Figure 1.
Nuclear hARD1 levels increase during the S phase.
A. hARD1 predominantly localizes to the cytoplasm of HeLa cells. HeLa cell lysate was subjected to nuclear/cytosolic fractionation, and hARD1 in each compartment was analyzed by immunoblotting. Histone H1 and α-tubulin were included as nuclear and cytoplasmic markers, respectively. Nuc, nuclear protein; Cyt, cytosolic protein. B and C. hARD1 translocates to the nucleus during S phase. B, HeLa cells were synchronized at the early S phase by double thymidine block, which was released for the indicated times, and then hARD1 subcellular localization was analyzed by performing western blot. Approximations of the cell cycle stages were based on the level of cyclins and were confirmed using flow cytometry. PARP and α-tubulin were included as nuclear and cytoplasmic markers, respectively. Asy, asynchronized cells. C, hARD1 subcellular localization was assessed by immunofluorescence staining. The nuclei were labeled with Hoechst (blue). Scale bar, 10 µm.
Figure 2.
The deletion of the putative NLS of hARD1 blocks nuclear localization.
A. The structure and sequence comparison of the putative NLS in ARD1. The reported structure of yeast ARD1 is shown. The region corresponding to the NLS in hARD1 (a.a. 79–84, MRSYRH in yeast [21]) is colored yellow. B. Schematic presentation of an NLS deletion mutant of hARD1. Wild type hARD1 was tagged with GFP at its N-terminus (GFP-hARD1 WT), and the putative NLS was deleted (GFP-hARD1ΔN). C. Deletion of NLS compromised nuclear translocation of hARD1. HEK293T cells transfected with GFP-hARD1 WT and ΔN were subjected to nuclear/cytosolic fractionation and GFP-hARD1 levels were detected by anti-GFP antibody. Note the slight decrease in size of ΔN relative to WT. D. NLS-deleted hARD1 was unable to access the nucleus. GFP-hARD1 WT- and ΔN-expressing HeLa cells were visualized by fluorescence microscopy. Scale bar, 10 µm.
Figure 3.
The hARD1 NLS mutant suppresses cell proliferation.
A. Constitutive expression of the NLS mutant of hARD1 leads to morphological alterations. HeLa cells transfected with hARD1 WT and ΔN were cultured in DMEM containing G418 to select for stable clones. After establishment, cell images were observed by phase-contrast microscopy. Arrows indicate cells with altered morphology (black, enlarged; red, rounded). Scale bar, 50 µm. B and C. hARD1ΔN-expressing cells showed a marked decrease in cell growth. B, cell growth of hARD1 WT and ΔN stable cell lines was monitored over time by MTS assay. The results are normalized to 0 day of each group and presented as mean ± S.D. (n = 5). C, HeLa cells stably expressing hARD1 WT and ΔN were subjected to anchorage-dependent (left) and -independent (right) colony formation assays. Number of colonies were presented as mean ± S.D. with representative well pictures. N = 3, ***P<0.005.
Figure 4.
The hARD1 NLS mutant causes alterations in cell cycle.
A. Deletion of NLS from hARD1 leads to moderate G2/M arrest in HeLa cells. Cell cycle profiles of hARD1 WT- and ΔN-expressing cells were determined by flow cytometry. Upper, representative cell cycle profiles; lower, the percentage of cells in each phase is presented as mean ± S.D. (n = 3). *p<0.05, **P<0.01 B. Deletion of NLS from hARD1 increased the protein levels involved in the G2/M phase and decreased those involved in the G0/G1 phase. The levels of Cyclin B1/E, Aurora kinase A/B (AURKA/B), and p53 were immunoblotted in the lysates extracted from hARD1 WT- and ΔN-expressing HeLa cells.
Figure 5.
An exogenous NLS can rescue the nuclear localization of the hARD1 NLS mutant.
A. The region of the exogenous NLS insertion in ARD1. On the reported structure of the yeast ARD1, the region corresponding to the exogenous NLS insertion site in hARD1 (next to a.a. 64) is indicated by a red arrow. B. Schematic presentation of an exogenous NLS insertion to the NLS deletion mutant of hARD1. The NLS of hARD1, KRSHRR, was inserted next to a.a. 64 of the NLS deletion mutant (GFP-hARD1+N). C. Insertion of NLS into hARD1 rescued the nuclear localization of the NLS deletion mutant. GFP-hARD1 WT, ΔN, and +N were transfected into HEK293T cells, and the localization of hARD1 was analyzed by nuclear/cytosolic fractionation. Note the slightly smaller size of ΔN recovered in +N. D. An exogenous NLS can redirect the hARD1 NLS mutant into the nuclei. GFP-hARD1 WT-, ΔN-, and +N-expressing HeLa cells were visualized under fluorescence microscopy. Scale bar, 10 µm.
Figure 6.
NLS insertion into hARD1 rescues the impaired cell cycle.
A. Insertion of NLS rescued hARD1ΔN-expressing HeLa cells from G2/M arrest. Cell cycle profiles of stable HeLa cells were determined by flow cytometry analysis. Upper, representative cell cycle profiles; lower, the percentage of cells in each phase. B. The exogenous NLS restored the altered protein expression involved in cell cycle regulation of the NLS mutant. The levels of Cyclin B1/E, AURKA/B, and p53 were determined in cell extracts from hARD1 WT-, ΔN-, and +N-expressing cells by western blot.
Figure 7.
NLS insertion into hARD1 rescues impaired cell growth.
A. The insertion of exogenous NLS into hARD1 restored the morphological alterations of ΔN-expressing cells. HeLa cells stably expressing hARD1+N were established, and cell morphology was observed under phase-contrast microscope. Scale bar, 50 µm. B and C. The exogenous NLS partially recovered the impaired growth of cells expressing the NLS deletion mutant. B, cell growth of hARD1+N stable cells with WT and ΔN stable cells was monitored by MTS assay. The results of each group were normalized to day 0. C, HeLa cells stably expressing hARD1 WT, ΔN, and +N were subjected to anchorage-dependent (left) and -independent (right) colony formation assays.