Ectopic expression of mutant p53 R163H cooperates with p53-KD to alter cyst morphology.

<p><b>A</b>, Generation of MCF-10A cell lines in which siRNA-resistant mutant p53-R163H was expressed along with knockdown of endogenous wild-type p53. The levels of wide-type p53 and mutant p53-R163H were determined by Western blotting. <b>B</b>, The level of wild-type p53 transcripts was determined by RT-PCR. <b>C</b>, Representative images of MDCK cells or MDCK cells with p53-KD-R163H in 2-D culture. <b>D</b>, Representative images of MDCK cells or MDCK cells with wild-type p53-KD and overexpression of mutant p53-R163H in 3-D culture for 12 d. Scale bar: 100 µM. <b>E</b>, Top panel: colony formation assay was performed with MDCK cells or MDCK cells with p53-KD and overexpression of R163H. Bottom panel: the number of colonies was counted and presented as Mean ± SD from three separate experiments. <b>F</b>, Wound healing assay was performed with MDCK cells, MDCK cells with p53-KD, or MDCK cells with p53-KD and overexpression of R163H. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. Bottom panel: the time required for wound closure was measured and presented as mean ± SD from three separate experiments.</p

Overexpression of mutant p53-R261H disrupted tubular formation in 3-D culture.

<p><b>A</b>, Generation of MDCK cell lines in which siRNA-resistant mutant p53-R261H was stably overexpressed (clones 1 and 2). The protein levels of mutant p53-R261H and actin were measured by Western blotting. <b>B</b>, The level of wild-type p53 transcripts was determined by RT-PCR. <b>C</b>, Representative images of MDCK cells, MDCK cells with p53 knockdown, or MDCK cells with mutant p53-R261H in 2-D culture (200×). <b>D</b>, Representative images of MDCK cells with mutant p53-R261H in 3-D culture. Scale bar: 100 µM. <b>E</b>, Top panel: colony formation assay was performed with MDCK cells or MDCK cells with mutant p53-R261H. Bottom panel: the number of colonies was counted and presented as Mean ± SD from three separate experiments. <b>F</b>, Wound healing assay was performed with MDCK cells, MDCK cells with p53-KD, or MDCK cells with mutant p53-R261H. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. Bottom panel: the time required for wound closure was measured and presented as mean ± SD from three separate experiments.</p

EMT markers are regulated upon ectopic expression of mutant p53, some of which are further enhanced by knockdown of endogenous wild-type p53 in MDCK cells.

<p><b>A</b>-<b>C</b>, Western blots were prepared with extracts from parental MDCK cells (lane 1), p53-KD MDCK cells (lane 2), MDCK cells in which a mutant p53 was ectopically expressed (lanes 3, 5) and MDCK cells in which a mutant p53 was ectopically expressed along with knockdown of endogenous wild-type p53 (lanes 4, 6). The blots were probed with antibodies against β-catenin (A), E-cadherin (A), Snail (B), Slug (B), Twist (B), c-Met (C) and actin (A-C). The protein levels of EMT markers were quantified and the ratios were labeled under the corresponding bands. <b>D</b>, Proposed model of mutant p53 in MDCK cell tubulogenesis. </p

Overexpression of mutant p53 R163H disrupted tubular formation in 3-D culture.

<p><b>A</b>, Generation of MDCK cell lines in which siRNA-resistant mutant p53-R163H was stably overexpressed (clones 3 and 5). The level of p53-R163H was determined by Western blotting. <b>B</b>, The level of wild-type p53 transcripts was determined by RT-PCR. <b>C</b>, Representative images of MDCK cells, MDCK cells with p53 knockdown, or MDCK cells with mutant p53 (R163H) in 2-D culture (200×). <b>D</b>, Representative images of MDCK cells, MDCK cells with p53 knockdown, or MDCK cells with mutant p53-R163H in 3-D culture for 6 d or 12 d. Scale bar: 100 µM. <b>E</b>, Top panel: colony formation assay was performed with MDCK cells, MDCK cells with p53 knockdown, or MDCK cells with mutant p53-R163H. Bottom panel: the number of colonies was counted and presented as Mean ± SD from three separate experiments. <b>F</b>, Wound healing assay was performed with MDCK cells, MDCK cells with p53-KD, or MDCK cells with mutant p53-R163H. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. Bottom panel: the time required for wound closure was measured and presented as mean ± S.D. from three separate experiments.</p

Ectopic expression of mutant p53 R261H cooperates with p53-KD to alter cyst morphology.

