Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 1. Tumor-associated soluble uPAR (s-uPAR) enhances HUVEC invasion, migration and angiogenesis. (a) Conditioned medium (CM) was collected from tumor cells (parental and stably expressing empty vector (EV), uPAR-cDNA (UR) and uPAR siRNA (UR-Si)). Immunoblot analyses were performed for s-uPAR and DDK using specific antibodies. (b) s-uPAR levels in CM were quantified using uPAR Quantikine Immunoassay kit. Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental control. (c) Cells were labeled (tumor cells: Qtracker-525-Green and HUVECs: Qtracker- 655-Red) and seeded into separate chambers of culture inserts. After 16 h, the culture inserts were removed and cells were allowed to migrate for a further 24 h. Images were captured at 0 and 24 h of incubation and cell migration was quantified using ImageJ software (NIH). The levels of HUVEC migration were normalized to HUVEC migration in parental cells and are represented as arbitrary units. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental control. (d) HUVEC invasion experiments were performed using ThinCertTM inserts as described in Materials and methods. The levels of HUVEC invasion was quantified and normalized to HUVEC invasion in parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (e, f) In vitro angiogenesis assay was performed as described in Materials and methods. The degree of angiogenic induction by CM was quantified by ImageJ software (NIH) for the numerical value of the product of the relative capillary length per microscopic field. Serum-free medium (SFM) and recombinant human uPAR (rh-uPAR) in SFM were used as controls (insets). Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM. uPAR antibody, uPAR-Ab; isotype control, NSp.IgG. (g) Migration assay was performed using CM. In this case, both chambers of culture inserts were seeded with HUVECs. After 16 h, the culture inserts were removed, CM was added and cells were allowed to migrate for 24 h. Invasion assay was performed as described above. uPAR-Ab. or Nsp.IgG were added to UR-CM before adding onto cells. rh-uPAR was added to SFM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Labeling, Incubation, Software, In Vitro, Angiogenesis Assay, Recombinant, Invasion Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 2. s-uPAR recruits onto HUVEC membrane. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured on CM for 24 h, labeled with anti-uPAR antibody, followed by Alexa Fluor-488-conjugated secondary antibody and were analyzed by fluorescence-activated cell sorting (FACS) for uPAR expression. Serum-free medium (SFM) and rh-uPAR were used as controls. Isotype control (Neg.). (b) HUVECs were cultured in chamber slides on CM for 24 h and fixed in 4% paraformaldehyde and 0.2% glutaraldedyde in phosphate-buffered saline for 1 h. Immunocytochemical analysis was performed as described in Materials and methods. Isotype control (Neg.; inset). Slides were mounted and photographed. (c) Equal amounts of proteins were used for the extraction of HUVEC membrane fractions and were subjected to immunoblot analysis for uPAR expression using specific antibodies. The blot was re-probed for DDK-tag expression.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Membrane, Cell Culture, Labeling, FACS, Expressing, Control, Saline, Extraction, Western Blot

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 3. s-uPAR colocalizes in lipid rafts on HUVECs. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured in chamber slides on CM for 24 h and incubated with anti-uPAR antibody followed by Alexa Fluor-488- conjugated secondary antibody at 4 1C. Cells were again labeled with Alexa Fluor-595-CTxB subunit. Slides were mounted and analyzed by confocal microscopy. Negative controls, using an isotype antibody, showed no staining (inset). Serum-free medium (SFM) and DDK-tag containing rh-uPAR were used as controls. To disrupt lipid rafts, HUVECs were pretreated with MBCD, as described in Materials and methods. (b) HUVECs lipid rafts were isolated as described in Materials and methods. Lipid raft-enriched fractions were analyzed for uPAR and DDK-tag levels using immunoblot analysis. Flotillin-1 and caveolin-1 served as controls. Protein band intensities were quantified by densitometric analysis using ImageJ software (NIH). The levels of uPAR protein were normalized to protein levels in HUVECs cultured on parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (c) Invasion and migration assays were performed as described in Figure 1d In vitro angiogenesis assay was performed as described in Figure 1. To deplete cholesterol, HUVECs were pretreated with MBCD as described in Materials and methods (c and d). Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Incubation, Labeling, Confocal Microscopy, Staining, Isolation, Western Blot, Software, Migration, In Vitro, Angiogenesis Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 4. s-uPAR induces ERK/Rac1-mediated migration and tube formation in HUVECs. Conditioned medium (CM) was collected from tumor cells, as described in Materials and methods. (a) HUVECs lysates were used to perform GST-Rac1 pull-down assay. The protein complexes were subjected to immunoblot analysis to detect active Rac1. Rac1 from total cell lysates was used as a control. (b) Total cell lysates were subjected to immunoblot analysis for phospho-ERK1/2 (pERK1/2) and total ERK1/2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. HUVECs grown on rh-uPAR were used as a control. (c) HUVECs were cultured on CM alone and/or supplemented with functional blocking anti-uPAR antibody (uPAR-Ab) or isotype control (Nsp.IgG.) or MEK inhibitor (U0126) for 24 h. Cell lysates or GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1, Rac1 pERK1/2 and ERK1/2. GAPDH served as a loading control. (d) HUVECs were transfected with dominant-negative mutant Rac1 (Dn-Rac1) for 24 h and cultured on UR-CM. Micrographs were captured for green fluorescent protein (GFP) expression (green) and phase contrast (gray) immediately after the addition of UR-CM (magnification 60). (e) HUVECs were transfected with Dn-Rac1 for 24 h, cultured on CM for another 24 h, collected and lysed. GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1. GFP and Rac1 from total cell lysates were used as controls. (f) HUVECs were transfected with Dn-Rac1 for 24 h and cultured on CM alone and/or supplemented with uPAR-Ab., or Nsp.IgG or U0126 for another 24 h. Invasion and migration assays were performed as described in Figure 1. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **Po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Pull Down Assay, Western Blot, Control, Cell Culture, Functional Assay, Blocking Assay, Transfection, Dominant Negative Mutation, Expressing

