Journal: Oncotarget

Article Title: Hyperactivated mTORC1 downregulation of FOXO3a/PDGFRα/AKT cascade restrains tuberous sclerosis complex-associated tumor development

doi: 10.18632/oncotarget.18963

Figure Lengend Snippet: ( A , B ) Tsc2−/− (A) or Tsc1−/− MEFs (B) were infected with lentivirus harboring a vector encoding PDGFRɑ (LV-PDGFRɑ) or the empty vector (LV). ( C ) Tsc2+/+ MEFs stably expressing shRNAs targeting PDGFRɑ (shPDGFRɑ) or a control shRNA (shSc). A–C. Cell lysates were subjected to immunoblotting with the indicated antibodies. ( D ) Proliferation of the indicated cells was examined using an MTT assay. ( E ) The colonies formed by the indicated cells were stained and counted. Representative images (left panels) and quantifications (right panels). ( F , G ) Tsc2−/− (F) or Tsc1−/− MEFs (G) transduced with LV-PDGFRɑ or LV lentiviruses were inoculated subcutaneously into nude mice, followed by monitoring for tumor growth. ( H , I ) Tumor tissues derived from Tsc2−/− (H) or Tsc1−/− MEFs (I) were fixed and embedded with paraffin, and then subjected to H&E and immunohistochemical staining. Representative images were presented. Scale bar, 50 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: GV248 lentiviral shRNA expression vectors targeting mouse PDGFRɑ, mouse Raptor, mouse Rictor, and the control scrambled shRNA (shSc) were obtained from Genechem (Shanghai, China).

Techniques: Infection, Plasmid Preparation, Stable Transfection, Expressing, Control, shRNA, Western Blot, MTT Assay, Staining, Transduction, Derivative Assay, Immunohistochemical staining

Journal: Oncotarget

Article Title: Hyperactivated mTORC1 downregulation of FOXO3a/PDGFRα/AKT cascade restrains tuberous sclerosis complex-associated tumor development

doi: 10.18632/oncotarget.18963

Figure Lengend Snippet: ( A , B ) Tsc2−/− (A) or Tsc1−/− MEFs (B) were transduced with LV-PDGFRɑ or LV lentiviruses. ( C , D ) Tsc2+/+ (C) or Tsc1+/+ MEFs (D) were transduced with shPDGFRɑ or shSc lentiviruses. A–D. Cells were starved in DMEM for 24 h, followed by stimulation with serum or PDGF (50 ng/ml) for 30 min, and then cell lysates were harvested and subjected to immunoblotting with the indicated antibodies. ( E , F ) Tumor tissues derived from Tsc2−/− (E) or Tsc1−/− MEFs (F) transduced with LV-PDGFRɑ or LV lentiviruses were subjected to immunoblotting with the indicated antibodies.

Article Snippet: GV248 lentiviral shRNA expression vectors targeting mouse PDGFRɑ, mouse Raptor, mouse Rictor, and the control scrambled shRNA (shSc) were obtained from Genechem (Shanghai, China).

Techniques: Transduction, Western Blot, Derivative Assay

Journal: Breast Cancer Research : BCR

Article Title: Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

doi: 10.1186/bcr3611

Figure Lengend Snippet: Runx2 specifically maintains pAkt in invasive mammary epithelial cells. A) The basal levels of pAkt (Serine 473) expression was examined in MDA-MB-231, SUM-159 and SUM-159-PT cells cultured in regular growth medium by Western blotting. B) The Runx2 protein expression was stably suppressed in SUM-159 cells by lentivirus-mediated Runx2 shRNA delivery. The Runx2 expression levels were analyzed by Western blotting while β-Actin was used as the loading control. C) The SUM-159 cells expressing Runx2-RNAi or control were serum-deprived and stimulated with 100 ng/ml of EGF in the presence of 20 μM LY294002. The whole cell lysates were analyzed for pAkt (Serine 473) and total Akt expression by Western blotting at 10 minutes post epidermal growth factor (EGF) stimulation. A quantification of normalized relative pAkt levels is given below respective lane. D) The SUM-159-PT cells were transfected with dsRNA targeting a different Runx2 mRNA sequence to transiently suppress Runx2 expression. E) The serum-deprived, EGF stimulated SUM-159-PT cells were analyzed for pAkt (Serine 473) and Akt levels by Western blotting. A quantification of normalized pAkt level is shown below the blot. F) The MDA-MB-231 cells expressing Runx2-RNAi or control were serum-deprived and stimulated with 100 ng/ml of EGF in the presence of 20 μM LY294002. The whole cell lysates were analyzed for pAkt (Serine 473) and total Akt expression by Western blotting at 10 minutes and 1 hour post EGF stimulation.

