Effects of p150^Sal2 expression on the growth of SKOV-3 cells. (A) Colony reduction assay. Shown are a Western blot of HOSE cells and three other ovarian tumor cell lines with various levels of p150 expression (arrow) (top left panel) and a Western blot of p150 expression in various cells with transfected empty vector or a p150^Sal2 expression construct (lower left panel). Tubulin was used as a loading control. Neomycin-resistant colonies were reduced in SKOV-3 and RUMGS cells transfected with a p150^Sal2 expression construct (p150) or empty vector (Vector) but not in HOSE or IGR-OV-1 cells (right panel). (B) BrdU incorporation assay. SKOV-3 cells were transfected with either empty vector or constructs expressing p150^Sal2 or p53 and incubated with BrdU-containing medium. Enhanced GFP construct was cotransfected in each experiment to identify the transfected cells. The cells were then stained for BrdU and DAPI. Results are presented as the percentage of BrdU-negative (BrdU⁻) cells among the total number of transfected cells (positive for cotransfected enhanced GFP). (C) Apoptosis analysis. The DAPI staining pattern of apoptotic SKOV-3 cells induced by actinomycin D was confirmed by TUNEL assay (top left panel). SKOV-3 cells were transiently transfected with p150 or empty vector together with GFP and stained with DAPI (bottom left panel). Arrows indicate cells that are p150 transfected and also apoptotic. Quantification is shown on the right as the percentage of apoptotic cells among all transfected cells.

Effect of p150 expression on tumorigenicity. (A) Western blot comparison of p150^Sal2 expression in normal HOSE cells, pooled neomycin-resistant SKOV-3 cells transfected with empty vector (SK-vector), and three independently derived SKOV-3 clones with various p150^Sal2 expression levels (SK-P150-1, -2, and -3). (B) Representative figure showing the reduction of anchorage-independent growth in SK-P150 cells with restored p150^Sal2 expression compared to SK-vector cells. (C) SCID mice inoculated by SKOV-3 cells with stably integrated empty vector (SK-vector) and SKOV-3 cells with stably integrated p150 expression construct (SK-P150-1). (D) Weights of tumors from SK-vector cells and from three independent SKOV-3 clones with human p150 (SK-P150-1, SK-P150-2, and SK-P150-3). (E) Apoptosis in SK-vector- and SK-P150-induced tumors. TUNEL staining of sections of SK-vector and SK-P150-1 tumors is shown at left (arrows indicate TUNEL-positive cells). Quantification of apoptotic cells in SK-vector and SK-P150-1 tumors is shown at right. Results are the averages for two independent tumors of each type, two sections of different areas, and three random fields of each section. (F) Mitosis in SK-vector and SK-P150 tumors. Hematoxylin and eosin staining of sections of SK-vector and SK-P150 tumors is shown at left (arrows indicate mitotic cells). Quantification of mitotic cells in SK-vector and SK-P150 tumors is shown at right. The sampling was the same as described for panel E.

p150^Sal2 regulates p21^Cip1/Waf1 expression in SKOV-3 and HOSE cells. (A) Western blots for p21 in empty vector-transfected SKOV-3 cells (SK-vector) and three independent SKOV3-p150^Sal2 clones (SK-P150-1, SK-P150-2, and SK-P150-3) is shown at left. α-Tubulin was used as a loading control. Quantification of p21 levels in SK-vector and SK-P150 clones is shown at right. (B) Semiquantitative RT-PCR comparison of p21 RNA levels using either SK-vector or SK-P150 total RNA. GAPD transcript (G3PDH) was used as an internal control. Relative levels of p21 transcripts of SK-vector and SK-P150 were assessed by comparing the 0.6-kb p21 RT-PCR products of the same cycle numbers within the linear range of amplification (34 to 40 cycles). The quantification was normalized by the levels of transcripts of GAPD within its own linear range of amplification (28 to 34 cycles). (C) p150^Sal2 stimulates p21 promoter activity (top). Increasing amounts of a human p150 cDNA expression construct (p150) were cotransfected into SKOV-3 cells along with a luciferase reporter driven by a 2.7-kb human p21 promoter (P21-Luc). An empty vector (Vector) and an unrelated (thymidine kinase) promoter-luciferase construct (PG-TK-Luc) were used as controls. Stimulation of p21 promoter activity by p150^Sal2 is presented as the increase (n-fold) in luciferase activity over that of the vector control normalized by the activity of cotransfected β-galactosidase. Also shown is the effect of p150 and p53 on P21-Luc induction (bottom). An additive effect was seen when p150^Sal2 and p53 were cotransfected (P53+p150) compared to what was seen with either p150 or p53 alone. (D) At left is a schematic presentation of p150^Sal2 protein showing the location of deleted zinc finger motifs in p150Δ3 (deletion of aa 631 to 711) and p150Δ2 (deletion of aa 911 to 956). Shown at top right are the results of a colony reduction assay using empty vector (Vector), p150^Sal2 (p150), and zinc finger domain deletions of the DNA binding triple zinc finger (p150Δ3) or the T-antigen-interacting last zinc finger pair (p150Δ2) in SKOV-3 cells. The graph at middle right shows induction of p21-Luc activities by vector, p150, p150Δ3, and p150Δ2 in SKOV-3 cells. Shown at bottom right are the expression levels of p150^Sal2 in SKOV-3 cells transfected with vector, p150, p150Δ3, or p150Δ2.

RNA interference analysis with HOSE cells. (A) Mock-transfected or p150 siRNA-transfected HOSE cells were cultured in BrdU-containing medium for 20 h (left panels). Nuclei with BrdU incorporation were stained with anti-BrdU antibody (red). Nuclei were also stained with DAPI (blue). The results were quantified by counting BrdU-positive cells in four random fields as the percentage of total cells (right). (B) Western blot analysis showing reduced p150^Sal2 levels after addition of siRNA (P150 siRNA) and reduced p21 levels in HOSE cells compared to what was seen with mock-transfected cells. The same blot was stripped and reprobed for α-tubulin as the loading control.

p150^Sal2 directly interacts with p21 promoter and regulates p21 transcription through a cis-acting element. (A) Schematic representation of the 2.7-kb human p21 promoter region and its digestion fragments used in the protein DNA immunoprecipitation assay (25) (sizes in base pairs are given under each fragment). (B) Immunoprecipitation of AvaI-digested, StyI-digested, or StyI/MspI double-digested fragments of p21 promoter by p150 antibody (AntiP150) with preimmune serum (Pre-Imm) as a control. Wild-type (Wt) p150 or p150 with a deletion of the putative DNA binding domain (Δ3) was from either P19 cell extract (Cell) or the in vitro translation (IVT) product of cloned human cDNA. Empty cloning vector was used as a negative control for in vitro-translated p150 in the immunoprecipitation of StyI/MspI fragments. (C) A p150-responsive cis-acting element is located 1.4 kb upstream of the human p21 promoter corresponding to the distal p150^Sal2 binding region. A luciferase assay was conducted in SKOV-3 cells to assess the responsiveness of various p21 promoter deletion constructs. Fold induction is the corresponding luciferase activity with cotransfected p150^Sal2 over that with cotransfected empty vector. The p53 binding sites are shown as landmarks. (D) p150 binds p21 promoter region in vivo. ChIP was performed using serum against p150 (Anti-p150) to precipitate chromatin cross-linked with p150 in P19 cells and amplified using p21 promoter-specific primers (p21) and primers for GAPD. The same sets of primers were also used for PCR amplification from preimmune serum-precipitated chromatin (PreImm) and total input chromatin extract (Total) as controls.