Fig 2: MiR-630 inhibits cell proliferation by inhibiting CDC7-mediated DNA synthesis. A549 cells were transfected with scrambled small interfering RNA (siRNA) (control), miR-630 mimic and miR-630 inhibitor for 48 and 72 h. (a) Western blotting for CDC7, MCM2 and phospho-MCM2. β-Actin was used as a loading control. (b) Flow cytometric analysis of BrdU-positive cells. After 48 and 72 h transfection, cells were labeled with 50 μM BrdU for 1 h before collection. Samples were stained with anti-BrdU FITC antibody and PI, and analyzed by flow cytometry. BrdU-positive cells were included in the gate region. (c) Relative amount of BrdU-positive cells in the gate region compared with total cells in (b) experiments. Data are presented as mean±S.D. (n=3); 48 h: *P=0.0396, ##P=0.0054; 72 h: **P=0.0037, #P=0.0171. (d) MTS assay showed the survival of A549 cells transfected with miR-630 mimic or inhibitor for 48 h. Data are presented as mean±S.D. (n=3); **P=0.0026 and #P=0.0223

Fig 3: SIRT1 suppresses replication-specific R-loops at rDNA loci. (A): IGV screenshot depicting SIRT1 and phosphorylated MCM2 binding at rDNA loci in fibroblast cells from aging individuals. ChIP-seq for pSIRT1 and pMCM2 was performed as described in Figure 3 legend. (B): Quantification of pSIRT1 and pMCM2 binding. With aging, fibroblast cells exhibited reduced SIRT1 binding and enhanced phosphorylated MCM2 association at rDNA loci. (C): Left, schematics of R-loop measurement from cancer cells and right quantification of R-loop signal over rDNA region using qPCR. Increased R-loop prevalence at rDNA loci in cancer cells deficient in SIRT1 activity. (D): Left, schematics of detection of replication-specific R-loops from cancer cells. Quantification of R-loops was associated with newly replicated DNA showing increased replication-specific R-loop at the 28S rDNA gene promoter in SIRT1 inactive cells.

Fig 4: Temporal course of differentially expressed phosphoproteins and phosphorylated phosphosites in myotubes during hyperammonemiaDifferentially expressed phosphoproteins (DEpP) and phosphorylated phosphosites (DPPS) were identified in murine C2C12 myotubes treated with10mM ammonium acetate (AmAc) for 6 and 24h compared to untreated controls.(A) Venn diagrams showing the number of shared and unique DEpP/DPPS.(B) Volcano plots comparing significance to expression of DPPS.(C) UpSet plot showing the number of DEpP in each group based on the direction of expression change (UP = increased expression, DOWN = decreased expression vs. untreated controls).(D) Representative immunoblots and densitometries for p-IKKBS672, total IKKB, p-MST2S316, total MST2, p-MCM2S139, total MCM2, p-S6S235/236, and total S6 in untreated and 6hAmAc and 24hAmAc myotubes. All myotube experiments were done in n = 3 biological replicates (one 24hAmAc phosphoproteomics data replicate was removed from downstream analyses because of outlier status). For densitometry, data shown as mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 on ANOVA with Bonferroni post-hoc analysis. Statistical significance cutoff for DEpP/DPPS was p-adj <0.05 (Student’s t test with Benjamini Hotchberg correction). IKKB = Inhibitor of nuclear factor kappa B kinase subunit beta, MST2 = Mammalian sterile 20-like kinase 2; MCM2 = mini chromosome maintenance protein 2.

Fig 5: MTBP and pRecQL4 association with dormant origins.A Effect of the MCM2S108A mutation on interactions with MTBP and pRecQL4. Experimental procedure: HCT116 cells with MCM2-mAID were complemented with WT or S108A MCM2 constructs. IAA treatment (500 μM for 16 h) depleted endogenous MCM2-mAID, enabling the incorporation of WT or S108A MCM2 into the pre-replication complex. Treatment with the SIRT1 inhibitor Ex527 (1 mM every 24 h for 2 days) activates dormant origins in cells complemented with the intact, but not the S108A mutated MCM215. B Replication origin binding of MTBP (left) and pRecQL4 (right) in unperturbed and Ex527 treated (1 mM every 24 h for 2 days) HCT116 cells harboring either WT-MCM2 or S108A-MCM2. Baseline and dormant origins were categorized as described in the legend to Fig. 1A, B.