Journal: International journal of oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells.

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: Figure 3. Effect of pUC and radiation alone and in combination on PKC and integrin levels in U251 and 5310 non-GICs and GICs. A, Western blot analysis of the cell lysates of U251 and 5310 non-GICs and GICs showing the protein expression levels of PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1, integrin α2, and integrin α5 after treatment with pUC and radiation alone or in combination. B, Total protein (300 µg) was collected from pSV, pUC, pSV + 10 Gy and pUC + 10 Gy samples of U251 and 5310 non-GICs and GICs and immunoprecipitated with integrin β1 antibody (2 µg) and protein A plus G agarose beads (20 µg) overnight at 4˚C. The precipitates were washed with lysis buffer and the integrin β1 pulled down protein was immunoblotted for pPKC θ/δ and PKC ζ. C, Co-localization of PKC ζ and integrin β1 was carried out with pSV and pUC with and without 10 Gy (24 h for non-GICs and 48 h for GICs). The cells were allowed to migrate on 4-well chamber slides for about 16 h after growing them in Ibidi culture inserts for 24 h after treatments. The cells were fixed, stained with primary antibody overnight at 4˚C, counter-stained with species-specific Alexa Fluor-conjugated secondary antibodies, nuclear stained with DAPI, mounted, and imaged under a confocal microscope. Arrows indicating the co-localization of PKC ζ and integrin β1.

Article Snippet: U251 and 5310 non-GICs or GICs were transfected with scrambled vector (pSV) or a bicistronic construct of uPAR and cathepsin B (pCU) or siRNA against integrin β1 (SCBT, Santa Cruz, CA).

Techniques: Western Blot, Expressing, Immunoprecipitation, Lysis, Staining, Microscopy

Journal: International journal of oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells.

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: Figure 4. PKCs regulate the ECM-integrin interaction signal and vice versa in U251 and 5310 non-GICs and GICs. A, SDS-PAGE was conducted with the cell lysates of control, DMSO-treated, rottlerin (200 µM) and integrin β1 siRNA-treated U251 and 5310 non-GICs and GICs grown in non-coated and collagen-coated culture dishes. The cell lysates were immunoblotted with PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1 and integrin α2. B, SDS-PAGE was conducted with the cell lysates of control, DMSO-treated, rottlerin (200 µM) and integrin β1 siRNA-treated U251 and 5310 non-GICs and GICs grown in non-coated and fibronectin- coated culture dishes. The cell lysates were immunoblotted for PKC θ, PKC δ, pPKC θ/δ, PKC ζ, integrin β1 and integrin α5.

Article Snippet: U251 and 5310 non-GICs or GICs were transfected with scrambled vector (pSV) or a bicistronic construct of uPAR and cathepsin B (pCU) or siRNA against integrin β1 (SCBT, Santa Cruz, CA).

Techniques: SDS Page, Control

Journal: International journal of oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells.

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: Figure 5. Depletion of uPAR and cathepsin B inhibits PKC/integrin signaling to FAK and the cytoskeletal molecules. A, Cell lysates from U251 and 5310 non-GICs and GICs were extracted using RIPA buffer after treatment with pUC and radiation alone or in combination, and western blot analysis was performed to determine the expression levels of FAK, pFAK (Tyr397), pFAK (Tyr925), vinculin, and α-actinin using their specific antibodies. GAPDH was used as a loading control. B, The total cell lysates of U251 and 5310 non-GICs and GICs as described above were immunoprecipitated with FAK antibody (2 µg). The protein precipitates were washed with lysis buffer and incubated with 1X loading dye at 90˚C for 10 min. SDS-PAGE was conducted and western blotting was performed with vinculin and α-actinin antibodies as described in Materials and methods.

Article Snippet: U251 and 5310 non-GICs or GICs were transfected with scrambled vector (pSV) or a bicistronic construct of uPAR and cathepsin B (pCU) or siRNA against integrin β1 (SCBT, Santa Cruz, CA).

Techniques: Western Blot, Expressing, Control, Immunoprecipitation, Lysis, Incubation, SDS Page

Journal: International journal of oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells.

