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First published online 22 May 2007
doi: 10.1242/jcs.03461

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Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells

¹ CREST Research Project, Kansai Advanced Research Center, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan
² Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043, Osaka, Japan

View larger version (46K): [in this window] [in a new window]   Fig. 1. Subcellular localization of BAF in various human cell lines. (A) Western blotting using unpurified (`before', left panel) and affinity-purified (`after', middle and right panels) anti-BAF-3273-6 antibody. Whole extract (total), the cytoplasmic fraction (cytosol), and the nuclear fraction (nucleus) from HeLa cells (left and middle panels) and GFP-BAF-expressing HeLa cells (right panel) were analyzed. (B) Indirect immunofluorescence staining of HeLa cells using affinity-purified anti-BAF antibody (upper panels) and the same antibody pretreated with epitope peptides (lower panels). Left panels are anti-BAF staining (green in merged images), middle panels are DAPI staining for DNA (blue in merged images), and right panels are merged images. (C) Indirect immunofluorescence staining of various human cell types using affinity-purified anti-BAF antibody. Arrows represent cells with prominent nuclear signals and arrowheads represent cells with prominent cytoplasmic signals. Asterisks indicate cells with uniform signals. Bar, 40 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Age-dependent dislocalization of nuclear BAF in normal cells. (A) Localization of BAF in TIG-1 cells with different ages (left panels). Right panels represent FACS profiles showing cell cycle stages of cells corresponding to the left panels. Scale, 40 µm. Arrows represent cells with prominent nuclear signals, the asterisk indicates cells with uniform signaling and arrowheads represent cells with prominent cytoplasmic signals. (B) Proportions of cell populations with different BAF localization: Nuc>Cyto, Nuc=Cyto and Nuc<Cyto represent cells with nucleus-positive, uniform and cytoplasm-positive BAF, respectively. The proportion of cell populations was determined from 19, 11 and 8 independent experiments for cells of PD15, PD47 and PD63, respectively. Data are mean ± s.d. of at least 100 cells for each experiment. (C) Western blotting for BAF in young (left) and old (right) TIG-1 cells. (D) Proportion of cell cycle stages of young proliferating cells with nuclear BAF (nucleus positive and uniform). The proportion of cells with no nuclear BAF (nucleus negative) was less than 1% in young cells, and thus ignored. The results were obtained from 15 independent experiments; at least 100 cells were examined in each experiment. (E) Cell cycle stages of old TIG-1 cells (PD63) with nuclear BAF (corresponding to the nucleus positive and uniform populations) were examined in six independent experiments; at least 100 cells were counted in each experiment. (F) Cell cycle stages of old TIG-1 cells (PD63) with no nuclear BAF (corresponding to the cytoplasm positive population) were examined in five independent experiments; at least 100 cells were counted in each experiment. No S-phase cells were observed in cells with nucleus-negative BAF, suggesting that nuclear BAF is required for S-phase. Data are mean ± s.d. for D-F.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Synchronization at S-phase causes accumulation of BAF in the nucleus. (A) Indirect immunofluorescence staining of endogenous BAF (upper panels) or exogenously expressed HA-BAF (lower panels) in non-synchronized (left panels) and S-phase synchronized (right panels) HeLa cells. Bar, 10 µm. (B) Endogenous BAF was stained in non-synchronized (control) and S-phase synchronized HeLa cells 0, 2, 4, and 6 hours after release from the S-phase block. Percentages of the cells with nucleus-positive (black bar), uniform (grey bar) and cytoplasm-positive (white bar) BAF localization patterns are shown. Number of cells examined: 297 for control, 629 for 0 hours, 548 for 2 hours, 714 for 4 hours and 599 for 6 hours. (C) Western blotting analysis using anti-BAF antibody. Whole cell extract (total), the cytoplasmic fraction (cytosol), and the nuclear fraction (nucleus) from non-synchronized (control) and S-phase synchronized (S-phase) HeLa cells were analyzed. Lactate dehydrogenase isoenzyme I (LDHI) and Lamin B were used as markers for cytosol and nucleus, respectively.