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 5. Diverse forms of tumor-associated s-uPAR in vitro and in vivo. (a) Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. CM was subjected to deglycosylation using a deglycosylation kit and analyzed by immunoblot for uPAR using specific antibodies. (b) Equal amount of proteins containing HUVEC lysates were used for extraction of cell membrane fractions and were subjected to deglycosylation, and analyzed by immunoblot for uPAR using specific antibodies. (c) In vivo angiogenic assay was performed by using the dorsal air sac model. 4910EV (EV), 4910UR (UR), 4910UR-Si (UR-Si) cells or a recombinant human uPAR (rh-uPAR) containing chamber was implanted in the dorsal cavity of mice. The micrographs for the presence of tumor-induced neovasculature (microvessels with curved thin structures and many tiny bleeding spots) and pre-existing vasculature (straight) were captured. Representative micrographs are shown. (d, e) Blood was collected from mice orthotopically xenografted with stably expressing EV, UR and UR-Si cells. Total uPAR levels were estimated using a commercial human uPAR Quantikine Immunoassay kit according to the manufacturer’s instructions. The data quantification for a set I (n ¼ 4; d) and set II (n ¼ 6; e), on day 15 and 40, respectively, after cell implantation are shown. Columns: mean; bars: s.d.; *Po0.01 vs parental control. (f) Blood serum (from mice 1–6; on day 40) was subjected to deglycosylation and analyzed by immunoblot for uPAR using specific antibodies. D2-D3, D2-D3 domain containing truncated s-uPAR; D3, D3 domain containing truncated s-uPAR; FL, full-length s-uPAR; .

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Western Blot, Extraction, Membrane, Recombinant, Stable Transfection, Expressing, Control

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 6. uPAR overexpression enhances tumor growth, vascularity and s-uPAR recruits onto endothelial cells in vivo. (a) Stably expressing EV, UR and UR-Si cells were injected intracerebrally into mice. Mice were euthanized and brains were collected and fixed as described in Materials and methods. Brain sections were stained with hematoxylin and eosin (H&E) solution, and representative micrographs are shown (upper panel). H&E-stained micrographs showing the tumor invasive front ( 20; lower panel). (b) Brain tumor areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Rockville, MD, USA). Columns: mean; bars: s.d.; n ¼ 6; *Po0.01 vs parental controls. (c) Immunohistochemical analysis of brain sections using anti-uPAR and anti-vascular endothelial growth factor (VEGF). Blood vessels in tumor sections were visualized with biotin-labeled tomato lectin. Inset: isotype control. (d, e) Fluorescence microscopy for colocalization of an endothelial cell marker (von Willebrand factor (vWF)/anti-CD31) and DDK-tag in tumor sections from mice that were implanted with 4910 EV (EV) and 4910UR (UR) cells. Inset, isotype control.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Injection, Staining, Software, Immunohistochemical staining, Labeling, Control, Fluorescence, Microscopy, Marker

Journal: Cancer research

Article Title: Elucidation of the roles of tumor integrin β1 in the extravasation stage of the metastasis cascade

doi: 10.1158/0008-5472.CAN-15-1325

Figure Lengend Snippet: (A) Schematic of our Luminex-based approach for multiplexing tail vein metastasis assays (see Methods). 4T1 cells were stably transduced with uniquely barcoded (BC) vectors expressing miR30-based shRNAs targeting one integrin subunit per cell population, and knock down was confirmed by flow cytometry (see Table 1). Uniquely barcoded 4T1 integrin knockdown cell populations were then mixed in equal numbers and injected into the tail veins of syngeneic Balb/C mice. After 17 days, Genomic DNA was isolated from the metastasis-containing lungs, and the relative amount of each barcode (i.e. the relative number cells expressing each shRNA) was quantified using streptavidin-conjugated APC (Strep-APC) and the Luminex FlexMap 3D system. (B) Luminex-based quantification of the relative metastatic burden of each 4T1 integrin knockdown cell population. Graphs show the relative signal + S.E.M. from each shRNA relative to the signal for that shRNA in the starting mixed population (n=10 mice / mix analyzed in duplicate).

Article Snippet: After 17 days, Genomic DNA was isolated from the metastasis-containing lungs, and the relative amount of each barcode (i.e. the relative number cells expressing each shRNA) was quantified using streptavidin-conjugated APC (Strep-APC) and the Luminex FlexMap 3D system. (B) Luminex-based quantification of the relative metastatic burden of each 4T1 integrin knockdown cell population.