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: Figure 7. Effect of pUC and radiation treatment on pre-established intracranial tumors. U251 non-GICs and GICs were implanted intracranially into nude mice and treated with 450 µg of pUC seven days after implantation. Radiation treatment was given in two doses (5 Gy at days 8 and 10). When chronic symptoms were observed, the mice were sacrificed, and their brains were collected and embedded in paraffin. Paraffin-embedded sections were deparaffinized, antigen retrieved, and co-localization studies were carried out. The brain sections were incubated overnight with primary antibodies in 10% goat serum at 4˚C in a humidified chamber, counterstained with Alexa Fluor-conjugated secondary antibodies, and incubated with DAPI for nuclear staining before mounting. A, Co-localization of integrin β1 (green), pPKC θ/δ (red) and DAPI (blue) in the paraffin sections of the nude mice established with U251 non-GICs and GICs and treated with shRNA and radiation alone or in combination. B, Interaction between integrin β1 (green) and PKC ζ (red) in the various in vivo combination treatments. DAPI (blue) was used for nuclear staining. C, In vivo brain sections of immunocompromised mice implanted with U251 non-GICs and GICs were labeled with FAK (green) and α-actinin (red) and processed for immunofluorescence. The sections were incubated with DAPI for a brief period of time for nuclear staining. D, Mock, pUC- treated, irradiated, and pUC + irradiated brain sections of the nude mice pre-established with U251 non-GICs and GICs were immunoprocessed and labeled with FAK (green), vinculin (red) and DAPI (blue) in order to observe the co-localization of FAK and vinculin in in vivo samples.

Article Snippet: U251 and 5310 non-GICs or GICs were transfected with scrambled vector (pSV) or a bicistronic construct of uPAR and cathepsin B (pCU) or siRNA against integrin β1 (SCBT, Santa Cruz, CA).

Techniques: Incubation, Staining, shRNA, In Vivo, Labeling, Immunofluorescence, Irradiation

Journal: International heart journal

Article Title: Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1.

doi: 10.1536/ihj.16-572

Figure Lengend Snippet: Figure 2. Reduction of TGFβ1 expression by downregulation of RyR2in cardiomyocytes under mechani- cal stretch. A: Measurement of RyR2 mRNA level in cultured cardiomyocytes by Real-Time PCR. B: Expres- sion of RyR2 protein level in cardiomyocytes with Western Blotting. C: TGFβ1 in supernates of cultured cardiomyocytes was detected with ELISA analysis. D: Expression of TGFβ1 protein level in cardiomyocytes with Western Blotting. E: Measurement of tgfb1 mRNA level in cultured cardiomyocytes using Real-Time PCR. Values are expressed as mean ± SEM obtained from 3 independent experiments. shRyR2 indicates RyR2 knockdown lentiviral particle-infected cardiomyocytes; MS, cardiomyocytes under mechanical stretch; MS + ryr2 KD, RyR2 knockdown cardiomyocytes under mechanical stretch. *P < 0.05 versus control.

Article Snippet: ELISA assay: Supernates of cultured cardiomyocytes were collected and examined by Rat Transforming Growth Factor β1 ELISA Kit (CUSABIO BIOTECH, China) following the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Infection, Control

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A and B ) HEK293T cells and MDA-MB-231 cells were serum starved and treated with TGFB1 (5 ng/mL) for the indicated durations, and whole-cell extracts (WCEs) were collected for IP with anti-SMAD3 antibody, followed by immunoblot (IB) analysis. ( C ) HEK293T cells were transfected with WT HA - SMAD3 or mutant plasmids as indicated and treated with TGFB1 (5 ng/mL). WCEs were then collected for IP with anti-HA antibody, followed by IB analysis. ( D ) SMAD3 K53/K333 site aa in different species. ( E ) HEK293T cells were transfected with WT HA - SMAD3 or K53/333R-mutant plasmids and then treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-HA antibody, followed by IB analysis. ( F ) ddH 2 O (10 μL) containing different peptides (0.1–0.75 μg) was added onto the PVDF membranes, followed by IB analysis using a K53-specific trimethylation antibody (anti–SMAD3 K53me3) and a K333-specific trimethylation antibody (anti–SMAD3 K333me3). ( G ) MDA-MB-231 cells were serum starved and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis with SMAD3 K53/K333 trimethylation–specific antibodies. ( H ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Western Blot, Transfection, Mutagenesis, Stable Transfection

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). Quantitative RT-PCR analysis of TGFB / SMAD3 signaling pathway downstream genes, including CTGF , PAI1 , PDGFB , and SMAD7 , in the indicated cells with or without TGFB1 (5 ng/mL) treatment. ( B ) Quantitative analysis of Transwell assay in the indicated cells. ( C ) IF and ( D ) IB analysis of EMT markers in the indicated cells. Scale bar: 50 um. ( E and F ) Representative lung image ( E ) and H&E-stained lung sections ( F ). Scale bars: 5 mm. ( G and H ) Scatter plots showing lung metastatic nodules ( G ) and lung weights ( H ). All immunoblots were performed 3 times, independently, with similar results. Data indicate the mean ± SD. ** P < 0.05, by 2-tailed Student’s t test ( A , B , G , and H ).