<p><b>A</b>, Generation of MDCK cell lines in which siRNA-resistant mutant p53 R261H was expressed along with knockdown of endogenous wild-type p53. The levels of wide-type p53 and mutant p53 R261H were determined by Western blotting. <b>B</b>, The level of wild-type p53 transcripts was determined by RT-PCR. <b>C</b>, Representative images of MDCK cells or MDCK cells with p53-KD-(R261H) in 2-D culture. <b>D</b>, Representative images of MDCK cells with p53-KD-R261H in 3-D culture for 12 d. Scale bar: 100 µM. <b>E</b>, Top panel: colony formation assay was performed with MDCK cells or MDCK cells with p53-KD-R261H. Bottom panel: the number of colonies was counted and presented as Mean ± SD from three separate experiments. <b>F</b>, Wound healing assay was performed with MDCK cells, MDCK cells with p53-KD, or MDCK cells with p53-KD-R261H. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. Bottom panel: the time required for wound closure was measured and presented as mean ± SD from three separate experiments.</p

Primers for RT-PCR and cloning.

<p>Primers for RT-PCR and cloning.</p

Identification of CUEs as a PCBP1-binding site in p63 3′UTR

<p>(A) Schematic presentation of <i>p63α</i> transcript and the location of probes. (B) REMSA was performed by mixing ³²P-labeled RNA probes (A, A1, or A2) with recombinant GST and GST-PCBP1proteins, respectively. (C) REMSA assay was performed by mixing a ³²P-labeled RNA probe A1 along with or without excess amount (50-fold) of unlabeled probe A1 or A2. (D) REMSA was performed by mixing a ³²P-labeled RNA probe (A1, CG1, or CG2) with recombinant GST or GST-fused PCBP1.</p

PUMA is necessary for morphogenesis of MCF10A cells.

<p><b>A</b>, Generation of MCF10A cells in which PUMA (clones #2 and 3) was stably knocked down. Western blots were performed with extracts from MCF10A cells untreated or treated with 0.2 µM doxorubicin for 24 h and then probed with antibodies against PUMA, ΔNp73 and actin, respectively. <b>B,</b> Representative images of MCF10A cells or MCF10A cells with PUMA-KD in 2-D culture (a and d, 200×) and 3-D culture (b and e, 40×; c and f, 100×). Black arrow indicates elongated spindle–liked MCF10A cells. <b>C,</b> Representative confocal images of cross-sections through the middle of acini stained with To-Pro-3 and antibody against E-cadherin in MCF10A cells with PUMA-KD. <b>D,</b> Representative confocal images of cross-sections through the middle of acini stained with To-Pro-3 and antibody against β-catenin in MCF10A cells with PUMA-KD. White arrows indicate the accumulation and translocation of β-catenin in acinus structure. <b>E</b>, Representative confocal images of cross-sections through the middle of acini stained with To-Pro-3 and antibody against laminin V in MCF10A cells with PUMA-KD. Scale bar, 20 µm.</p

<i>p63</i> mRNA stability is regulated by PCBP1.

<p>(A–C) The level of <i>p63</i> transcript is decreased by PCBP1-knockdown. HaCaT (A), MIA PaCa2 (B), and ME180 (C) cells were transduced with a lentivirus expressing a control luciferase (Luc) shRNA or PCBP1 shRNA, selected by puromycin for 3 d. Total RNAs were isolated and RT-PCR was performed to measure the levels of <i>PCBP1</i>, <i>GAPDH</i>, <i>ΔNp63</i> (A), and <i>p63α</i> (B–C) transcript. (D–E) PCBP1-knockdown destabilizes <i>p63</i> transcript. MIA PaCa2 (D) and ME180 (E) cells were transduced with a lentivirus expressing a control luciferase (Luc) shRNA or PCBP1 shRNA, selected by puromycin for 3 d, followed by treatment with actinomycin D (5 µg/ml) for various times. The level of <i>p63α</i> and <i>GAPDH</i> transcript was measured by RT-PCR and quantified by densitometric analysis. The relative half-life of <i>p63α</i> transcript was calculated.</p

The CUE in p63 3′UTR is sufficient for PCBP1 to regulate p63 expression.

<p>(A) Schematic presentation of luciferase reporters that carry either wild-type p63-CUE or a mutated CUE (CG) derived from p63 3’UTR. (B) MCF7 cells were transduced with a lentivirus expressing control luciferase shRNA (Ctrl) or PCBP1 shRNA (PCBP1-KD) for 24 h, then transiently transfected with pGL3-FSUT/p63-WT or pGL3-FSUT/p63-CG for 48 h. Cell lysates were collected and the luciferase activity was measured. The experiment was performed in triplicate. Error bars indicate standard deviation. *<i>P</i><0.05 by two-tailed <i>t</i>-test.</p