Techniques: Luminex, Multiplexing, Stable Transfection, Transduction, Expressing, Flow Cytometry, Injection, Isolation, shRNA

Journal: American journal of physiology. Cell physiology

Article Title: Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

doi: 10.1152/ajpcell.00442.2008

Figure Lengend Snippet: Fig. 2. Nox4 expression was paralleled with SMC markers expression and translocated into the nucleus during SMC differentiation. Undif- ferentiated ES cells were plated in dishes coated with 5 g/ml of collagen IV and cultured for 4, 8, and 12 days in the DM. Total RNA and protein from undifferentiated ES cells or differ- entiating ES cells were harvested and subjected to real-time PCR analysis with a set of specific primers for Nox4 (A) and Western blot analysis with specific antibodies for SMA, smooth mus- cle myosin heavy chain (SM-MHC), and Nox4 (B). -Tubulin was included as internal control. Double immunofluorescence staining was con- ducted on undifferentiated ES cells, days 4 and 8 differentiating ES cells with antibodies against Nox4 and SMC-specific markers (SMA and SM-MHC). Isotype IgG substituted primary an- tibody as negative control during staining pro- cess (C–G). The data presented here were rep- resentative or an average of three independent experiments. *P 0.05 vs. day 0.

Article Snippet: Antibodies against Nox4 for immunostaining and flow cytometric analysis (goat, N-15, sc-21860), serum-response factor (SRF, rabbit, G-20, sc-335), and myocardin (goat, N-16, sc-21559) were purchased from Santa Cruz Biotech.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Control, Staining, Negative Control

Journal: American journal of physiology. Cell physiology

Article Title: Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

doi: 10.1152/ajpcell.00442.2008

Figure Lengend Snippet: Fig. 3. Nox4 is essential for SMC differentiation from ES cells. Undifferentiated ES cells were nucleofected by nucleofector II with different amounts of Nox4 expression plasmids pcDNA3.1-Nox4. Nucleofected cells were plated in dishes coated with 5 g/ml of collagen IV and cultured for 3–4 days in DM. Total RNA and protein were harvested and subjected to real-time PCR analysis for gene expression and Western blot analysis for protein levels (A). Appropriate amount of empty vector pcDNA3.1 were included as plasmid amount compensation. Nox4-specific small interfering RNA (siRNA) and random siRNA control were transfected into day 3 or 4 differentiating ES cells; after additional 2–3 days of culture, total RNA and protein were harvested and subjected to real-time PCR analysis and Western blot analysis (B). -Tubulin was included as internal control. *P 0.05 vs. control. The data presented here were representative or an average of six independent experiments.

Article Snippet: Antibodies against Nox4 for immunostaining and flow cytometric analysis (goat, N-15, sc-21860), serum-response factor (SRF, rabbit, G-20, sc-335), and myocardin (goat, N-16, sc-21559) were purchased from Santa Cruz Biotech.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Plasmid Preparation, Small Interfering RNA, Control, Transfection

Journal: American journal of physiology. Cell physiology

Article Title: Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

doi: 10.1152/ajpcell.00442.2008

Figure Lengend Snippet: Fig. 4. Nox4 activates SMC-specific transcrip- tion factors. A: total RNA from undifferentiated ES cells or differentiating ES cells at day 4, 8, and 12 were subjected to real-time PCR to examine serum-response factor (SRF) and myo- cardin gene expression. *P 0.05 vs. day 0. B: RNA and protein samples as Fig. 3A were subjected to real-time PCR analysis and Western blot analysis to examine gene expression and protein levels of SRF and myocardin. C: RNA and protein samples as Fig. 3B were subjected to real-time PCR analysis and Western blot analy- sis to examine gene expression and protein lev- els of SRF and myocardin. -Tubulin was in- cluded as internal control in Western blot anal- ysis. *P 0.05 vs. control. The data presented here were representative or average of six inde- pendent experiments.

Article Snippet: Antibodies against Nox4 for immunostaining and flow cytometric analysis (goat, N-15, sc-21860), serum-response factor (SRF, rabbit, G-20, sc-335), and myocardin (goat, N-16, sc-21559) were purchased from Santa Cruz Biotech.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

