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

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

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

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

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

Pirh2 physically associates with mutant p53 protein for polyubiquitination.

<p>(A) HaCaT and MIA PaCa-2 cells were treated with 5 or 7.5 µM ATO for 6 h. Cell extracts from HaCaT (left) and MIA PaCa-2 (right) cells were immunoprecipitated with anti-p53 or control IgG. The immunocomplexes were then used to detect mutant p53 and Pirh2 along with whole-cell lysates as input control. (B) The experiment was performed as described in (A), except that anti-Pirh2 antibody was used in immunoprecipitation. (C–E) In vitro synthesized ³⁵S-labeled wild-type p53 (C), R175H (D), and R273H (E) were mixed with GST, GST-tagged Pirh2, Pirh2-DN, or Pirh2-ΔRING. The complexes were then mixed with a buffer containing E1, E2 (UbcH5b), and Ub and then incubated at 30°C for 2 h. Ubiquitinated p53 was analyzed by SDS-PAGE and detected by autoradiography. (F) A model of Pirh2-mediated degradation of mutant p53 induced by ATO.</p

Knockdown ofΔNp73 mitigates EMT induced by PUMA-KD or p21-KD.

<p><b>A–C</b>,MCF10A cells were grown in Matrigel for 20 days. Western blots were prepared using extracts from MCF10A cells (lane 1), MCF10A cells with ΔNp73-KD (lane 2), with p21-KD (lane 3), with PUMA-KD (lane 4), with ΔNp73&p21-KD (lane 5) or with ΔNp73&PUMA-KD (lane 6). The blots were probed with antibodies against laminin V (A), Twist (A), E-cadherin (B), Snail-1 (B), β-catenin (C), Slug (C), and actin (A-C), respectively. <b>D,</b> Top panel: Colony formation assay was performed with MCF10A cells and MCF10A cells with ΔNp73&p21-KD or with ΔNp73&PUMA-KD. Bottom panel: the number of colonies was counted and presented as Mean ± SD from three separate experiments. <b>E,</b> Top panel: Wound healing assay was performed with MCF10A cells and MCF10A cells with ΔNp73&p21-KD or with ΔNp73&PUMA-KD. Cell migration was determined by visual assessment of cells migrating into the wound for a period of 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. <b>F,</b> A model of PUMA, p21 and ΔNp73 in cell polarity.</p

Arsenic-induced degradation of mutant p53 protein is inhibited by MG132, an inhibitor of 26S proteasome.

<p>Western blots were prepared with extracts from HaCaT (A) and MIA PaCa-2 (B) cells, which were untreated or pretreated with 4 µM MG132 for 2 h, and then untreated or treated with ATO for 6 h.</p

Knockdown of PUMA and p21 enhances EMT.

<p><b>A-B</b>, Western blots were prepared with extracts from MCF10A cells (lane 1), and MCF10A cells with p21-KD (lane 2), PUMA-KD (lane 3), or PUMA&p21-KD (lane 4). MCF10A cells were grown in Matrigel for 20 days. The blots were probed with antibodies against E-cadherin (A), β-catenin (A), laminin V (A), Snail-1 (B), Slug (B), Twist (B), and actin (A–B), respectively. <b>C,</b> Top panel: Colony formation assay was performed with MCF10A cells, or MCF10A cells with p21-KD, PUMA-KD or PUMA&p21-KD. Cells were cultured for a period of 12 days, then fixed and stained with crystal violet. Bottom panel: The number of colonies was counted and presented as Mean ± SD from three separate experiments. <b>D,</b> Top panel: Wound healing assay was performed with MCF10A cells and MCF10A cells with p21-KD, PUMA-KD or PUMA&p21 -KD. Cell migration was determined by visual assessment of cells migrating into the wound for a period of 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