Figure 1: H2A.X is a key player in the regulation of EMT and in colon cancer metastasis signalling.

HCT116 cells were infected with shRNAs against H2A.X (shH2A.X) or scrambled sequences (shCTRL). (a,b) Compared with controls, shH2A.X-infected HCT116 cells exhibited decreased H2A.X protein levels (a, top, immunoblot), mesenchymal-like morphology (a, bottom, photomicrographs, scale bar, 100 μm) and increased invasion in transwell invasion assays (b, top, photomicrograph, scale bar, 100 μm; bottom, quantification). Error bars represent the means±s.d. of three independent experiments. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (c) Ingenuity Pathway Analysis (IPA) revealed differentially expressed genes (DEGs) in three canonical pathways. Blue bars represent the top three canonical pathways overrepresented in the DEGs. The height of the bar represents the P value. The yellow line indicates the −log (P value) threshold of significance (1.3), which corresponds to P value of 0.05. Statistical significance was determined by Fisher’s test. (d) Venn diagram of DEGs assigned to the invasion, migration and metastasis pathways using IPA. DEGs were generated using Partek Genomics Suite software, version 6.6. (e) Heat map of the 18 DEGs common to the invasion, migration and metastasis pathways. The numbering refers to independent replicates for either shCTRL sample or shH2A.X sample. (f) Verification of differential expression of genes by real-time PCR). Expression values are relative fold change for gene transcripts normalized to 18S RNA (gene/18S ratio). Error bars represent s.d. (n=3). Statistical significance was determined by a two-tailed, unpaired Student’s t-test.