doi: 10.1152/ajpcell.00442.2008

Figure Lengend Snippet: Fig. 5. Nox4-derived H2O2 mediates SMC dif- ferentiation from ES cells. A: reactive oxygen species (ROS) generated during stem cell differ- entiation is derived from Noxs. Undifferentiated ES cells or differentiating ES cells at day 4, 8, and 12 were harvested and homogenized. Cell lysate were subjected to lucigenin-enhanced chemiluminescence assay to measure Noxs ac- tivity. The enzymatic source of NADPH-stimu- lated superoxide production in undifferentiated ES cells or differentiating ES cells was deter- mined using various inhibitors (see text for de- tailed information). Relative AUC unit (RAUC) was defined as the ratio of various treatments versus vehicle-treated undifferentiated ES with that of the vehicle control set as 1.0. *P 0.001 (inhibitors vs. vehicle). B: Nox4-derived H2O2 was analyzed by FACS. Undifferentiated ES cells were nucleofected with different amount of pcDNA3.1-Nox4 and pcDNA3.1 (0, 0.5, 1.0 and 2 g/106 cells). Nucleofected cells were plated in collagen IV-coated dishes and cultured for 3–4 days in the DM. Cells were treated with 3,000 U/ml of catalase or vehicle for 4 h, incu- bated with oxyBURST Green H2DCFDA, and subjected to flow cytometry analysis for the fluorescence intensity. Left: representative of flow cytometric graph; right: quantitative analy- sis for the intensive of fluorescence signal. The 5-(and-6)-chloromethyl-29,79-dichlorodihy- drofluorescein diacetate (DCF) fluorescence in- tensive of control is set as 100%. P 0.05 (catalase vs. vehicle). C: exogenous H2O2 pro- motes SMC differentiation in a dose-dependent manner. D: high amount (100 M) of H2O2 causes cell apoptosis. Day 4 or 5 differentiating ES cells were treated with 10 M of H2O2 for 5 h in the absence or presence of 3,000 U/ml catalase. Total RNA and protein were harvested and subjected to real-time PCR (E) and Western blot (F) analysis. -Tubulin was included as internal control in Western blot analysis. *P 0.05 vs. control. &P 0.05 (catalase vs. vehi- cle). The data presented here were representative or average of three (A, C–F) or five (B) indepen- dent experiments. DPI, diphenylene iodonium; L- NAME, NG-nitro-L-arginine methyl ester; TTFA, thenoyltrifluoracetone; Allo, allopurinol.

Article Snippet: Antibodies against Nox4 for immunostaining and flow cytometric analysis (goat, N-15, sc-21860), serum-response factor (SRF, rabbit, G-20, sc-335), and myocardin (goat, N-16, sc-21559) were purchased from Santa Cruz Biotech.

Techniques: Derivative Assay, Generated, Chemiluminescence Immunoassay, Control, Cell Culture, Cytometry, Real-time Polymerase Chain Reaction, Western Blot

Journal: American journal of physiology. Cell physiology

Article Title: Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

doi: 10.1152/ajpcell.00442.2008

Figure Lengend Snippet: Fig. 6. Nox4-derived H2O2 phosphorylates SRF and drives activated SRF to shift into the nucleus. A: undif- ferentiated or day 8 of differentiating ES cells were subjected to immunofluorescence staining with SRF antibody. The intracellular localization of SRF was examined by confocal microscopy. Representative im- ages were taken and presented here. B: day 4 or 5 of differentiating ES cells were treated with 10 M of H2O2 for 5 h in the absence or presence of 3,000 U/ml catalase. Cytosol and nuclear fraction were harvested and subjected to Western blot analysis to examine the cellular localization and protein levels of phosphory- lated SRF, total SRF, and myocardin. -Tubulin and histone 4 were included as internal control for cytosol and nuclear fraction, respectively. Top: representative data; bottom: means SE of densitometric analysis (n 3), *P 0.05 (H2O2 vs. vehicle); #P 0.05 (catalase vs. vehicle). Note that one lane in B was removed from the original image but all samples were run on the same gel.

Article Snippet: Antibodies against Nox4 for immunostaining and flow cytometric analysis (goat, N-15, sc-21860), serum-response factor (SRF, rabbit, G-20, sc-335), and myocardin (goat, N-16, sc-21559) were purchased from Santa Cruz Biotech.

Techniques: Derivative Assay, Staining, Confocal Microscopy, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

doi: 10.1152/ajpcell.00442.2008

Figure Lengend Snippet: Fig. 7. Transforming growth factor (TGF)-1 promotes SMC differentiation through activa- tion of TGF- receptor 1 and Nox4. Data from ELISA measurement demonstrated that protein levels of TGF-1 were increased in conditioned medium (A) and cell lysate (B) during SMC differentiation. Exogenous TGF-1 enhances SMC differentiation as well as Nox4 activation in a dose-dependent (C) and time-dependent (D) manner. *P 0.05 vs. control. Nox4 siRNA ablated the effects of TGF-1 on Nox4 activa- tion and SMC differentiation (E). *P 0.05 (TGF-1 vs. vehicle); #P 0.05 (Nox4 siRNA vs. control siRNA). Blockdown of TGF- re- ceptor 1 inhibits Nox4 activation and SMC dif- ferentiation (F). *P 0.05 vs. control. The data presented here were representative of an average of three independent experiments.

Article Snippet: Antibodies against Nox4 for immunostaining and flow cytometric analysis (goat, N-15, sc-21860), serum-response factor (SRF, rabbit, G-20, sc-335), and myocardin (goat, N-16, sc-21559) were purchased from Santa Cruz Biotech.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Control

Journal: American journal of physiology. Cell physiology

Article Title: Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.

doi: 10.1152/ajpcell.00442.2008

Figure Lengend Snippet: Fig. 8. Nox4 is required for maintenance of differentiated phenotype of ES-derived SMCs. A: stable long-term Nox4 gene modified ES-derived SMC cell lines were generated by nucleofection and G418 selection. Nox4 colony were amplified and defined as stable long-term Nox4 gene-modified ES-derived SMC cell lines in our study. B: cells (passage 5) from stable cell lines harboring pcDNA3.1 or pcDNA3.1-Nox4 were harvested and subjected to Western blot analysis. Data were representative of two independent experiments. -Tubulin serves as loading control. Cells (passage 5) from stable cell lines harboring pcDNA3.1-Nox4 were analyzed by immunofluorescence staining (C) and flow cytometric analysis (D). Isotype IgG were applied as negative control. E: functional characteristic, cell contractibility in response to muscatinic agonist carbachol (1 mM), were conducted with cells (passage 5) from stable cell lines harboring pcDNA3.1-Nox4. Photographs were taken before (0 min) and after treatment with 1 mM carbachol for 1.0, 5.0, and 15 min (clockwise). Arrows show representative contracted cells in response to carbachol. The data presented in C–E were representative images of three independent experiments.

