Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 1 Autophagy stimulation by ABT737. (a, b) Levels of autophagy induced by ABT737 in U2OS cells stably expressing GFP- LC3 were measured in time-dependent manner as described in Materials and methods section. Representative microphotographs of cells (size bar 50 mm) cultured in the presence of ABT737 (1 mM) are shown in (a) and the number of GFP-LC3 puncta per cell (means±s.e.m., n ¼ 3 separate experiments) are quantified in (b). (c, d) Effect of Bafilomycin A1 (Baf A1) on ABT737-induced autophagic vacuolization. GFP-LC3-expressing HCT116 cells were treated with Baf A1 (1 nM) for further 24 h. Panel c depicts the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) (means±s.e.m., n ¼ 3). (d) Representative immunoblots (n ¼ 3) of HCT116 cells treated with ABT737 and BafA1 for the indicated time. (e) Involvement of Vps34 in ABT737- induced autophagy. HeLa cells were transfected with a control siRNA or a siRNA specific for Vps34. After 48 h, the cells were treated for 12 h with ABT737 (1 mM). Each experiment has been repeated at least three times, yielding similar results. (d, e) Actin levels were assessed to ensure equal loading of lanes.

Article Snippet: The profile of phospho-kinase proteins was performed on HeLa cells treated with ABT737 (1 mM) and HA14-1 (20mM) for 2, 6 and 12 h using a Proteome Profiler array–PhosphoKinase array kit (ARY003) according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA).

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

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 2 Implication of AMPK, mTOR and IKK in the autophagy induction by ABT737. Immunoblot detection of the levels of p62, LC3I/II, p53 and Sirtuin1, as well as the phosphorylation status of AMPKa (Y172), ACCa (S79), mTOR (S2448), p70S6K (Y421/S424), IkBa (S32/36), IKKa/b (S176 or S180, respectively) and NEMO (S376) in different human cell lines (a–c) stimulated with ABT737 (1 mM) for the indicated time. Glyceraldehyde-3-phosphate dehydrogenase levels were monitored to ensure equal loading. Data are representative of three independent experiments.

Article Snippet: The profile of phospho-kinase proteins was performed on HeLa cells treated with ABT737 (1 mM) and HA14-1 (20mM) for 2, 6 and 12 h using a Proteome Profiler array–PhosphoKinase array kit (ARY003) according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Phospho-proteomics

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 3 Involvement of AMPKa and mTOR in ABT737-induced autophagy. (a) Dose-dependent induction of autophagy by ABT737. Representative immunoblots of LC3, p62 and phosphorylated AMPKa (Y172) or ACCa (S79) were performed on lysates from HCT116 cells that were treated with ABT737 at the indicated concentrations for 12 h. (b) Immunoblot detection of LC3 lipidation and p62 degradation in HCT116 depleted from AMPKa. At 48 h after the transfection with the indicated siRNAs, the cells were treated with ABT737 (1 mM) for 12 h. The efficiency of siRNA-mediated downregulation of the protein is also shown. Actin levels were assessed to ensure equal loading of lanes. Data in (a) and (b) are representative of three independent experiments. (c) Implication of mTOR in ABT737-induced autophagy. HeLa cells were simultaneously transfected with GFP-LC3 and equivalent amounts of plasmids coding for control vector, Rheb D60K (dominant-negative construct) or Rheb Q64L (constitutively active construct). After 24 h, the cells were treated with ABT737 (1 mM) for 12 h. The percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is shown (means±s.e.m., three independent experiments).

