Journal: Oncogene

Article Title: Oncogenic suppression of PHLPP1 in human melanoma.

doi: 10.1038/onc.2013.420

Figure Lengend Snippet: Figure 4. Knockdown of PHLPP1 results in anchorage-independent growth of melanocytes, whereas overexpression of PHLPP1 inhibits melanoma growth in a xenograft mouse model. (a) HEMa-LP melanocytes were transduced with the control shRNA, PHLPP1 shRNA1 or PHLPP1 shRNA2. After 24 h, whole-cell lysates were subjected to western blot analysis. Data shown are representative of three individual western blot analyses. (b) Left panel: anchorage-independent growth assay of HEMa-LP melanocytes transduced with the control, PHLPP1 shRNA1, or PHLPP1 shRNA2. Scale bar, 0.5 mm. Right panel: quantitation of colony formation as shown in left panel. Data shown are the numbers of colonies in 10 random phase-contrast microscopic fields (n ¼ 3, mean±s.e.m; Student’s t-test, *Po0.05). (c) HEMa-LP melanocytes transduced with the control shRNA, PHLPP1 shRNA1, PHLPP1 shRNA2 were counted on days 3, 6 and 9 using an automated cell counter (n ¼ 3, mean±s.e.m; Student’s t-test, *Po0.05). (d) A total of 5 104 HEMa-LP melanocytes transduced with the control shRNA, PHLPP1 shRNA1 or PHLPP1 shRNA2 with or without exposure to the vehicle control (DMSO) or LY294002 (20 mM) were counted on day 9 using an automated cell counter (n ¼ 3, mean±s.e.m; Student’s t-test, *Po0.001). (e) MM200.PHLPP1 and Mel-RM.PHLPP1 cells and their corresponding control counterparts carrying vector alone were xenografted into flanks of nu/nu mice via subcutaneous (s.c.) injection. The data shown are photographs of representative melanomas harvested at day 36 after intraperitoneal (i.p.) injection. Scale bars, 5 mm. (f) MM200.PHLPP1 and Mel-RM.PHLPP1 cells and their corresponding control counterparts carrying vector alone were xenografted into flanks of nu/nu mice via s.c. injection. The data shown are growth curves of melanoma tumors represented by the volume calculated with the modified ellipsoidal formula (tumor volume ¼ 1/2(length width2)), which are mean±s.e.m. of all tumors in each experimental group (*Po 0.001, Student’s t-test). (g) MM200.PHLPP1 and Mel-RM.PHLPP1 cells and their corresponding control counterparts carrying vector alone were xenografted into flanks of nu/nu mice via s.c. injection. The data shown tumor weight (mean±s.e.m.) generated by PHLPP1-overexpressing melanoma sub-lines (MM200.PHLPP1 and Mel-RM.PHLPP1) in comparison with that formed corresponding control lines carrying vector alone. (h) Whole-cell lysates of crude tumor tissues from randomly sampled tumors generated by PHLPP1-overexpressing melanoma sub-lines (MM200.PHLPP1 and Mel- RM.PHLPP1) and those formed by corresponding control lines carrying vector alone were subjected to western blot analysis. Data shown are representative of three individual western blot analyses. (i) MM200.PHLPP1 cells with or without co-transduced with Akt/S473D were xenografted into flanks of nu/nu mice via s.c. injection. The data shown are photographs of representative melanomas harvested at day 36 after i.p. injection. Scale bars, 5 mm. (j) MM200.PHLPP1 cells with or without co-transduced with Akt/S473D were xenografted into flanks of nu/nu mice via s.c. injection. The data shown are tumor volume at 36 days after i.p. injection (mean±s.e.m; Student’s t-test, *Po0.05).

Article Snippet: Human PHLPP1 shRNA kit (TG302501) and scramble shRNA kit (TG300130) were purchased from Origene (Karrinyup, WA, Australia).

Techniques: Knockdown, Over Expression, Transduction, Control, shRNA, Western Blot, Growth Assay, Quantitation Assay, Plasmid Preparation, Injection, Generated, Comparison

Journal: Molecular Cancer

Article Title: Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

doi: 10.1186/1476-4598-10-51

Figure Lengend Snippet: LPP3 regulates glioblastoma cell proliferation . (A) The time line indicates when glioblastoma cell lines were plated, shRNA (retrovirus) mediated knockdown was conducted, cells were growth factor and serum starved, fresh medium was added, BrdU (1.0 μg/ml) was added, and the proliferation assay was performed. (B) Quantification of glioblastoma cell proliferation assay. The data represent the mean ± s.e.m. n = 5-7 from five to seven independent experiments, ¥ P < 0.05 vs. control untreated; ¶ P < 0.05 vs. control shRNA treated group. (C-F) Representative images of the BrdU assay. Arrows indicate BrdU positive cells. Scale bar, 150 μm. (G) The efficiency of knockdown of LPP3 was assessed by western blotting with the indicated antibodies. Figure 3G (top panel, last lane, anti-LPP3), a partial band is likely produced by over flow from the neighboring well. All experiments are representative of those obtained in at least three separate experiments with similar results.

