ZONAB regulates cell proliferation and density of MCF-10A cells. (A) Equal amount of proteins of independent clones of MCF-10A cells transfected with empty vector or a plasmid that was driving ZONAB-B expression were analyzed by immunoblotting by using anti-ZONAB antibodies. Nontransfected cells express comparatively small amounts of ZONAB that cannot be seen on short exposures (see panel F for a comparison). (B) Expression of the HA-tagged SH3 domain of ZO-1 in MCF-10A cells was analyzed by immunofluorescence. Only clone S7 is shown, which is representative of the three clones used. (C) Pull-down assays using SH3 domains of the indicated proteins as glutathione S-transferase (GST) fusions were bound to glutathione-agarose and incubated with equal amounts of His₆-ZONAB. The pull downs were analyzed by immunoblotting (IB) by using either anti-His tag or anti-GST antibodies. (D) MCF-10A cells transfected with the empty vector, overexpressing ZONAB or expressing the HA-tagged SH3 domain of ZO-1 were grown for 4 days (low confluence) or 14 days (high confluence) in growth factor-containing medium. Where specified, cells had been synchronized and were then stimulated to enter S phase for 20 h. S-phase cells were detected by BrdU incorporation. Shown are means ± 1 standard deviation (error bars) of three clones for each transfection that were analyzed twice. *, samples with P values of <0.05 in t tests. (E) Cell density of clonal and lentivirus-transduced MCF-10A-95 cells were analyzed after culturing to full confluence for 14 days. Cells were then trypsinized and counted. Error bars indicate standard deviations. *, samples with P values of <0.05 in t tests. (F) The lentivirally transduced MCF-10A cells were analyzed for expression of ZONAB and ZO-1. Equal amounts of protein were loaded and confirmed by immunoblotting with antitubulin (α-tubulin) antibodies.

Regulation of PCNA expression by ZONAB. (A) MCF-10A cells transduced with lentivirus (lenti) expressing ZONAB-B or stably expressing the HA-tagged SH3 domain of ZO-1 were either grown for four days in growth factor-containing medium or synchronized and then analyzed without or with stimulation with growth factors for 20 h. Equal amounts of proteins were analyzed for PCNA expression by immunoblotting using anti-PCNA monoclonal antibody. (B) Equal amounts of proteins of transduced ARPE-19 cells were analyzed for PCNA expression as described for panel A. lenti, lentivirus. (C) Double-stranded oligonucleotides from the PCNA, erbB-2, and NF-κB promoters were labeled with ³²P, incubated with GST or GST-ZONAB as indicated and analyzed by gel electrophoresis. For competition experiments, unlabeled oligonucleotides (none [0] or 500 pmols of PCNA, erbB2, or NF-κB) were used to quench binding of the PCNA or erbB-2 probe (5 pmols) as indicated. (D) Reporter gene assays were performed by cotransfecting ARPE-19 cells with a wild-type PCNA promoter reporter plasmid encoding firefly luciferase, a control renilla luciferase plasmid, and different concentrations of pCB6-ZONAB-B expression plasmid (DNA concentrations were equalized with empty expression plasmid). Expression of the luciferases was analyzed, and the values obtained for renilla luciferase were used for normalization. Shown are means ± 1 SD (error bars) of a typical experiment performed in triplicate.

ZONAB binds to the PCNA promoter in vivo. (A) MCF-10A cells were cross-linked, extracted, and immunoprecipitated with anti-ZONAB antibodies or negative control beads. The precipitated ZONAB-chromatin complexes were then analyzed by PCR for the presence of PCNA and erbB-2 promoter sequences. β-actin primers were used as a negative control. Input label PCRs in which total nuclear cell extracts were used as template. Plasmids containing the respective promoter sequences were used as positive controls. (B) Reporter gene assays were performed by cotransfecting ARPE-19 cells with a control renilla luciferase plasmid and pCB6-ZONAB-B, pCB6-HA-SH3, or empty pCB6, together with a firefly luciferase reporter plasmid containing the wild-type PCNA promoter or a promoter in which the inverted CCAAT box sequence had been substituted. Shown are means ± 1 standard deviation (error bars) of a typical experiment performed in triplicate. *, samples with P values of <0.05 in t tests.

