Fig 2: ?H2AX positivity increase in IM lesions as compared to normal controls in the GCEP cohort. (A) Samples from the GCEP cohort comprising of six cases of normal gastric tissue, seven cases of NAG and six cases of IM were subjected to multiplex IHC staining with antibodies against EpCAM/pan-Cytokeratin (CK), MCM2 and ?H2AX. Immunofluorescence images of ?H2AX or MCM2 with EpCAM/pan-Cytokeratin (CK) are shown. Scoring map depicts epithelial cells positive for ?H2AX (magenta), MCM2 (green), ?H2AX/MCM2 double-positive (DP) (red) and ?H2AX/MCM2 double-negative (grey). (B) Plot shows percent ?H2AX positivity across the three stages, normal, NAG and IM. Line within the vertical scatter plot shows the mean value. Error bar shows ±SD. Statistical significance was analysed by the Mann-Whitney non-parametric test. * p<0.05, n.s-not significant. GCEP, gastric cancer epidemiology programme; IHC, immunohistochemistry; IM, intestinal metaplasia; NAG, non-atrophic gastritis.

Fig 3: Changes in HEK293 cells expressing p.R44C variant MCM2.(a-b) HEK293 cells were treated with liposome transfection reagent only (lipo), empty vector transfection (empty vector), wild-type MCM2 plamid transfection (WT), and variant MCM2 plasmid transfection (M) respectively. After transfection for 48 h, apoptotic cell ratios were determined with annexin-V-PE-staining. Data represent the mean and SD of 3 experiments. The asterisks (**) indicate significant differences between the control and experimental groups or differences between the WT group and the M group (**p<0.001). (c-d) The western blot analysis of whole cell lysates from HEK293 cells showed a level of endogenic expression of MCM2 (as shown in lipo and empty vector groups). The upper bands in lane 3 and 4 (from the left) represent the transfected exogenous MCM2 with a GFP tag. The endogenous level of MCM2 is shown in the lower band. The ratio of cleaved caspase3 densitometry to exogenous MCM2 densitometry is compared. In the histogram, whole cell lysates from the variant MCM2 (M) transfected cells showed an upper regulated expression of cleaved caspase-3 compared with wild-type MCM2 (WT) transfected cells. Data represent the mean and SD of 3 experiments. The asterisks (*) indicate significant differences between the WT group and the M group (*p<0.01). (e) Cell cycle phases of HEK293 cells transiently transfected with wild-type or c.130C>T variant MCM2 cDNA plasmid for 48 h. Cell cycle phases were measured by flow cytometry using a cell cycle assay kit. In cells transfected with wild-type MCM2 cDNA plasmid, G1, S, and G2 phases were 27.08±4.99, 50.69±2.14, 22.21±6.04, respectively; N = 3. In cells transfected with the variant MCM2 cDNA plasmid, G1, S, and G2 phases were 28.61±1.29, 44.80±0.64, 26.57±0.80, respectively; N = 3.

Fig 4: miR-195-5p/497–5p could suppress the tumorigenesis and stem-like properties of CCSCs in vivo by targeting MCM2. CCSCs (SW620 CSCs and LoVo CSCs) with overexpressed miR-195-5p/497–5p and/or MCM2 were subcutaneously injected into nude mice to establish a subcutaneous xenograft tumor model. a & b, The volume and weight of xenografted tumors were observed and recorded. c, The expression of MCM2, miR-195-5p and miR-497-5p detected by RT-qPCR. d, The protein expression of MCM2, CD133, and EpCAM in tumor tissues as detected by Western blot analysis. e, The protein expression of apoptosis-related proteins (Bcl-2 and Bax) in tumor tissues as detected by Western blot analysis. Measurement data were expressed as mean ± s.d. Data among multiple groups were compared using one-way analysis of variance and then analyzed with Tukey’s post-hoc test. Repeated measures analysis of variance was used for comparing data among multiple groups at different time points, followed by Bonferroni post-hoc test. Cell experiment was repeated three times. * p < 0.05 vs. miR-NC. # p < 0.05 vs. miR-195-5p/497–5p agomir + pcDNA-3.1

Fig 5: BRD4-E2F2-cell cycle regulation circuit is highly activated in human HCC tissues and high E2F2 expression is associated with poor prognosis of HCC patientsA. The relative E2F2 gene expression levels in non-cancerous tissue (Non-tumor) and HCC patients’ tissue (Tumor) was illustrated by scatter blot using TCGA data set. The median expression level of each group was indicated by horizontal lines. B. Overall survival dependent on E2F2 expression was shown by Kaplan-Meier survival curves. P-values were obtained with the log-rank test. C. The relative level of represented core genes of BRD4 inhibition and E2F target including CDC25A, MCM2, MCM3, MCM4, MCM6 MCM7, PNCA and PKMYT1 were illustrated with box plot using TCGA dataset. D. The correlation analysis was performed between E2F2 and 8 core genes expression and represented by correlation heatmap. The numbers indicate R values which calculated based on Pearson correlation coefficient. E. BRD4, E2F2, MCM2, MCM3, PCNA and GAPDH protein levels were analyzed by western blot in three human HCC tissues paired with histologically normal liver tissue.