Article Snippet: Antibodies against Nox4 for immunostaining and flow cytometric analysis (goat, N-15, sc-21860), serum-response factor (SRF, rabbit, G-20, sc-335), and myocardin (goat, N-16, sc-21559) were purchased from Santa Cruz Biotech.

Techniques: Derivative Assay, Generated, Selection, Stable Transfection, Western Blot, Control, Staining, Negative Control, Functional Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Hela, HepG2, and Huh7 cells were seeded in a 12-well plate for overnight, then left untreated or treated with human IFN-α (1,000 IU/ml) for 18 or 36 h. The untreated cells (control) were harvested together with the cells treated by IFN-α for 36 h. ISG20 expression was detected by Western blot. Hela cells transfected with plasmid F-ISG20 expressing the FLAG-tagged ISG20 were used as positive control for ISG20 Western blot (lane 2). β-actin expression was presented as loading control. (B) PHHs and HepG2 cells were cultured in 12-well-plate and treated with type I IFN-α (1,000 IU/ml), type II IFN-γ (100 ng/ml) or type III IFN-λ (100 ng/ml), or left untreated (control) for 2 days. The levels of ISG20 expression were determined by Western blot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Control, Expressing, Western Blot, Transfection, Plasmid Preparation, Positive Control, Cell Culture

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with either pHBV1.3 and F-ISG20 or empty vector, or pCMVHBV and F-ISG20 or empty vector, as indicated. Cells were harvested at day 5 post-transfection, and the levels of viral RNA and DNA were determined by Northern (top) and Southern (middle) blot hybridization, respectively. For RNA analysis, each lane was loaded with 10 μg of total RNA and probed with a genome-length, plus-strand-specific HBV riboprobe. Ribosomal RNAs (28S and 18S) are presented as loading controls. The positions of HBV pgRNA (3.5kb) and subgenomic surface RNAs (2.4kb and 2.1kb) are indicated. For DNA analysis, HBV core DNA was probed with genome-length, minus-strand-specific HBV riboprobe. The positions of relaxed circular (RC) and single-stranded (SS) DNAs are indicated. The relative pgRNA, sRNA or total DNA replicative intermediate level in each sample is expressed as the percentage of RNA or DNA of the cells transfected with empty vector. ISG20 overexpression was confirmed by Western blot using monoclonal antibodies against FLAG-tag. β-actin expression was presented as protein loading control (bottom panels). (B) The same experiment was done in Huh7 cells with pHBV1.3 as HBV expression vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Northern Blot, Hybridization, Over Expression, Western Blot, Bioprocessing, FLAG-tag, Expressing, Control

