Fig. 6: NFIB promotes S-phase progression and cell proliferation.

a ssDNA formation in control or NFIB-depleted U2OS cells after HU treatment. BrdU (20 µg/ml) was added during the last 24 h before cells were harvested and removed by a brief wash prior to HU treatment (4 mM, 2 h), pre-extraction, and fixation. BrdU in ssDNA patches and PCNA were detected without DNA denaturation. Scale bar, 10 µm (left). Diagrams show the frequency of PCNA-positive BrdU foci (right). The data are presented as the means ± SD for triplicate experiments. P values were determined by one-way ANOVA. b, c Synchronized U2OS cells were analyzed at different time points after release from G₁/S transition for EdU incorporation by flow cytometry. The data are presented as the means ± SD for triplicate experiments. P values were determined by two-way ANOVA followed by the Dunnett test. NS, not significant. d U2OS cells were treated with or without 200 μM HU for CCK-8 assays. The data are presented as the means ± SD for three independent experiments. P values were determined by an unpaired two-tailed T test. e U2OS cells were infected with lentiviruses carrying the indicated shRNA and cultured in regular medium or medium supplemented with 200 μM HU for 14 days. Cells were then stained with crystal violet and counted for colony numbers. The data are presented as the means ± SD for three independent experiments. P values were determined by two-way ANOVA followed by the Dunnett test. f CCK-8 assays in MCF-10A cells infected with lentiviruses carrying control vector or NFIB expression construct. The data are presented as the means ± SD for three independent experiments. P values were determined by an unpaired two-tailed T test. g MCF-10A cells were infected with lentiviruses carrying control or NFIB expression construct. Colony formation assays were performed. The data are presented as the means ± SD for three independent experiments. P values were determined by an unpaired two-tailed T test. h FACS analysis of EdU incorporation and DNA content after 1-h pulse with EdU. Re-replicated DNA ( >4 N) contents are presented as gates, and their percentages are plotted as bars. The data are presented as the means ± SD for three independent experiments. P values were determined by an unpaired two-tailed T test. i CsCl density gradient analysis for the replication pattern of BrdU-labeled genomic DNAs in NFIB-overexpressing MCF-10A cells. Data were presented as DNA concentration of the indicated fraction from the bottom of the CsCl gradient. The fractions showing light: light (L:L), heavy: light (H:L), or heavy: heavy (H:H) DNA content were indicated. A representative result from three independent repeat experiments is shown.