Regulation of cyclin D1 expression by ZONAB. (A) Control cells, ZONAB-B-overexpressing cells, and HA-SH3-expressing MCF-10A cells were grown for 4 days in growth factor-containing medium or synchronized and then stimulated with growth factors for the indicated times. Equal amounts of protein were then analyzed by immunoblotting using an anti-cyclin D1 antibody. (B) Immunoprecipitated ZONAB-chromatin complexes were tested for the presence of cyclin D1 promoter sequences by using PCR. The used input and precipitates were derived from the same experiment as the one described in the legend for Fig. 3A. (C) Transcriptional regulation of the cyclin D1 promoter by ZONAB requires an inverted CCAAT box. Reporter gene assays were performed by cotransfection of ARPE-19 cells with a control renilla luciferase plasmid and either pCB6-ZONAB-B, pCB6-HA-SH3 or empty pCB6 plasmid and a firefly luciferase reporter plasmid containing the wild-type cyclin D1 promoter or a promoter in which the inverted CCAAT box had been substituted. Shown are means ± 1 standard deviation (error bars) of a typical experiment performed in triplicate. *, samples with P values of <0.05 in t tests. (D) Double-stranded oligonucleotides from the cyclin D1, erbB-2, and NF-κB promoters labeled with ³²P were incubated with GST or GST-ZONAB as indicated and analyzed by gel electrophoresis. For competition experiments, unlabeled oligonucleotides (500 pmols for cyclin D1, erbB2, or NF-κB) were used to quench binding of the cyclin D1 or erbB-2 probe (5 pmol) as indicated.

ZONAB increases cyclin D1 expression in MDCK cells. (A) MDCK cells conditionally expressing either control, ZO-1 or ZONAB-directed RNA duplexes were plated and grown in the absence or presence of tetracycline (TC) for 3 or 2 days. Total cell extracts were then analyzed by immunoblotting with antibodies against ZO-1, ZONAB or tubulin (α-tubulin). (B) Equal numbers of conditional control or ZONAB RNAi MDCK cells were plated and then grown with or without tetracycline (TC) for 3 days, and phase-contrast pictures were taken to monitor proliferation. (C) MDCK wild-type cells, two independent clones of ZONAB RNAi cell lines, a ZO-1 RNAi line, and a cell line overexpressing ZONAB-A were plated at low confluence with tetracycline; after 36 h, they were synchronized by serum starvation. S-phase entry was then stimulated by adding 5% serum for 12 or 24 h. Total cell extracts were analyzed by immunoblotting with antibodies against ZO-1, ZO-2, ZONAB, cyclin D1 or tubulin (α-tubulin).

ZONAB regulates cyst formation of MDCK cells grown in 3-D cultures. Control, ZONAB, and ZO-1 RNAi lines as well as ZONAB-A and B overexpressing MDCK lines were plated in the absence or presence of tetracycline (TC) in collagen-Matrigel cultures for 5 days. Cells were then fixed with 3% paraformaldehyde and processed for epifluorescence by using a Cy3-conjugated secondary antibody to detect beta-catenin (red), FITC-phalloidin (green) to detect actin, and Hoechst 33528 (white) to stain DNA. Wild-type and control RNAi cells formed normal cysts without or with tetracycline as the shown control cultures. Shown are overview images of larger fields (a1, a3, b1, b3, c1, d1, and d2) and larger magnifications of characteristic structures (a2, a4, b2, b4, and c2). (B) In two independent experiments, as in the one shown in panel A, pictures from 10 different areas were taken and quantified by counting the number and types of structures. The formed structures were divided into the following classes: spherical structures with no lumen as in a4, a single lumen as in a2, multiple lumens or structures that were not spherical and had a generally disorganized appearance as the ones shown in panels b4, d1, and d2.