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Cell Culture, Transfection, Control, Plasmid Preparation, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepDES19 cells were transfected with 100 nM of control siRNA (Lane 1–3) or ISG20 siRNA (siISG20) (lane 4–6) twice with a 24 h interval after tet being withdrawn. Culture medium was replaced 12 h after the 2nd siRNA transfection, and cells were either left untreated as controls (lane 1 &4) or treated with 100 IU/ml (lanes 2 & 5) or 1,000 IU/ml (lanes 3 & 6) of IFN-α. Cells were harvested 5 days after 2nd transfection. Viral total RNA (top panel), encapsidated pgRNA (upper middle panel), and core DNA (lower middle panel) were subjected to Northern and Southern analyses, respectively. ISG20 protein expression was revealed by Western blot, and β-actin served as loading control (bottom panels). The relative levels of viral nucleic acids and ISG20 expression in the siISG20 transfected or IFN-α treated samples (lanes 2–6) are expressed as the percentage of the control sample (lane 1). The data presented here are representative of two independent experiments.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2-NTCP12 cells stably transduced by control lentiviral shRNA (shcontrol) or ISG20 lentiviral shRNA (shISG20) were spinoculated with HBV at 100 vge/cell. 16 h later, the infected cells were mock treated or treated with 1,000 IU/ml of IFN-α for 6 days, and the cells were subjected to the following analyses: (A) The expression of ISG20 was analyzed by Western blot. (B) HBV infectivity was assessed by HBcAg immunofluorescence, and the percentage of HBcAg-positive cells were calculated from multiple microscopic field of view (mean±SD, n = 5). Nuclei were stained with DAPI. (C) HBV total RNA were quantified by qPCR and the relative expression levels to β-actin mRNA were plotted as fold change to control samples (HBV infected shcontrol cells without IFN-α treatment) (mean±SD, n = 3).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Stable Transfection, Control, shRNA, Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were transfected with pHBV1.3 and equal amount of control vector (lanes 1 & 2) or F-ISG20 (lanes 3 & 4) or F-ISG20 D94G (lanes 5 & 6). Cells were harvested 5 days post-transfection and levels of HBV RNA (1st panel from the top) and encapsidated pgRNA (4th panel from the top) were determined by Northern blot hybridization. The assembled HBV capsid was revealed by native capsid gel EIA assay (3rd panel from the top) and the viral DNA in capsid was detected in situ by hybridization (5th panel from the top). HBV core DNA replicative intermediates were extracted and analyzed by Southern blot (6th panel from the top). Expression of FLAG-tagged ISG20 proteins was revealed by Western blot and β-actin served as loading control (bottom two panels). Results from duplicate experiments are presented.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Hybridization, Enzyme Immunoassay, In Situ, Southern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were co-transfected with plasmid pCMVHBVΔCΔP and either control vector (lane 1) or FLAG-Pol (lanes 2&3), or pCMVHBV with either control vector (lane 4) or F-ISG20 D94G (lanes 5&6). Cells were harvested 4 days post-transfection. Input HBV RNA was determined by Northern blot (top panels). Input FLAG-Pol and F-ISG20 D94G proteins were determined by Western blot using FLAG Ab (top panels). Cell lysates were immunoprecipitated with beads coated with FLAG Ab, the immunoprecipitated Pol and ISG20 D94G were revealed by Western blot using FLAG Ab (lanes 3&6, bottom panel), and the bound RNA was extracted by Trizol and analyzed by Northern blot (lanes 3&6, bottom panel). HA Ab pull-down served as negative controls (lanes 2 & 5, bottom panel). See for more experimental details.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Control, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells in 6-well-plate were co-transfected with 1 μg of pCMVHBVΔCΔP and 5 μg control vector (lane 1) or 1 μg of FLAG-Pol in the absence of HA-ISG20 D94G (lane 2) or increased amount of HA-ISG20 D94G (1 μg, 2 μg, 4 μg; lanes 3–5). The total amount of transfected DNA was kept constant (6 μg/well) by adding control vector plasmid (lanes 2–4). 5 days later, total cellular HBV RNA and protein (FLAG-Pol and HA-ISG20 D94G ) were determined by Northern and Western blot, respectively, as input controls (top panels). Immunoprecipitation was performed by using antibodies against HA or FLAG epitopes, followed by Northern blot analysis of HBV RNA (bottom panels).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Control, Northern Blot, Hybridization, Construct, Luciferase, Expressing, Activity Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic stem-loop structure of HBV ε RNA. Ribonucleotide sequences (nt 1847–1991, genotype D, subtype ayw) are presented with base paring indicated by dotted line. (B) Verification of the purified recombinant 6×His-tagged ISG20 by SDS-PAGE Coomassie staining. (C) EMSA assay of ISG20-ε binding. The indicated amount of ISG20 proteins were incubated with 100 ng 32 P-end-labeled ε RNA in binding buffer to form nucleoprotein complexes. Monoclonal anti-His antibody was used for supershifting of the His-ISG20/ HBV ε complex. Excessive amount of cold unlabeled HBV ε RNA (10×, 20×, 40×) were used to compete with the binding of ISG20 to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native PAGE and the shifted bands were detected by autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Purification, Recombinant, SDS Page, Staining, Binding Assay, Incubation, Labeling, Clear Native PAGE, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustrations of the wildtype HBV ε RNA and mutants. The substructural domains of ε, including the lower stem, bulge, upper stem, and apical loop, are marked on the full-length form. Shorter versions of ε include the upper stem loop (US+L), lower stem with wildtype or mutant bulge sequence as loop (LS+B, LS+Bm), and LS+B with bottom 4 base-pairs removed from the lower stem (LSΔ4+B). These RNA fragments were chemically synthesized and 5’ end radiolabeled for ISG20 EMSA. (B) EMSA of ISG20 binding with full-length (FL) ε, US+L, and LS+B. (C) EMSA of ISG20 binding with LS+B, LS+Bm, and LS+B. (D) HepG2 cells were transfected with plasmid pMS transcribing the 2.1kb HBV RNA, or pMSΔ4bp transcribing the 2.1kb HBV with 4 nucleotides removed from the bottom right arm of the lower stem of ε, in the absence or presence of F-ISG20. HBV RNA and FLAG-tagged ISG20 were analyzed by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Mutagenesis, Sequencing, Synthesized, Binding Assay, Transfection, Plasmid Preparation, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Labeling, Mutagenesis, Purification, SDS Page, Staining, Recombinant, Binding Assay, Transfection, Control, Plasmid Preparation, Construct, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: 0.1 µg of 5’-radiolabeled synthetic RNA substrates, specifically (A) the intact ε, upper stem-loop region (US+L), and lower stem with bulge serving as loop (LS+B); and (B) the intact ε, single-stranded left arm portion of ε, and 30-mer poly(rA), were incubated with the indicated amount of RNase A or purified His-ISG20 in nuclease reaction buffer for 15 min, then the reactions were terminated and the mixtures were fractionated through 10% TBE-Urea denaturing polyacrylamide gel, and the dried gel was subjected to autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Incubation, Purification, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: The major viral intermediates and products at each major HBV replication steps are illustrated. cccDNA (or other HBV transcription template)-derived 3.5kb RNA (including precore mRNA and pgRNA) and other shorter subgenomic RNA species (2.4/2.1kb surface mRNA and 0.7kb X mRNA) are aligned to show the location of ε on different RNA species. The black circle dots indicate the 5’ cap of mRNA, the zigzag lines represent the polyA tails. ISG20 is shown as a rectangle box and its targeting sites on HBV RNA are indicated by arrowheads. As a consequence of ISG20-mediated HBV RNA degradation, the illustrations and labels of viral proteins/antigens, pgRNA encapsidation and DNA replication are shown in gray color schemes. The solid gray triangles indicate capsid proteins, viral polymerase is shown in an oval shape before ε binding and then a gray circle dot in the nucleocapsids after pgRNA encapsidation.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Derivative Assay, Binding Assay