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Stable Transfection, Transfection, Quantitative RT-PCR, Transwell Assay, Staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) WCEs of MDA-MB-231 and MCF-7 cells were collected and subjected to co-IP and IB assays. ( B ) HEK293T and MCF-7 cells were serum starved and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( C ) MDA-MB-231 and MCF-7 cells were treated with TGFB1 (5 ng/mL) and the EZH2 inhibitors GSK126 or GSK503, and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( D ) HEK293T cells were transfected with WT HA-SMAD3 or mutant plasmids and a Flag-EZH2 plasmid as indicated/WCEs were then collected for IP with anti-HA antibody, followed by IB analysis. ( E ) Immunoprecipitated EZH2 from HEK293 cells was incubated with SAM along with SMAD3 protein for in vitro methylation of SMAD3 . The methylated proteins were separated by SDS-PAGE, and SMAD3 methylation was analyzed by IB using anti–SMAD3 K53/K333 trimethylation–specific antibodies. ( F ) MDA-MB-231 cells silenced with control (shNC) or EZH2 shRNA (nos. 1 and 2) were treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( G ) HEK293T cells were transfected with vector, EZH2 WT , or EZH2 H689A and then treated with TGFB1 (5 ng/mL). WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( H ) HEK293T cells were transfected with vector, EZH2 WT , or EZH2 Y641H , and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Incubation, In Vitro, Methylation, SDS Page, Control, shRNA, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) Quantitative analysis of Transwell assay in the indicated MDA-MB-231 cells treated with TGFB1 (5 ng/mL). ( B ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and silenced with control or EZH2 shRNA (nos. 1 and 2). WCEs were collected for IB analysis. ( C ) WT Flag - EZH2 or a Flag - EZH2 H689A plasmid was transfected into MDA-MB-231 SMAD3–/– cells ectopically expressing WT SMAD3 or SMAD3 K53/333R, and WCEs were collected for IB analysis. ( D and E ) A Transwell cell invasion assay was performed using MDA-MB-231 SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and transfected with a vector, EZH2 WT , or EZH2 H689A . Representative images ( D ) and quantitative analysis ( E ). Original magnification, ×200. ( F – H ). Representative lung image ( F ),H&E-stained lung sections ( G ), and scatter plot showing lung weights ( H ). Scale bars: 5 mm. All immunoblotting was performed 3 times, independently, with similar results. Data indicate the mean ± SD. ** P < 0.05, by 2-tailed Student’s t test ( A , E , and H ).

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Transwell Assay, Stable Transfection, Transfection, Control, shRNA, Plasmid Preparation, Expressing, Invasion Assay, Staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) Membrane and cytosolic fractions from MDA-MB-231 SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids were collected and subjected to IB analysis. ( B ) Membrane fractions from MDA-MB-231 SMAD3–/– cells stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL) were collected and subjected to IB analysis. ( C ) MDA-MB-231 SMAD3–/– cells were stably transfected with WT SMAD3 or SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL). IF images show the cellular localization of SMAD3 . Scale bar: 10 μm. ( D ) Membrane and cytosolic fractions from MDA-MB-231 cells silenced with control or EZH2 shRNA (no. 1) were collected and subjected to IB analysis. ( E ) HEK293T cells were transfected with WT HA- SMAD3 or HA- SMAD3 K53/333R plasmids and treated with TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-HA antibody, followed by IB analysis. ( F ) HEK293T cells were silenced with control or EZH2 shRNA (nos. 1 and 2), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( G ) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2 WT or EZH2 H689A , followed by IB analysis. ( H ) Co-IP of endogenous SMAD3 from MDA-MB-231 cells transfected with EZH2 WT or EHZ2 Y641H , followed by IB analysis. All immunoblotting was performed 3 times, independently, with similar results.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Membrane, Stable Transfection, Transfection, Control, shRNA, Co-Immunoprecipitation Assay, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: EZH2 -triggered methylation of SMAD3 promotes its activation and tumor metastasis

doi: 10.1172/JCI152394

Figure Lengend Snippet: ( A ) The aa sequence of different TAT peptides. ( B and C ) HEK293T cells were treated with different TAT peptides (Pep-1, Pep-2) and TGFB1 (5 ng/mL), and WCEs were collected for IP with anti-SMAD3 antibody, followed by IB analysis. ( D ) MDA-MB-231 were silenced with TAT peptides and treated with TGFB1 (5 ng/mL), and WCEs were collected for IB analysis. ( E ) Quantitative analysis of Transwell cell migration and invasion assays using MDA-MB-231 cells treated with different TAT peptides. ( F and G ) Representative lung image ( F ) and H&E-stained lung sections ( G ). Scale bars: 10 mm. ( H and I ) Scatter plots show the number of lung metastatic nodes ( I ) and lung weights ( H ). All immunoblotting was performed 3 times, independently, with similar results. Data indicate the mean ± SD. ** P < 0.05, by 2-tailed Student’s t test.

Article Snippet: The following other small-molecule materials were used: GSK126 (T2079, Topscience); GSK503 (T1775,Topscience); TGFB1 (HY-P7118, MedChemExpress); and DiO (C1038, Beyotime).

Techniques: Sequencing, Migration, Staining, Western Blot