Article Snippet: The profile of phospho-kinase proteins was performed on HeLa cells treated with ABT737 (1 mM) and HA14-1 (20mM) for 2, 6 and 12 h using a Proteome Profiler array–PhosphoKinase array kit (ARY003) according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Dominant Negative Mutation, Construct

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 4 Involvement of IKK in ABT737-induced autophagy. (a, b) Pharmacological inhibition of IKK pathway reduces autophagy increased by ABT737. (a) LC3 maturation and p62 degradation in HCT116 treated with ABT737 (1 mM) in the presence of Bay11-7082 (1 mM), GA (geldanamycin; 2 mM), 17-AAG (17-allylamino-geldanamycin; 2 mM) for 12 h. Actin levels were assessed to ensure equal loading of lanes. Data are representative of three independent experiments. (b) GFP-LC3-expressing HeLa, U2OS, HCT116 cells were treated under the same conditions of (a) and the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is shown (means±s.e.m., n ¼ 3). (c–e) Immunoblot detection of LC3 lipidation, as well as p62 degradation in HCT116 subjected to knockdown of IKKa, IKKb, NEMO. At 48 h after the transfection, the cells were treated with ABT737 (1 mM) for 12 h. The efficiency of siRNA-mediated downregulation of the protein is also shown. Glyceraldehyde-3-phosphate dehydrogenase levels were monitored to ensure equal loading (n ¼ 4). (f) HCT116 were transfected with unrelated (UNR) or a siRNA specific for the IKK complex subunits and subsequently (after 24 h) with a plasmid for the expression of GFP-LC3, followed by culture in the presence of ABT737 (1 mM) for 12 h (means±s.e.m., n ¼ 3). (g) Immunoblot detection of phosphorylation status of IkBa, IKKa/b and NEMO. HCT116 cells were transfected with UNR or siRNAs specific for Atg5 and Beclin 1 as mentioned previously and treated with ABT737 (1 mM) for 12 h. The efficiency of siRNA-mediated downregulation of the proteins is also shown. Actin levels were assessed to ensure equal loading of lanes. Data are representative of three independent experiments.

Article Snippet: The profile of phospho-kinase proteins was performed on HeLa cells treated with ABT737 (1 mM) and HA14-1 (20mM) for 2, 6 and 12 h using a Proteome Profiler array–PhosphoKinase array kit (ARY003) according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA).

Techniques: Inhibition, Expressing, Western Blot, Knockdown, Transfection, Plasmid Preparation, Phospho-proteomics

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 5 Involvement of p53 and SIRT1 in ABT737-induced autophagy. (a, b) Levels of LC3I/II and p62 expression in HCT116 treated with ABT737 (1 mM) for 12 h in the presence or absence of pharmacological inhibitors of MDM2 (RITA or Nutlin-3, both used at 10 mM), proteasome (MG132, 10 mM) and SIRT1 (EX527, 100 mM) (n ¼ 3). Actin and glyceraldehyde-3-phosphate dehydrogenase levels were assessed to ensure equal loading of lanes. (c) GFP-LC3-expressing HeLa, U2OS or HCT116 cells were treated under the same conditions as in (a, b) and the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (means±s.e.m., n ¼ 3). (d, e) Immunoblot detection of LC3 lipidation, as well as p62 degradation in HCT116 subjected to knockdown of SIRT1, HDM2 and p53. At 48 h after the transfection, the cells were treated with ABT737 for 12 h. The efficiency of siRNA- mediated downregulation of the proteins is also demonstrated. Glyceraldehyde-3-phosphate dehydrogenase levels were monitored to ensure equal loading (n ¼ 5). (f) Quantification of the percentage of GFP-LC3vac in HCT116 transfected with siRNAs specific for SIRT1, HDM2 and p53, as described in Materials and methods section, and stimulated for 12 h with ABT737 (1 mM) (means±s.e.m., n ¼ 3).