Journal: Scientific Reports

Article Title: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

doi: 10.1038/srep37227

Figure Lengend Snippet: BEAS-2B cells were exposed to arsenic (0 to 10 μM) for 12 h. Arsenic induced generation of the ROS radicals O 2 − and H 2 O 2 were identified by DHE ( A ) and DCFDA ( B ) staining, respectively. Upper panels show representative images obtained by fluorescence microscopy and the graphs (lower panels) demonstrate fluorescent intensity determined by flow cytometry. ( C ) Generation of • OH as determined by electron spin resonance. The generation of a 1:2:2:1 quartet ESR signal is shown. ( D ) NOX activity, measured after 6, 12 and 24 h arsenic exposure, utilized the lucigenin chemiluminescence assay. Activity increased in a time- and dose-dependent manner. ( E ) Western blot demonstrates an apparent increase in protein levels of the NOX subunit, p47 phox with arsenic exposure. ( F–H ) BEAS-2B cells exposed to increasing concentrations (0–10 μm) of arsenic for 24 h. ( F ) The relative miR-21 level was determined by Taqman real-time PCR. ( G ) Immunoblot analysis of PDCD4 protein levels after acute arsenic treatment. Arsenic induced increases in miR-21 and decreases in PDCD4 levels. ( H ) Representative images of fluorescence immunostaining demonstrate decreased PDCD4 expression with arsenic treatment. Dapi: blue-nuclear; Phalloid: red-cytoplasmic actin; PDCD4, green ( I ) BEAS-2B cells were transfected with the renilla reporter construct (pGL3-PDCD4_3′-UTR), miR-21 inhibitor (100 nM), negative control (100 nM), or pGL3-promoters and treated with 10 μM arsenic for 6 h. Cellular lysates were subjected to a luciferase reporter analysis as described in Materials and Methods and results are expressed as a relative activity (relative luminescence units, (RLU) normalized to the luciferase activity in the vector control cells without treatment. Arsenic increased the binding of miR-21 to the 3′-UTR of PDCD4. Exogenous addition of ROS inhibitors catalase or NAC inhibited the acute arsenic-induced ( J ) miR-21 increase and ( K ) PDCD4 suppression. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference from control cells with p < 0.05.

Article Snippet: Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry, Electron Paramagnetic Resonance, Activity Assay, Chemiluminescence Immunoassay, Western Blot, Real-time Polymerase Chain Reaction, Immunostaining, Expressing, Transfection, Construct, Negative Control, Luciferase, Plasmid Preparation, Control, Binding Assay

Journal: Scientific Reports

Article Title: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

doi: 10.1038/srep37227

Figure Lengend Snippet: BEAS-2B cells were maintained in a medium containing various concentrations of arsenic (0.1, 0.25 and 0.5 μM) for 6 months. ( A,B ) Cells were cultured in 0.35% soft agar for 5 weeks and number of colonies in the entire dish counted. ( A ) Representative images of control (left panel) and arsenic-treated (right panel) colonies. ( B ) Colony number increased in a dose-dependent manner. ( C ) The relative miR-21 level, determined by Taqman real-time PCR, increased in a time- and dose-dependent manner. ( D ) Total cell lysates were prepared for western blot analysis after 2, 4 and 6 months exposure to arsenic using specific antibodies against PDCD4, p47phox, pSTAT3 and STAT3. Apparent protein levels for PDCD4 decreased and P47phox and pSTAT3 increased in a time- and dose-dependent manner. ( E ) Representative images of fluorescence immunostaining for PDCD4 and pSTAT3 after 2, 4 and 6 months exposure to arsenic, and confirm results from western blot analysis. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.

Article Snippet: Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Cell Culture, Control, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Immunostaining

Journal: Scientific Reports

Article Title: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

doi: 10.1038/srep37227

Figure Lengend Snippet: ( A–D ) BEAS-2B cells with stable overexpression of catalase or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. ( A ) Catalase overexpression was verified by western blot analysis. ( B ) The relative miR-21 level was determined by Taqman real-time PCR and decreased with catalase overexpression. ( C ) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased with catalase overexpression. ( D ) Anchorage-independent colony growth, assessed as previously described, decreased with catalase overexpression. ( E–H ) p47phox expression was stably knocked down in BEAS-2B cells and exposed to arsenic (0 or 0.5 μM) for 6 months. (E ) P47phox knockdown was confirmed by western blot analysis. (F ) Relative miR-21 level, determined by Taqman real-time PCR was decreased. ( G ) Cell lysates were prepared to evaluate PDCD4 protein levels by western blot analysis. An apparent restoration in PDCD4 levels was observed. ( H ) Anchorage-independent colony growth, assessed as previously described, was also decreased compared with respective vector controls. ( I–K ) BEAS-2B cells with stable knockdown of STAT3 or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. ( I ) The Relative miR-21 level, determined by Taqman real-time PCR, decreased compared with respective vector controls. ( J ) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased. ( K ) Anchorage-independent colony growth, assessed as previously described, decreased compared with respective vector controls. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.