Article Snippet: Control and LPP shRNA retroviral constructs were purchased from Origene (Rockville, MD).

Techniques: shRNA, Knockdown, Proliferation Assay, Control, BrdU Staining, Western Blot, Produced

Journal: Molecular Cancer

Article Title: Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

doi: 10.1186/1476-4598-10-51

Figure Lengend Snippet: LPP3 regulates glioblastoma cell migration . (A) The time line indicates when glioblastoma cell lines were plated, shRNA (retrovirus) mediated knockdown was conducted, medium was added, and the migration assay was performed as described in materials and methods. (B) Quantification of glioblastoma cell migration through the Boyden chamber. The data represent the mean ± s.e.m. n = 5-7, ¥ P < 0.05 vs. control shRNA treated group. (C) Efficiency of knockdown of LPP3 was assessed by western blotting with the indicated antibodies. All experiments are representative of those obtained in at least three separate experiments with similar results.

Article Snippet: Control and LPP shRNA retroviral constructs were purchased from Origene (Rockville, MD).

Techniques: Migration, shRNA, Knockdown, Control, Western Blot

Journal: Molecular Cancer

Article Title: Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

doi: 10.1186/1476-4598-10-51

Figure Lengend Snippet: LPP3 regulates glioblastoma tumor growth . (A) Stable clones of U87 and U118 were selected and efficiency of knockdown was determined by western blotting with the indicated antibodies. LPP3 -knockdown decreased total β-catenin, CYCLIN-D1, and CD133 proteins, whereas it had no effect on LPP2 or GAPDH. (B) Conditioned media were collected and subjected to ELISA for VEGF, IL-8, and TIMP-2. LPP3 -knockdown in U87 and U118 cells reduced the concentration of VEGF and IL-8, whereas TIMP-2 was unaffected. (C) In the xenograft assay, mice receiving cells that expressed control shRNA showed increased tumor growth (n = 12, ¥ P < 0.05) after 14 and 28 days. LPP3 -knockdown reduced U87 and U118 tumor growth (n = 12, ¶ P < 0.05). (D) Representative images from the xenograft assay. Dotted circles indicate the location of tumor implants.

Article Snippet: Control and LPP shRNA retroviral constructs were purchased from Origene (Rockville, MD).

Techniques: Clone Assay, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Xenograft Assay, Control, shRNA

Journal: Molecular Cancer

Article Title: Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

doi: 10.1186/1476-4598-10-51

Figure Lengend Snippet: Forced expression of LPP3 potentiates SW480 tumor growth in the xenograft assay . (A) pLNCX2-based retroviral constructs expressing vector alone, hLPP1, hLPP2, hLPP3 or mLpp3 cDNAs. (B) The comparable levels of hLPP1, hLPP2, hLPP3 and mLpp3 proteins were confirmed by western blotting with anti-HA antibody. (C) Xenograft assay: there was minimal tumor growth after 14 and 28 days in mice receiving LPP3-deficient SW480 cells (2 × 10 4 tumor cells/site) that expressed vector alone, hLPP1, or hLPP2 constructs. In contrast, tumor volume was significantly increased in mice receiving SW480 cells that expressed hLPP3 or mLpp3 (n = 12, ¥ P < 0.01 vs. hLPP1 group; n = 12, ¶ P < 0.005 vs. hLPP1 group) at 14 and 28 days.

Article Snippet: Control and LPP shRNA retroviral constructs were purchased from Origene (Rockville, MD).

Techniques: Expressing, Xenograft Assay, Retroviral, Construct, Plasmid Preparation, Western Blot

Journal: Molecular Cancer

Article Title: Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

doi: 10.1186/1476-4598-10-51

Figure Lengend Snippet: Domains involved in the regulation of SW480 tumor growth . (A) Schematics of pLNCX2-based retroviral constructs (a-e). Empty red and blue circles indicate the relative positions of lipid phosphatase and RGD cell adhesion domains, respectively. Filled red and blue circles indicate phosphatase-inactive (PI) and RAD (adhesion defective) domains, respectively. Black boxes represent transmembrane segments. Three copies of hemagglutinin (HA) epitopes were fused to the N-terminal and in-frame to the open reading frame of the cDNAs. (B) Equivalent protein expression and phosphorylation states of β-catenin and CYCLIN-D1 proteins were analyzed by immunoblotting with anti-HA, anti-β-catenin, anti-p-β-catenin, and anti-CYCLIN-D1 antibodies. Equal loading of proteins across the lanes were determined with the anti-GRB-2 antibody. All blots are representative of those obtained in at least three separate experiments with similar results. (C) LPP3 expression potentiates SW480 tumor growth. Nude mice were injected subcutaneously with SW480 cells (~ 2 × 10 4 ) expressing the indicated constructs. After 21 days, the tumor outgrowths were measured and photographed. (n = 12, ¶ P < 0.005 vs. vector alone group). (D) Representative pictures of primary tumors in nude mice injected with SW480 cells expressing the indicated constructs.

Article Snippet: Control and LPP shRNA retroviral constructs were purchased from Origene (Rockville, MD).

Techniques: Retroviral, Construct, Expressing, Phospho-proteomics, Western Blot, Injection, Plasmid Preparation