Fig. 3: Transcriptome analysis revealed that overexpression of EZH2 catalytic function (elevated H3K27me3) increases KRT14 transcription.

A The schematic representation of experimental planning. B The immunoblot analysis of EZH2, H3K27me3 expression in 4T-1 cells harvested from the primary tumor and metastatic organs; GAPDH was used as a loading control. C The in vivo imaging of mice having a primary tumor and splenic metastatic cell inoculation (n = 5). The color scale indicated the photon flux (photon/s) emitted from each group. C Quantitative bar graph representation of total photon flux calculated from the region of flux (ROI), data are represented as mean, error bar ± SD, *P = 0.0038, compared to the primary tumor, two-way ANOVA, Sidak’s multiple comparisons tests. D The Kaplan-Meier survival curve of mammary tumor onset in the nude mice with inoculation of the primary tumor and spleen metastatic cells (n = 6). *P = 0.0150, compared to the primary tumor, log-rank test. E Volcano plot represents the significant differential expression of genes between control and Y641-F cells. Red and blue dots represent the upregulated and downregulated genes with (Log₂FC +1, −1, and p < 0.05), respectively. F Heat map of top 100 ranked genes differentially expressed between control and Y641-F phenotype. Expression values are represented as colors, where the range of colors (red, pink, light blue, dark blue) shows the range of expression values (high, moderate, low, lowest), respectively. G Heat map shows differential expression of cell migration and metastasis-related genes between control and Y641-F. H RNA was isolated from control and EZH2 Y641-F 4T-1 cells and subjected to real time-PCR for gene expression analysis. Data points are mean of triplicate readings of samples; error bars, ±S.D, ^*,#P = 0.0001, ^@P = 0.0134, ^$P = 0.001, ^{%,+,&,^}P = 0.0001, two way ANOVA, Sidak’s multiple comparisons test. I RNA was isolated from 4T-1 cells isolated from the primary tumor, metastatic lung, liver, and spleen and subjected to real time-PCR for gene expression analysis. Data points are the mean of triplicate readings of samples; error bars, ±S.D, left panel, ^*P = 0.0162, ^#P = 0.0101, ^XP = 0.0031, ^@P = 0.0002, ^$P = 0.0065, ^%P = 0.0039, ⁺P = 0.0001, ^&P = 0.001, ^{^}P = 0.0001, right panel, ^*P = 0.0001, ^#P = 0.0009, ^@P = 0.0001, ^$P = 0.0088, ^%P = 0.0009 and ⁺P = 0.0493, compared to isolated cells from primary tumors, two way ANOVA, Dunnett’s multiple comparisons test. Source data are provided as a Source Data file.