Fig 1: Mcm2 chromatin localization largely depends on its ability to bind H3-H4.(A) Representative heatmaps of Mcm2, H3K4me3, H3K27me3 CUT&RUN, and ATAC-seq peaks in wild type (WT) embryonic stem (ES) cells (left) and neural precursor cells (NPCs) (right). The density of H3K4me3 and H3K27me3 CUT&RUN and ATAC-seq surrounding Mcm2 CUT&RUN peaks [–5k, 5k] was calculated. Color scales represent reads per million. (B) Peak distribution of H3K4me3, H3K27me3, and bivalent domains based on their co-localization with Mcm2 peaks. The percentile of ‘with Mcm2 peaks’ for each marker were labeled on top. Y-axis represents the peak number. (C) Density profiles of Mcm2 CUT&RUN (RPM, reads per million) surrounding transcription starting sites (TSS) and transcription termination sites (TTS). Genes were separated into four groups based on their expression in mouse ES cells or NPCs (Q1=lowest quartile, Q4=highest quartile). (D) Volcano plot of differential Mcm2 protein CUT&RUN peaks between WT Mcm2 and Mcm2-2A ES cells (left) and NPCs (right) from two independent replicates, with the total number of significantly up-regulated and down-regulated peaks (|log2 fold change|>1) shown. (E) Mcm2 CUT&RUN density in WT and Mcm2-2A ES cells at the reduced and increased Mcm2 CUT&RUN peaks in Mcm2-2A NPCs identified in D. (F) Mcm2 CUT&RUN density in WT and Mcm2-2A ES cells at the promoters ([–3k, 3k] of TSS) of down-regulated and up-regulated genes in Mcm2-2A mutant ESCs (ES cells) identified in Figure 3A, left. (G) Mcm2 CUT&RUN density in WT and Mcm2-2A NPCs at the promoters ([–3k, 3k] of TSS) of down-regulated and up-regulated genes in Mcm2-2A mutant NPCs (Figure 3A, right). (H) Mcm2 CUT&RUN density in WT and Mcm2-2A ES cells at the promoters ([–3k, 3k] of TSS) of down-regulated genes in Mcm2-2A mutant NPCs (Figure 3A, right). (E–H) The Y-axis represents the log2 ratio of CUT&RUN density (reads per kilobase per million reads [RPKM]), with p values calculated using Wilcoxon signed-rank test from two independent replicates. (I) Snapshots displaying Mcm2 CUT&RUN density at Pou5f1 and Pax6 loci of WT and Mcm2-2A ES cells and NPCs. One representative result from two independent replicates is shown. Shadows indicate CUT&RUN signals around the TSS.
Fig 2: Mcm2-2A mutation in mouse embryonic stem cells (ESCs) impairs differentiation.See also Figure 1—source data 1. (A) A diagram showing the embryoid body (EB) formation assay. (B) Representative images of wild type (WT) and Mcm2-2A cells during the process of EB formation. Scale bar: 20 µm. (C) RT-PCR analysis of the expression of Pou5f1 (a gene involved in pluripotency) and three lineage-specific genes in WT and Mcm2-2A cells during EB formation. GAPDH was used for normalization. Data are presented as means ± SD from three independent experiments. (D) Representative immunofluorescence images for detection of Pou5f1 and MERVL-Gag proteins in WT and Mcm2-2A mouse ESCs. Scale bar: 20 µm. (E) Quantification of Gag+ Pou5f1- cells in (D). At least n=1500 cells were counted for each cell line. Data are presented as means ± SD from three independent experiments. Statistical analysis in C and E was performed by two-tailed unpaired Student’s t test with p values marked on the graphs (N.S., no significant difference). Figure 1—source data 1.Relative mRNA level of pluripotency and lineage-specific genes during embryoid body (EB) formation in wild type (WT) and Mcm2-2A cells, and 2-cell (2C)-like ratio in WT and Mcm2-2A embryonic stem cells (ESCs).
Fig 3: HB-EGF+EGF infusion increases the proliferation of NPCs and GFAP+ MCM2+ neural stem cells in the V-SVZ.A, Density of mitotic NPCs (PCNA+ cells) in the V-SVZ of Nestin:YFP mice at 6 weeks recovery after cuprizone withdrawal assessed across the rostocaudal axis for all age groups. B, Experimental timeline for Nestin:YFP mice indicating time points for tamoxifen gavage, cuprizone challenge, growth factor infusion, EdU injection and tissue collection. C, Representative confocal micrograph of a coronal brain section following immunohistochemistry to detect GFAP, MCM2, EdU and Hoechst. Labeling was performed to identify putative neural stem cells (NSCs) (GFAP+ MCM2+ cells) that passed through the S phase of the cell cycle within 2 hours after EdU injection. C, Absolute number of EDU+ GFAP+ MCM2+ cells (dividing NSCs) in the dorsolateral of the V-SVZ in rostral, middle and caudal segments. D, Absolute number of EDU− GFAP+ MCM2+ cells (non-dividing NSCs) in the dorsolateral of the V-SVZ. Data represent mean ± SEM. Data were analyzed by two-way ANOVA with Sidak’s multiple comparisons test: *p<0.05, ***p<0.001, ****p<0.0001. Scale bar: 200 μm (C), 50 μm (C’). CC, corpus callosum; LV, lateral ventricle; Sep, septum; Str, striatum.
Fig 4: Kaplan-Meier curves for OS and EFS according to the expression levels of MCM2/5/6 in the GWCMC dataset (n = 130)(A–F) Plots for OS show (A) MCM2, (B) MCM5, and (C) MCM6 in NB patients. Plots for EFS show (D) MCM2, (E) MCM5, and (F) MCM6 in NB patients.OS, overall survival; EFS, event-free survival; HR, hazard ratio; CI, confidence interval; MCM, minichromosome maintenance; NB, neuroblastoma.
Fig 5: Photomicrographs of the immunohistochemical staining of MCM2 (arrows) in the different studied groups. (A), Benign neoplasm, 10% of cells stained (original magnification × 200); (B), PTC + 0, 20% of cells stained (original magnification × 200); (C), PTC + E, 25% of cells stained (original magnification × 200); (D), PTC + M, 25% of cells stained (original magnification × 200).
Supplier Page from Abcam for Anti-MCM2 antibody