak2 mRNA levels in the livers of 8.5-month-old untreated (Control) and DEN-treated mice. Data are presented as in (B) (n = 5).
See also Figure S3.
Cancer Cell  , DOI: ( /j.ccell )
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6. Figure 4 Smyd3 Is Required for EMT in Liver Cancer
(A) Representative immunohistological staining of liver sections from 8.5-month-old untreated and DEN-treated wild-type (WT) and Smyd3-KO mice with E-cadherin and Ki67 antibody.
(B) mRNA levels of the key EMT regulators in the livers of 8.5-month-old untreated (Control) and DEN-treated mice. Bars represent mean mRNA levels normalized to Gapdh mRNA and ±SEM from n=5 individual mice. ∗p < 0.01; ∗∗∗p <
(C) Western blot analysis of Snai1 and Fn1 protein levels in liver extracts of 8.5-month-old untreated (Control) and DEN-treated wild-type (WT) and Smyd3-KO mice.
(D) mRNA levels of matrix metalloproteases (Mmp 14, 9, 7, 2) in the livers of 8.5-month-old untreated (Control) and DEN-treated mice. Data are presented as in (B) (n = 5).
(E) Zymogram showing Mmp9 activity in liver extracts of 8.5-month-old untreated (Control) and DEN-treated wild-type (WT) and Smyd3-KO mice. Panel at right: Quantitation of Mmp9 activity signals by ImageJ software. Bars represent average fold difference of activity over wild-type control and SEM from four experiments. ∗∗p <
See also Figure S4.
Cancer Cell  , DOI: ( /j.ccell )
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7. Figure 5 Smyd3 Is Required for EMT in Colon Cancer
(A) Representative immunohistological staining of colon sections from untreated and DMH/DSS-treated wild-type (WT) and Smyd3-KO mice with E-cadherin and Ki67 antibody.
(B) mRNA levels of Snai1, Fn1, Timp1, Mmp7, Mmp9, and Mmp13 in the colons of untreated (Control) and DMH/DSS-treated mice. Bars represent mean mRNA levels normalized to Gapdh mRNA and ±SEM from n=5 individual mice. ∗p < 0.01; ∗∗p <
(C) Zymogram showing Mmp9 activity in colon extracts of untreated (Control) and DMH/DSS-treated wild-type (WT) and Smyd3-KO mice. Panel at right: Quantitation of Mmp9 activity signals by ImageJ software. Bars represent average fold difference of activity over wild-type control and SEM from four experiments. ∗∗p <
See also Figure S5.
Cancer Cell  , DOI: ( /j.ccell )
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8. Figure 6
Comparative Analysis of Transcriptomic and Genome-Wide Smyd3 Binding Profiles in Liver Cancer
(A) Multidimensional scaling (principal coordinate analysis) plots of the mRNA expression levels in the livers of 8.5-month-old untreated wild-type (WT), Smyd3-KO (Smyd3KO), and DEN-treated wild-type (DEN-WT) or DEN-treated Smyd3-KO (DEN-Smyd3KO) mice. The proportion of variability (or dissimilarity) explained by each principal coordinate is indicated in parentheses.
(B) Hierarchical clustering analysis and corresponding heatmap of differentially expressed genes in the livers of DEN-treated versus untreated 8.5-month-old wild-type (WT) and Smyd3-KO mice. The color scale bar indicates the fold-change ranges for the comparisons (ratios) indicated on the top of the heatmap. Representative genes whose differential expression was validated by RT-PCR are indicated on the right.
(C) Binding-intensity heatmaps showing the genomic occupancy distribution of Smyd3 and RNA Pol-II, as well as H3K4Me3 modifications from 2 kb downstream to 2 kb upstream from the Smyd3 peak summits (PS, base pair showing the highest Smyd3 read pileup) of Smyd3-occupied genes in the livers of 8.5-month-old DEN-treated wild-type (first four panels from the left) and Smyd3-KO (last panel) mice. The depicted regions were ranked by decreasing Smyd3 binding strength, as measured by the normalized fold enrichment of reads under Smyd3 peaks to the respective input regions.
(D) Venn diagram showing the overlap between RNA Pol-II and Smyd3 occupancy at the core promoter regions in the livers of 8.5-month-old DEN-treated wild-type mice.
(E) Correlation between RNA Pol-II and Smyd3 coverage in the livers of 8.5-month-old DEN-treated wild-type mice. RNA Pol-II and Smyd3 binding densities were calculated on an average genomic region normalized coverage basis (average mapped reads per base pair −1 kb to +1 kb from TSS of Smyd3-occupied genes) and ranked by increasing RNA Pol-II levels and mRNA levels (color bar at bottom). The read counts of input sample are also indicated (solid black line). The dashed line represents the median normalized RNA Pol-II coverage, which was chosen as a cut-off value of RNA Pol-II occupancy for silent and actively transcribed genes.
See also Figure S6 and Tables S1 and S2.
Cancer Cell  , DOI: ( /j.ccell )
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9. Figure 7
Distribution of Smyd3 Binding at the Core Promoters and Association with Trimethylated H3K4
(A) Average binding profiles of Smyd3, RNA Pol-II, and H3K4Me3 around the TSS of Smyd3 occupied genes in 8.5-month-old DEN-treated wild-type and Smyd3-KO mice. The profiles were calculated on the basis of average normalized coverage (reads per base pair).
(B) Average binding profile of Smyd3, RNA Pol-II, and H3K4Me3 modifications of Smyd3 peak summits for Smyd3 peaks located at −1 kb to TSS (panel at left) and at TSS to +1 kb regions (panel at right). Red line, Smyd3 reads; green line, RNA Pol-II reads; yellow line, H3K4Me3 reads in DEN-WT mice; blue line, H3K4Me3 reads in DEN-Smyd3KO mice.
(C) In vitro association of Smyd3 with modified histone tail peptides. Extracts from CMV-Flag-Smyd3-transfected HEK-293 cells were incubated with the indicated histone-3-tail peptides immobilized to streptavidin-agarose beads. Interaction with Smyd3 was evaluated by western blot analysis using Flag antibody.
(D) Venn diagram showing the overlap between H3K4Me3 modifications and Smyd3 occupancy at the core promoter regions in the livers of 8.5-month-old DEN-treated wild-type mice.
(E) Schematic presentation of Smyd3-regulated cancer-promoting genes and pathways.
See also Figure S7 and Tables S1 and S2.
Cancer Cell  , DOI: ( /j.ccell )
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