Fig 2: MCM3 phosphorylation in NSCs as a trigger of FTLD phenotypes.(A) Western blots of phosphorylated MCM3 and MCM2 with NSCs prepared from VCPT262A-KI mouse embryos at E14. Right graphs show quantitative analyses. (B) Molecular network of proteins connected to MCM3, MCM2, and Lig1 (“DNA duplication” group in Fig S7B). One-edge and two-edge networks were generated using the PPI database (see the Materials and Methods section). Red nodes indicate MCM3, MCM2, and Lig1. Yellow nodes indicated proteins that changed in the phosphoproteome analysis (Fig S7A). (C) Candidate kinases for MCM3 phosphorylation at Thr719 predicted by NetworKIN. (D) Mass analysis of in vitro phosphorylation of MCM3 peptide by candidate kinases. “Detected” in upper tables indicates detection of phosphorylated or non-phosphorylated peptide fragments of MCM3 at Thr719 by mass spectroscopy at >95% confidence. Lower images show raw data. (E) Western blots revealed distinct levels of various DNA damage response kinases in developmental cortex at E15. Right graphs show quantitative analyses of three C57BL/6J and three VCPT262A-KI mice. (F) Immunohistochemistry of the active form of DNA-PK, the active form of CDK1, phosphorylated MCM3, and DNA damage markers (?H2AX and 53BP1). The active form of DNA-PK, phospho-MCM3 and DNA damage markers are detected in apical NSCs, whereas the active forms of CDK1 and DNA damage markers were detected in basal NSCs at E15. The right graph shows average signal intensities of 30 cells per field from each mouse (N = 3). (G) Effect of siRNA-mediated knockdown of each kinase on pThr719-MCM3 in primary NSCs prepared from VCPT262A-KI mouse E14 embryos. Box plots show medians, quartiles, and whiskers, which represent data outside the 25th–75th percentile range.Source data are available for this figure.

Fig 3: MYH4 ensures replication licensing and promotes faithful replication.(A) QIBC plot indicating EdU incorporation of U2OS cells transfected with indicated siRNAs for 48 hours. Boxes indicate cell cycle phases: blue, G1; green, S; and red, G2. Color threshold indicates mean γH2AX intensity in different cell cycle phases. Blue, minimum; red, maximum. (B) A pie chart indicating EdU- and DAPI-based cell cycle distribution of cells. (C) Statistics for mean EdU intensity in S phase. Data presented as mean ± SD. ***P < 0.001 by 2-tailed, paired t test. (D) QIBC plot of cells transfected with the indicated siRNAs and stained for chromatin-bound (CB) MCM2. Top panels: MCM2 distribution in cell cycle phases. Bottom panels: Mean intensity of CB MCM2. Color threshold: gray, minimum; blue, average; red, maximum. (E) A Western blot analysis of indicated proteins in soluble (SOL) and CB extracts from cells transfected with indicated siRNAs, n = 3. Note: Some of the blots were stripped and reblotted with different antibodies. (F) U2OS cells stably expressing DOX inducible, siRNA-resistant MYH4-GFP WT or ΔABD mutant, transfected with indicated siRNAs for 48 hours. The bar plots illustrate relative mean intensity of CB MCM2. Data presented as mean ± SD, n = 3. *P < 0.05, **P < 0.001 by 1-way ANOVA with Šidák’s multiple-comparison test. NS, not significant. (G) QIBC of cells transfected with the indicated siRNAs and stained for CB-PCNA. Top panel: PCNA distribution in cell cycle phases. Bottom panel: Mean intensity of γH2AX in the same set of cells, indicated with a box. (H) Representative confocal images showing PCNA and EdU staining after 48 hours of indicated siRNA transfection (n = 3). Scale bar: 20 μm. (I) Quantification of DNA fibers from cells transfected with indicated siRNAs and labeled with indicated analogs for indicated time. Error bars indicate ±SEM, n = 3. **P < 0.001 by 2-tailed, paired t test.

Fig 4: MYH4 ensures replication licensing and promotes faithful replication.(A) QIBC plot indicating EdU incorporation of U2OS cells transfected with indicated siRNAs for 48 hours. Boxes indicate cell cycle phases: blue, G1; green, S; and red, G2. Color threshold indicates mean γH2AX intensity in different cell cycle phases. Blue, minimum; red, maximum. (B) A pie chart indicating EdU- and DAPI-based cell cycle distribution of cells. (C) Statistics for mean EdU intensity in S phase. Data presented as mean ± SD. ***P < 0.001 by 2-tailed, paired t test. (D) QIBC plot of cells transfected with the indicated siRNAs and stained for chromatin-bound (CB) MCM2. Top panels: MCM2 distribution in cell cycle phases. Bottom panels: Mean intensity of CB MCM2. Color threshold: gray, minimum; blue, average; red, maximum. (E) A Western blot analysis of indicated proteins in soluble (SOL) and CB extracts from cells transfected with indicated siRNAs, n = 3. Note: Some of the blots were stripped and reblotted with different antibodies. (F) U2OS cells stably expressing DOX inducible, siRNA-resistant MYH4-GFP WT or ΔABD mutant, transfected with indicated siRNAs for 48 hours. The bar plots illustrate relative mean intensity of CB MCM2. Data presented as mean ± SD, n = 3. *P < 0.05, **P < 0.001 by 1-way ANOVA with Šidák’s multiple-comparison test. NS, not significant. (G) QIBC of cells transfected with the indicated siRNAs and stained for CB-PCNA. Top panel: PCNA distribution in cell cycle phases. Bottom panel: Mean intensity of γH2AX in the same set of cells, indicated with a box. (H) Representative confocal images showing PCNA and EdU staining after 48 hours of indicated siRNA transfection (n = 3). Scale bar: 20 μm. (I) Quantification of DNA fibers from cells transfected with indicated siRNAs and labeled with indicated analogs for indicated time. Error bars indicate ±SEM, n = 3. **P < 0.001 by 2-tailed, paired t test.