Article Snippet: Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Over Expression, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Knockdown, Control

Journal: Scientific Reports

Article Title: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

doi: 10.1038/srep37227

Figure Lengend Snippet: ( A–C ) BEAS-2B cells with stable knockdown of miR-21 or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. ( A ) The relative miR-21 level, determined by Taqman real-time PCR, decreased. ( B ) Cell lysates were prepared to evaluate PDCD4 protein levels by western blot analysis. PDCD4 levels showed an apparent increase. ( C ) Anchorage-independent colony growth was assessed as previously described. ( D–E ) BEAS-2B cells stably overexpressing PDCD4 or their corresponding vehicle vector were exposed with arsenic (0 or 0.5 μM) for 6 months. ( D ) Cell lysates were prepared for western blot analysis and confirm expression of PDCD4 protein. ( E ) Anchorage-independent colony growth, assessed as previously described, demonstrate decreased colony formation when compared with respective vector controls. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.

Article Snippet: Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Stable Transfection, Expressing, Control

Journal: Scientific Reports

Article Title: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

doi: 10.1038/srep37227

Figure Lengend Snippet: BEAS-2B cells exposed to indicated concentration of arsenic for 6 months were injected sc into nude mice. After 4 weeks, mice were euthanized using CO 2 and tumor was isolated for further examination. ( A ) Representative images of excised tumors and ( B ) graphic representation of tumor volume demonstrate increased growth with arsenic treatment. ( C ) The relative miR-21 level, determined by Taqman real-time PCR, increased with arsenic treatment. ( D ) Immunohistochemical analysis of PDCD4 protein showed decreased expression with increasing arsenic concentration. ( E ) Protein levels for PDCD4 and pSTAT3, analyzed by western blot, showed an apparent decrease in PDCD4 and apparent increase in pSTAT3 expressions with arsenic exposure. ( F ) Increased STAT3 phosphorylation with arsenic exposure was verified by immunohistochemistry. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.

Article Snippet: Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Concentration Assay, Injection, Isolation, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Expressing, Western Blot, Phospho-proteomics, Immunohistochemistry, Control

Journal: Scientific Reports

Article Title: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

doi: 10.1038/srep37227

Figure Lengend Snippet: Multiple sets of BEAS-2B cells were exposed to arsenic (0 or 0.5 μM) for 6 months: those stably silenced with either miR-21 or STAT3 shRNA, or transfected and overexpressing either PDCD4 or catalase, or the respective vector controls. Cells from different treatments were injected into the flanks of 6-week old athymic nude mice (2 × 10 6 cells per mouse), and tumor volume was measured after 30 days. ( A ) Shown are the representative images of excised tumors and ( B ) graphic representation of tumor volume for each treatment group. ( C ) The relative miR-21 level was determined by Taqman real-time PCR. ( D ) PDCD4 protein expression was detected by immunohistochemistry. Results indicate that arsenic-induced tumorigenicity is associated with an increased levels of miR-21 and STAT3 while PDCD4 is decreased. Data presented in the bar graphs are the mean ± SD of three independent experiments. * # Indicates a statistically significant difference from respective control cells with p < 0.05.

Article Snippet: Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Stable Transfection, shRNA, Transfection, Plasmid Preparation, Injection, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry, Control

Journal: Scientific Reports

Article Title: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

doi: 10.1038/srep37227

Figure Lengend Snippet: BEAS-2B cells were maintained in a medium containing arsenic (0.5 μM) for 6 months, and then cells were cultured in 0.35% soft agar for 5 weeks. ( A ) The arsenic-transformed cells (AsT) from anchorage-independent colonies were selected and maintained in DMEM. Passage-matched cells without arsenic treatment were used as the control. ( B ) The relative miR-21 level, determined by Taqman real-time PCR, was increased. ( C ) Total cell lysates were prepared for western blot analysis; apparent PDCD4 protein levels decreased. ( D–E ) Clonogenic assays were used to determine tumor cell proliferation. Cells (300) from indicated treatments were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet. Colony numbers in the entire dish were counted. ( D ) Images of representative plates for each treatment showing clonogenic activity, and ( E ) graphic representation of colony counts. Cells from indicated treatments were injected into the flanks of 6-week old athymic nude mice (2 × 10 6 cells per mouse) and tumor volume was measured after 30 days. ( F ) Images of representative tumors excised from mice from each treatment group and ( G ) the associated tumor volumes were obtained. Knockdown of miR-21 or STAT3 and overexpression of PDCD4 inhibited tumor growth. ( H ) The relative miR-21 level for each treatment, determined by Taqman real-time PCR, was decreased relative to levels obtained from vector control tissues. ( I ) Representative images immunohistochemical staining show increased PDCD4 protein expression and ( J ) STAT3 phosphorylation. Data presented in the bar graphs are the mean ± SD of three independent experiments. * # Indicates a statistically significant difference from respective control cells with p < 0.05.

Article Snippet: Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Cell Culture, Transformation Assay, Control, Real-time Polymerase Chain Reaction, Western Blot, Staining, Activity Assay, Injection, Knockdown, Over Expression, Plasmid Preparation, Immunohistochemical staining, Expressing, Phospho-proteomics