View larger version (35K): [in this window] [in a new window]   Fig. 4. BAF nuclear-positive cells correlate with BrdU-positive cells. Two fields from the same experiment are shown in this figure. Non-synchronized cells were labeled with BrdU, and stained for chromosomes (with DAPI, far left), BAF (second column) and BrdU (third column). The correlation of BAF nuclear-positive cells with BrdU-positive cells was assessed. Of the 467 cells analyzed, 173 cells were BAF nuclear positive; 142 of these cells were BrdU positive. Therefore, 82% of BAF nuclear-positive cells in a non-synchronized population were in S phase (BrdU positive). Merged image (far right) represents chromosomes in blue, anti-BAF in green and anti-BrdU in red. Bar, 10 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Loss of BAF by RNAi causes a delay in S-phase progression. (A) Indirect immunofluorescence staining of HeLa cells treated with Luciferase RNAi (control RNAi) or BAF RNAi (BAF RNAi) using Oligofectamine. Cells were stained with anti-BAF antibody-PU38143 for endogenous BAF (upper panels) and DAPI for chromosomes (lower panels). Bar, 20 µm. (B) Quantitative western blotting. 25 µg (lane 1, extract corresponding to 5x104 cells), 12.5 µg (lane 1/2), and 5 µg (lane 1/5) of whole cell extract from cells treated with luciferase RNAi (control RNAi, left three lanes) or BAF RNAi (right three lanes). Endogenous BAF was reduced to approximately 10% of the original amount by RNAi treatment. (C) Cells were treated with luciferase RNAi (control RNAi) or treated with BAF RNAi (BAF RNAi). Cells were then labeled with BrdU. The percentage of cells positive for BrdU (black bars) and negative for BrdU (white bars) is shown. The proportion was represented as a percentage obtained from 19 and 13 independent experiments for control RNAi and BAF RNAi, respectively; more than 260 cells were examined in each experiment. Error bars represent s.d. (D) For long-term RNAi treatment experiments, HeLa cells (5x104 in a 35 mm dish at day 0) were transfected with 5-10 µg luciferase RNAi (control; closed circles) or BAF RNAi (BAF RNAi; open circles) on day 1 and again on day 2. The cells were subsequently transfected every 3 to 4 days: day 6, 9, 13, 16, 20, 23, 28 and 31. Cell numbers (y-axis) were counted on day 4, 7, 11, 14, 18, 21, 25, 29, 32. Arrows represent the time of RNAi treatment. (E) Western blotting corresponding to Fig. 5D for luciferase RNAi treated cells (left three lanes, control RNAi) and BAF RNAi treated cells (right three lanes, BAF RNAi). 25 µg (lane 1, extract corresponding to 5x104 cells), 6.25 µg (lane 1/4), and 2.5 µg (lane 1/10) of whole cell extract were applied. Amounts of BAF remaining in BAF RNAi cells are represented on the right as percentages of the control.

View larger version (43K): [in this window] [in a new window]   Fig. 6. Loss of BAF causes an increase in the number of cells present in the early stages of S phase. HeLa-S3 cells were treated with luciferase RNAi (control RNAi; black bar) or BAF RNAi (BAF RNAi; white bar) using Lipofectamine PLUS and labeled with BrdU. These BrdU-positive cells were classified into five types (I, II, III, IV and V) based on patterns of replication foci as described in O'Keefe et al. (O'Keefe et al., 1992): I for early stage, II for middle, III middle-late, IV for late, and V for late: typical examples are shown in the top panels. The proportion of BrdU-positive cells in each stage was presented as a percentage. Approximately 200 cells were counted for each of 13 independent experiments. The means of the 13 experiments for cells treated with luciferase RNAi (control RNAi) or BAF RNAi (BAF RNAi) are shown. Error bars represent s.d.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Western blotting analysis for cell-cycle-related proteins. HeLa cells were treated with luciferase RNAi as control (left lane) or BAF RNAi (right lane) using Oligofectamine. 25 µg cell extract (corresponding to 5x104 cells) was analyzed by western blotting using the antibodies indicated on the left. Amounts of corresponding protein in BAF RNAi cells are represented on the right as percentages of the control. Cell extracts were prepared in the homogenizing buffer without (A) or with (B) phosphatase inhibitors.

View larger version (52K): [in this window] [in a new window]   Fig. 8. Loss of BAF causes disassembly of emerin and lamin A from the nuclear envelope. (A) Quantitative western blotting using the antibodies indicated on the left. 25 µg (lane 1, extract corresponding to 5x104 cells), 12.5 µg (lane 1/2), 5 µg (lane 1/5), 2.5 µg (lane 1/10) of whole cell extract from cells treated with luciferase RNAi (control RNAi, left) or BAF RNAi (right) using Oligofectamine. Upper and lower bands in lamin A/C are lamin A and lamin C, respectively. (B) Indirect immunofluorescence staining using anti-BAF, anti-emerin, anti-lamin A, and anti-lamin B antibodies of cells treated with luciferase RNAi (control RNAi; left panels) or BAF RNAi (BAF RNAi; right panels). Chromosomes were stained with DAPI. Bar, 20 µm.