Fig. 2: NFIB facilitates the Pre-RC assembly by increasing chromatin accessibility.
From: NFIB facilitates replication licensing by acting as a genome organizer
a U2OS cells were infected with lentiviruses carrying control shRNA, NFIB shRNA, and/or FLAG-NFIB and synchronized at G1 phase, G1/S boundary or S phase. Whole cell extracts (WCE, left) or chromatin fraction (Chromatin, right) were prepared and analyzed by western blotting with antibodies against the indicated proteins. GAPDH or H3 was used as loading control as indicated. The experiment was performed three times with similar observations. b U2OS cells were infected with lentiviruses carrying control shRNA, NFIB shRNA, and/or FLAG-NFIB and synchronized at G1 phase or G1/S boundary for immunofluorescent staining (IF) using the indicated antibodies with pre-extraction. DAPI staining was included to visualize the nucleus (blue). Scale bar, 10 μm. Representative images from triplicate experiments are shown. The relative IF intensity was quantified by ZEN software. Data were presented as mean ± SD of ten sections from each slice for triplicate experiments. P values were determined by two-way ANOVA followed by Tukey test. NS, not significant. c U2OS cells were synchronized at G1 phase for CUT&Tag experiments. Venn diagrams of the overlapping regions of NFIB binding and ORC1 binding identified by CUT&Tag of NFIB and ORC1 (left). Heatmaps showed the CUT&Tag signal distribution of 4752 N1 peaks (peaks with a decreased signal intensity of both NFIB and ORC1 in NFIB-depleted cells) (right). d Heatmaps showing the H3K4me3 and H3K9me3 signal distribution surrounding N1 peaks in U2OS cells synchronized at G1/S phase. e The efficiency of knockdown was verified by western blotting in U2OS cells. The experiment was performed three times with similar observations. f U2OS cells without (siCTR) or with NFIB depletion (siNFIB) were synchronized at G1/S or released into S phase for ATAC-seq. Differential accessibility between siCTR and siNFIB U2OS was plotted against the mean reads per region. Values aside brackets indicate the number of significantly changed peaks (P value < 0.01). g Heatmaps showing the distribution of chromatin accessibility signal in G1/S phase surrounding N1 peaks in U2OS cells without (siCTR) or with NFIB depletion (siNFIB).