Article Snippet: The profile of phospho-kinase proteins was performed on HeLa cells treated with ABT737 (1 mM) and HA14-1 (20mM) for 2, 6 and 12 h using a Proteome Profiler array–PhosphoKinase array kit (ARY003) according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Knockdown, Transfection

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 6 Pro-autophagic activity of the BH3 mimetic HA14-1. (a, b) U2OS cells stably expressing GFP-LC3 were treated with HA14- 1 and the kinetics of puncta formation was determined by videomicroscopy (means±s.e.m., n ¼ 3 separate experiments). (c) Alternatively, HeLa cells were cultured in the presence of HA14-1 (20 mM) at the time points mentioned, and the lipidation of LC3 and the expression of p62 were monitored by immunoblot. Actin levels were assessed to ensure equal loading of lanes. Results are representative of three independent determinations. (d) WT and Bax/Bak/ MEF were cultured in the presence of ABT737 (1 mM) or HA14-1 (20 mM) overnight, and the lipidation of LC3 and the expression of p62 were monitored by immunoblot. Actin levels were assessed to ensure equal loading of lanes. Results are representative of three independent determinations. (e) In the same conditions as shown in d, the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (means±s.e.m., n ¼ 3).

Article Snippet: The profile of phospho-kinase proteins was performed on HeLa cells treated with ABT737 (1 mM) and HA14-1 (20mM) for 2, 6 and 12 h using a Proteome Profiler array–PhosphoKinase array kit (ARY003) according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA).

Techniques: Activity Assay, Stable Transfection, Expressing, Cell Culture, Western Blot

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 7 Profile of phospho-proteome phosphorylation status elicited in HeLa cells after ABT737 treatment. HeLa cells were treated with ABT737 (1 mM) for 2, 6 or 12 h. Cell extracts were then probed on human phosphoprotein arrays following manufacturer’s instructions (see Materials and methods section). (a) Representative pictures of proteome profiler array. (b) Clustering analysis for the effect of BH3 mimetic on protein kinase phosphorylation. (c, d) Representative immunoblots of selected kinases whose phosphorylation status was affected by ABT737 (1 mM) or HA14-1 (20 mM) treatment (Pyk2, Akt, STAT3 and p53) validating the phosphoprotein arrays (n ¼ 3). (e) Comparison of the effects of ABT737 (1 mM) or HA14-1 (20 mM) on the mTOR, ACC, AMPK and p70S6K activation status (n ¼ 3). (f) Effect of Vps34 and Beclin 1 downregulation on the ABT737-mediated activation of AMPKa and inhibition of mTOR, as determined by immunoblot experiments (n ¼ 3). (c–f) Actin levels were monitored to ensure equal loading (n ¼ 3).

Article Snippet: The profile of phospho-kinase proteins was performed on HeLa cells treated with ABT737 (1 mM) and HA14-1 (20mM) for 2, 6 and 12 h using a Proteome Profiler array–PhosphoKinase array kit (ARY003) according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Western Blot, Comparison, Activation Assay, Inhibition

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: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Journal of applied microbiology

Article Title: Lactobacillus salivarius Ren prevent the early colorectal carcinogenesis in 1, 2-dimethylhydrazine-induced rat model.

doi: 10.1111/jam.12499

Figure Lengend Snippet: Figure 3 Structural segregation analysis of gut microbiota in rats from different group. (a) 16s rRNA gene V3 region PCR-denaturing gradient gel electrophoresis (DGGE) profiles of faecal samples obtained from healthy rats (No treatment group), 1, 2-dimethylhydrazine (DMH)-treated rats (DMH alone group) and Lactobacillus salivarius Ren (LS)-treated group (DMH + LS high group). M represents the marker for DGGE analysis. (b) Principal component analysis (PCA) score plots (PC1 vs PC2) based on the PCR–DGGE profiles among no treatment ( ), DMH alone ( ) and DMH +LS high ( ) groups. The first and the second axes explained 1 95 and 122% of total variation, respectively.

Article Snippet: PCR products were examined by denaturing gradient gel electrophoresis (DGGE) (D-code Universal Mutation Detection System, Bio-Rad, Hercules, CA).

Techniques: Denaturing Gradient Gel Electrophoresis, Marker