Fig 2: NFIB is physically associated with components of the Pre-RC.a The relative protein level of NFI family members in different tissues from the Human Protein Atlas. b Analysis of the level of mRNA (upper) or protein (lower) of the NFI family members in different human cancer cell lines, by qPCR and western blotting, respectively. The heatmap was generated by log transformation of the data based on -?CT values. c Immunopurification and mass spectrometric analysis of NFIB-associated proteins. Cellular extracts from U2OS cells stably expressing FLAG-NFIB were affinity-purified. The eluates were resolved by SDS-PAGE and silver-stained. The protein bands were retrieved and analyzed by mass spectrometry. The experiment was performed twice with similar observations. d U2OS cells were synchronized at G1/S boundary by a double-thymidine block. Co-immunoprecipitation assays were performed with whole cell extracts (WCE, left). The level of NFIB was examined by western blot (bottom). Co-immunoprecipitation assays were performed with chromatin fraction using antibodies against NFIB followed by immunoblotting (IB) with antibodies against the indicated proteins (Chromatin, middle), or with antibodies against the indicated proteins followed by IB with anti-NFIB (Chromatin, right). e GST pull-down assays with GST-fused full-length NFIB and in vitro transcribed/translated proteins as indicated. f GST pull-down assays with deletion mutants of NFIB and in vitro transcribed/translated pre-RC components as indicated. Schematic diagrams of NFIB and NFIB deletion mutants are shown. Coomassie brilliant blue staining of GST-fused proteins is marked (arrow). g U2OS cells were synchronized at G1/S boundary by double-thymidine block and released for different hours to allow cells to enter different phases of the cell cycle as indicated for cell cycle analysis by cytometry (left) or for western blotting analysis of whole cell extracts (WCE) or chromatin fraction (Chromatin) with antibodies against the indicated proteins. GAPDH or H3 was used as a loading control as indicated. (b, d–g) The experiment was performed three times with similar observations.

Fig 3: 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.

Fig 4: Depletion of NFIB alters chromosome contacts/compartments and replication profile.a Left: Heatmap of NS-seq signals in NFIB-depleted versus control U2OS cells. The fold change cutoff of signal intensity was set to 1.2. S1, peak signals decreased; S2, peak signals increased; S3, peak signals unaltered. Right: Heatmap of NS-seq signal distribution surrounding N1 peaks in NFIB-depleted versus control U2OS cells. b Different time points of synchronized NFIB-depleted U2OS cells were analyzed by Repli-seq. Control or NFIB-depleted U2OS cells were synchronized via thymidine/aphidicolin block followed by release for 0.5 h, 4 h, or 6 h to enrich cells in the early, middle, or late S phase, respectively. Graph showing the replication timing of NFIB-associated active origins (S1 peaks overlapped with N1 peaks, S1.N1, n = 415), other S1 peaks (S1.non-N1, n = 14,203), S2 peaks (n = 24,410) and total NS-seq peaks (all, n = 48,273). c Comparison between replication initiation patterns after NFIB was depleted in U2OS cells. Genome tracks showing the signals of Repli-seq, NFIB ORC1, and H3K4me3 at selected regions of chromosome 5. d Scatter plot of PC1 eigenvector of G1/S boundary or S phase U2OS cells without (shCTR) or with NFIB depletion (shNFIB) for each 250 kb genome bin. Red points: changed regions. Black points: unchanged regions. e Histogram showing the number of regions (250 kb bin) where the PC1 eigenvector significantly changed. f Knight-Ruiz (KR) balanced Micro-C contact map of chromosome 1: 162 to 183 Mb at a 100-kb resolution. g Violin plot showing the distribution of N1 peaks in different chromatin regions in G1/S phase (bin size 250 kb).

Fig 5: NFIB overexpression provokes genomic aberrations that mimics genomic aberrations in breast cancer.a Bar plotting of loci with copy number gain, copy number loss, insertion, or deletion in MCF-10A cells infected with lentiviruses carrying control vector or NFIB expression construct. b Fraction of NFIB-associated CNAs observed in breast cancer samples. The union represents events that are observed in any of the tested datasets. c Fraction of the alteration in cancer samples of the indicated genes identified from NFIB overexpression-associated amplification that also significantly co-varied with NFIB CNAs in breast cancer samples. d Copy number of the indicated genes in MCF-10A cells stably expressing empty vector or NFIB was measured by Taqman copy-number assay. The data are presented as the means ± SD for three independent experiments. P values were determined by an unpaired two-tailed T test. e Control or MCF-10A cells stably expressing NFIB were treated with the indicated anti-neoplastic compounds for 3 days. The growth of cells was measured with the CCK-8 assay. Error bars represent mean ± SD for triplicate experiments. IC50 (half maximal inhibitory concentration) was listed. P values of lgIC50 were calculated based on the extra sum-of-squares F-test. f Bar plotting of loci with copy number gain, copy number loss, insertion, or deletion in control MCF-10A cells, NFIB-overexpressing MCF-10A cells cultured for 75 days, and NFIB-overexpressing/drug-resistant MCF-10A cells. g Venn diagrams of the overlapping and differential sets of amplified CNAs by comparing the data from control MCF-10A cells, NFIB-overexpressing MCF-10A cells cultured for 75 days, and NFIB-overexpressing/drug-resistant MCF-10A cells.