Fig 2: DN-PPAR? does not up-regulate phospho-Rb, cyclin A, or MCM7 in PPAR?-deficient embryonic stem cells. PPAR? -/- ES cells were differentiated and then growth arrested in serum-deprived medium for 24 hours, then infected with or without Ad-GFP, -DN, or WT recombinant adenovirus (MOI = 80) for 48 hours, with or without serum stimulation. Western blot analyses were performed on whole cell extracts of these samples using antibodies specific for the indicated regulatory cell cycle proteins, then stripped and rehybridized with a ß-actin specific antibody to assess loading variability. Western blots shown are representative of at least three independent experiments.

Fig 3: Cell cycle and chromatin distributions of preRC subunits.(A–C) G0-arrested CHO cells were released into G1, and samples were collected at the indicated times after release. Total cell extracts (TCE) were compared to cytosolic/nucleosolic (soluble) and chromatin-bound (chromatin) extracts by immunoblotting. Asynchronous (Log) cultures were processed identically. Extract from an equivalent amount of cells was loaded in each case, and analyzed with the indicated antibodies. BrdU labeling was ~50% at 9 hr (G1-S transition), ~95% at 12 and 15 hrs, and ~80% at 18 hrs (data not shown). (D) Quantitation of Mcm7 levels on total and soluble/chromatin fractions in synchronized CHO cells 8 hrs post release.

Fig 4: Replisome components limit the MCM accessibility at actively progressing replication forks.a Representative MIP confocal images of chromatin-bound CDC45-GFP, MCM4-Halo and immunostained MCM7 in S5 stage of S phase sub-stratified as in (Supplementary Fig. 1b). Cells were transfected by control siRNA or siRNA against TIMELESS (5 nM; 24 h) as indicated and treated with ATR inhibitor (ATRi). Scale bar, 10 µm. b Colocalization analysis of endogenously tagged CDC45-GFP and immunostained MCM7 in the S5 stage of S phase in cells treated as in a. Lines are medians; n = 20 cells per condition; P value was calculated by two-tailed unpaired t-test; ****P > 0.0001. c Colocalization analysis of endogenously tagged CDC45-GFP and immunostained MCM4 in the S5 stage of S phase in cells treated as in a. Lines are medians; n = 20 cells per condition; P value was calculated by two-tailed unpaired t-test; ****P > 0.0001. d Colocalization analysis of endogenously tagged CDC45-GFP and immunostained MCM5 in the S5 stage of S phase in cells treated as in a. Lines are medians; n = 20 cells per condition; P value was calculated by two-tailed unpaired t-test; **P = 0.0096. e Colocalization analysis of endogenously tagged CDC45-GFP and MCM4-Halo in the S5 stage of S phase in cells treated as in a. Lines are medians; n = 20 cells per condition; P value was calculated by two-tailed unpaired t-test; not significant (n.s.) denotes P > 0.05. f A model clarifying the MCM paradox. Upon conversion of inactive MCMs to active replisomes, antibody binding sites in the MCM2-7 proteins are masked by replisome factors, hindering MCM immunodetection. Thus, MCM antibodies primarily detect the highly abundant pool of inactive MCMs (in MCM-DHs). In contrast, endogenous tagging of MCM subunits enables visualization of entire pool of MCMs, including those in fully assembled active replisomes (see text for details). Source data are provided as a Source data file.

Fig 5: Overexpression of miR-99a-3p regulates cell growth and EMT associated proteins in LUAD cell lines.A, B qRT-PCR was conducted to confirm the overexpression efficiencies of miR-99a-3p in A549 and H1299 cells. C, D The protein levels of MTHFD2, MCM4, MCM7 and Cyclin D1 in miR-99a-3p overexpressed A549 and H1299 cells. a-tubulin was used as an internal control. E, F The protein levels of Zeb1, E-cadherin, Vimentin and Snail in miR-99a-3p overexpressed A549 and H1299 cells. a-tubulin was used as an internal control. ***P < 0.001.