Fig 1: DNA re-replication requires the components of the MLL complexes. (A) HCT116 cells were transfected with 50 nM siRNAs of luciferase, Geminin, Geminin+RBBP5, and RBBP5 for 48 h. The nuclei were stained with DAPI. Scale bar: 50 μm. (B) The percentages of enlarged nuclei in various siRNA-ablated cells in A. Enlarged nuclei were examined, quantified, and error bars calculated in each sample for standard deviations as in Fig. 1B. Left plot: the statistical differences in enlarged nuclei between control siRNA-treated and each specific siRNA-treated cells were calculated using the two-tailed Student's t-test (P1=3.17E-04 and P2=1.39E-03). Right plot: significant difference was observed between Geminin siRNA-treated and Geminin+RBBP5 double siRNA-treated cells (P=0.00328<0.01) as indicated. (C) FACS analysis of DNA contents in A. The polyploid cell populations (>4N) are indicated. Three independent experimental repeats were conducted with similar results, and only one representative experiment is shown. (D) The statistical differences in polyploid DNA (>4N) in Geminin siRNA- treated and Geminin+RBBP5 double siRNA-treated cells were calculated using the two-tailed Student's t-test (P1=1.06E-04, P2=2.45E-02). (E) The proteins in the lysates from siRNA-treated cells in A were analyzed by immunoblotting with specific antibodies as indicated. The changes of histone H3K4 methylations were quantified on the right plot. Error bars indicate standard deviation of triplicated samples. The statistical differences were evaluated between control (luciferase) and specific siRNA samples using the two-tailed Student's t-test (P3=0.0335, P4=0.00397, P5=0.0233, P6=0.0168, P7=7.61E-04 and P8=0.0020). (F) Reduction of DPY30 or ASH2L suppresses the formation of polyploid nuclei in Geminin-deficient cells. HCT116 cells were transfected with 50 nM siRNAs of luciferase, Geminin, DPY30 or ASH2L, and Geminin+DPY30 or ASH2L for 48 h as in A. The statistical significance between single Geminin siRNA- treated and Geminin+DPY30/ASH2L double siRNA-treated cells was analyzed by the two-tailed Student's t-test (P9=0.00663 and P10=0.0171). (G) FACS analysis of DNA content from cells in F. The polyploid DNA fractions of cells were indicated. Three independent experimental repeats were conducted with similar results, and only one representative experiment is shown. (H) The statistical differences in polyploid DNA (>4N) in Geminin siRNA- treated and Geminin+DPY30 or ASH2L double siRNA-treated cells were calculated as in 2D. (I) Immunofluorescence staining of H2AX in A. Scale bars: 50 μm.
Fig 2: Analysis of gene expression profile alterations upon DPY30 knockdown in HT29 cells based on RNA-Seq. (A) Venn diagram showing the genes expressed in HT29-shCtrl and HT29-shDPY30 cells. Among these genes, 10814 are co-expressed, 2741 genes up-regulated (diagram with red bar) and 2906 genes down-regulated (diagram with green bar). The number of specifically expressed genes between the two groups is 874 and 643, respectively. (B) Volcano plot showing on the x-axis changes in gene expression levels in HT29-shCtrl and HT29-shDPY30 cells and on the y-axis the p values associated with these changes. (C, D) Heatmaps representing unsupervised hierarchical clustering of mRNA expression level depicting the top 50 downregulated (panel C) and upregulated genes (panel D) with FDR < 0.05. Each column represents the indicated sample. The genes tracked red were found and selected. (E) The relative mRNA expression levels of the other cell cycle related genes found in top 50 regulated genes (ANKRD53, CUZD1, MAGI2 and ABCB1) when DPY30 knockdown were detected by qRT-PCR. Data were expressed as mean ± SEM. ***P < 0.001. (F) Gene ontology (GO) biological process analysis for genes downregulated by DPY30's knockdown in HT29 cells (FDR < 0.05 and fold change ≥ 2).
Fig 3: DPY30 knockdown suppresses the establishment of H3K4me3 to PCNA, Ki67 and cyclin A2 genes then downregulates transcriptional expression. (A) Western blotting to detected the endogenous levels of histone H3 when tri-methylated on Lys4 in KM12C and HT29 shDPY30 cells. (B) qPCR was performed to evaluate the ChIP analysis of IgG and H3K4me3 interaction status with candidate DPY30 target genes (PCNA, Ki67 and cyclin A2) after knockdown assay. H3K4me3 bindings were monitored at the promoters of PCNA, Ki67 and cyclin A2 genes. IgG antibody was included as a negative control. H3K4me3 was normalized to total H3. Values were presented as percentage of input. The results were presented as the means ± SEM of values obtained in three independent experiments. **P < 0.01, ***P < 0.001. (C) The correlations between DPY30 with PCNA, Ki67 and cyclin A2 (CCNA2) were evaluated in the GEPIA2 database. R, correlation coefficient. Positive number indicated positive correlation and negative number indicated negative correlation.
Fig 4: Graphical Abstract. Overexpression of DPY30 promotes CRC cell proliferation and cell cycle progression through facilitating the transcriptional expression of PCNA, Ki67 and cyclin A2 via mediating the establishment of H3K4 trimethylation on gene promoter regions, consequently promoting the tumorigenesis and progression of color
Fig 5: DPY30 knockdown inhibits CRC tumor growth in vivo. (A) The tumor volume growth curve of nude mice was measured from xenograft tumour models established with HT29-shDPY30 and its control cells (n = 6) every 2 days. (B) Tumors formed by subcutaneous injection photographed (C) Tumors weight were measured for statistical analysis at harvest time. (D) The changes of protein expression of PCNA, Ki67, cyclin A and H3K4me3 after DPY30 knockdown in xenograft tumor tissues by western blotting. (E) Representative IHC staining of tissue sections from shCtrl and shDPY30 tumors. Magnification, 400×. (F) The correlation between DPY30 protein expression levels with Ki67, PCNA and cyclin A protein level were measured in the animal tumor tissues respectively. The IHC score values were subjected to Pearson correlation analysis (n = 15). Data were expressed as means ± SEM of values. ***P < 0.001, between the indicated subgroups.
Supplier Page from Abcam for Anti-DPY30 antibody