Article Snippet: The lentivirus vector expressing Rictor shRNA was obtained from Addgene (plasmid #1853) (Cambridge, MA, USA) [ ].

Techniques: Expressing, Cell Culture, Western Blot, Stable Transfection, shRNA, Control, Transfection, Sequencing

Journal: Breast Cancer Research : BCR

Article Title: Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

doi: 10.1186/bcr3611

Figure Lengend Snippet: Reducing PHLPP1 phosphatase and pERK kinase activity rescues Runx2-mediated inhibition of pAkt in MDA-MB-231 cells. A) The PHLPP1 mRNA expression was transiently suppressed in MDA-MB-231 cells and gene expression was analyzed by real-time PCR. A quantification of GAPDH normalized PHLPP1 mRNA expression is shown. B) A quantification of the relative phosphorylation levels of pAkt (Serine 473) were determined by Western blotting in PHLPP1 knockdown MDA-MB-231 cells. C) The levels of pAkt (Serine 473) and total Akt proteins were analyzed by Western blot in PHLPP1 knock out control or Runx2 suppressed MDA-MB-231 cells as indicated. A quantification of pAkt levels normalized to total Akt protein is shown below the respective lane. D) The control or Runx2 knockdown MDA-MB-231 cells were stimulated with EGF for indicated times in the presence or absence of ERK inhibitor U0126. The level of pAkt and Akt was determined by Western blotting and quantification is shown below the respective lane. E) The Runx2 gene expression was conditionally suppressed by doxycycline in MDA-MB-231 cells stably expressing tTR-KRAB and Runx2-shRNA. The serum-deprived cells were stimulated with EGF in the presence of ERK inhibitor PD184161 for indicated times. The pAkt and total Akt expression was analyzed by Western blotting and a quantification of normalized expression is shown below the respective lanes.

Article Snippet: The lentivirus vector expressing Rictor shRNA was obtained from Addgene (plasmid #1853) (Cambridge, MA, USA) [ ].

Techniques: Activity Assay, Inhibition, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Knockdown, Knock-Out, Control, Stable Transfection, shRNA

Journal: Breast Cancer Research : BCR

Article Title: Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

doi: 10.1186/bcr3611

Figure Lengend Snippet: Runx2 knockdown alters expression levels of mTORC-2 proteins. A) The MDA-MB-231 cells’ transient Runx2 suppression was analyzed for mTOR and Runx2 levels. B) A quantification of mTOR protein expression normalized to β-Actin is shown. C) The stable Runx2 knockdown MDA-MB-231 cells were serum-deprived, epidermal growth factor (EGF) stimulated and examined for pmTOR (Serine 2481) and mTOR (total) protein. D) The Runx2 knockdown cells were assayed for mTOR gene expression by RT-PCR (normalized to GAPDH ). E) The Runx2 knockdown and Ad-GFP- or WT-Runx2-treated MDA-MB-231 cells were tested for Runx2 recruitment on mTOR promoter by ChIP assays. A schematic diagram of the mTOR promoter region is bars indicating potential Runx binding sites (black bars) and location of PCR primers (small arrows). F) and G) The mRNA (F) and protein (G) expression levels of Rictor were analyzed in control or Runx2 knockdown MDA-MB-231 cells by RT-PCR and Western blotting, respectively. H) The MDA-MB-231 cells stably over-expressing WT-Runx2 were assayed for Runx2 and Rictor gene expression by RT-PCR. I) The MDA-MB-231 cells with stable Rictor knockdown were stimulated with EGF and were analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor protein levels by Western blotting. J) The stably Runx2 knockdown MDA-MB-231 cells together with Rictor suppression were stimulated with EGF and analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor proteins by Western blotting. K) The glucose and serum-deprived (24 h) MDA-MB-231 cells with Runx2 knockdown (Runx2-RNAi) together with Rictor knockdown (Rictor -shRNA) were stained for Annexin V and AAD by flow cytometry. L) A quantification of positive cells with or without Rictor knockdown is shown. M) A schematic diagram depicting the function of Runx2 in regulating Akt via mTORC2 complex and its effect on survival of invasive cancer cells.

Article Snippet: The lentivirus vector expressing Rictor shRNA was obtained from Addgene (plasmid #1853) (Cambridge, MA, USA) [ ].

Techniques: Knockdown, Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Control, Western Blot, Stable Transfection, shRNA, Staining, Flow Cytometry