Journal: British Journal of Cancer

Article Title: Enhanced progression of human prostate cancer PC3 cells induced by the microenvironment of the seminal vesicle

doi: 10.1038/sj.bjc.6604169

Figure Lengend Snippet: Effects of treatment with extract of either prostate or seminal vesicle (SV) on malignant phenotypes, including cell growth, motility and invasion, in human prostate cancer PC3 cells. ( A ) PC3 cells were treated with various concentrations of the prostate or SV extract diluted with serum-free DMEM/F12. After 48 h of incubation, the number of viable cells was determined by the MTT assay. Columns, mean of three independent experiments; bars, s.d. ( B ) PC3 cells seeded at 1 × 10 5 per well in Boyden chambers were treated with various concentrations of the prostate or SV extract diluted with serum-free DMEM/F12. Chambers were incubated for 48 h in serum-free DMEM/F12, and then cells that had migrated to the lower surface of filters were stained with crystal violet stain solution. After the elution of crystal violet, the absorbance value in each well was measured with a microculture plate reader. Columns, mean of three independent experiments; bars, s.d. ( C ) PC3 cells seeded at 1 × 10 5 per well in Boyden chambers were treated with various concentrations of the prostate or SV extract diluted with serum-free DMEM/F12. Chambers were incubated for 48 h, and then cells that had migrated to the lower surface of filters through reconstituted basement membrane Matrigel were stained with crystal violet stain solution. After the elution of crystal violet, the absorbance value in each well was measured with a microculture plate reader. Columns, mean of three independent experiments; bars, s.d. * , differs from control ( P <0.01).

Article Snippet: PC3, derived from human prostate cancer, was purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Incubation, MTT Assay, Staining, Membrane, Control

Journal: British Journal of Cancer

Article Title: Enhanced progression of human prostate cancer PC3 cells induced by the microenvironment of the seminal vesicle

doi: 10.1038/sj.bjc.6604169

Figure Lengend Snippet: Effects of treatment with growth factors and cytokines on invasive potential in human prostate cancer PC3 cells. ( A ) PC3 cells seeded at 1 × 10 5 per well in Boyden chambers were treated with 1 ng ml −1 transforming growth factor- β 1 (TGF- β 1 ), 1 ng ml −1 epidermal growth factor (EGF), 10 ng ml −1 basic fibroblast growth factor (bFGF), 10 ng ml −1 hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), 10 ng ml −1 granulocyte colony-stimulating factor (G-CSF), 10 ng ml −1 granulocyte monocyte colony-stimulating factor (GM-CSF), 100 U ml −1 tumour necrosis factor- α (TNF- α ), 100 U ml −1 interkeukin-1 β (IL-1 β ) or 10 ng ml −1 interleukin-6 (IL-6) diluted with serum-free DMEM/F12. Chambers were incubated for 48 h, and then cells that had migrated to the lower surface of filters through reconstituted basement membrane Matrigel were stained with crystal violet stain solution. After the elution of crystal violet, the absorbance value in each well was measured with a microculture plate reader. No Tx, untreated cells. Columns, mean of three independent experiments; bars, s.d. * , differs from control ( P <0.01). ( B ) PC3 cells seeded at 1 × 10 5 per well in Boyden chambers were treated with various doses of TGF- β 1 or EGF diluted with serum-free DMEM/F12. Chambers were incubated for 48 h, and then cells that had migrated to the lower surface of filters through reconstituted basement membrane Matrigel were stained with crystal violet stain solution. After the elution of crystal violet, the absorbance value in each well was measured with a microculture plate reader. Columns, mean of three independent experiments; bars, s.d. ** and * , differs from control ( P <0.01 and P <0.05, respectively). ( C ) PC3 cells seeded at 1 × 10 5 per well in Boyden chambers were treated with 10 μ g ml −1 seminal vesicle extract and various doses of anti-TGF- β 1 or anti-EGF antibody diluted with serum-free DMEM/F12. Chambers were incubated for 48 h, and then cells that had migrated to the lower surface of filters through reconstituted basement membrane Matrigel were stained with crystal violet stain solution. After the elution of crystal violet, the absorbance value in each well was measured with a microculture plate reader. Columns, mean of three independent experiments; bars, s.d. * , differs from control ( P <0.01).

Article Snippet: PC3, derived from human prostate cancer, was purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Derivative Assay, Incubation, Membrane, Staining, Control

Journal: British Journal of Cancer

Article Title: Enhanced progression of human prostate cancer PC3 cells induced by the microenvironment of the seminal vesicle

doi: 10.1038/sj.bjc.6604169

Figure Lengend Snippet: Regulation of urokinase-type plasminogen activator production in human prostate cancer PC3 cells by transforming growth factor- β 1 (TGF- β 1 ). ( A ) PC3 cells were treated with various concentrations of TGF- β 1 diluted with serum-free DMEM/F12. After 48 h of incubation, serum-free DMEM/F12 was collected, and the concentration of uPA in each sample was determined with a quantitative sandwich enzyme immunoassay kit for human uPA. Columns, mean of three independent experiments; bars, s.d. * , differs from control ( P <0.01). ( B ) PC3 cells were treated with various concentrations of seminal vesicle (SV) extract and anti-TGF- β 1 antibody diluted with serum-free DMEM/F12. After 48 h of incubation, serum-free DMEM/F12 was collected, and the concentration of uPA in each sample was determined with a quantitative sandwich enzyme immunoassay kit for human uPA. Columns, mean of three independent experiments; bars, s.d. ** and * , differs from control ( P <0.01 and P <0.05, respectively). ( C ) PC-3 cells were treated with various concentrations of SV extract and/or anti-TGF- β 1 antibody diluted with serum-free DMEM/F12. After 48 h of incubation, protein was extracted from culture cells, and uPA and β -tubulin protein levels were analysed by western blotting.

Article Snippet: PC3, derived from human prostate cancer, was purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: British Journal of Cancer

Article Title: Enhanced progression of human prostate cancer PC3 cells induced by the microenvironment of the seminal vesicle

doi: 10.1038/sj.bjc.6604169

Figure Lengend Snippet: Comparison of disease progression in vivo following the injection of PC3 cells into the prostate or SV

Article Snippet: PC3, derived from human prostate cancer, was purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Comparison, Biomarker Discovery, In Vivo, Injection

Journal: Oncotarget

Article Title: Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis

doi: 10.18632/oncotarget.18621

Figure Lengend Snippet: (A) Western blot analysis of SHH and Gli-1 expression in OC cells. GAPDH served as the loading control. (B) ELISA of SHH ligand secreted by OC cells. (C) ELISA of SHH ligand secreted by TOV-112D cells stably transfected with either shNC or SHH-shRNA lentivirus. (D) ELISA of SHH ligand secreted by Caov3 cells stably transfected with either NC or SHH-OE lentivirus. (E) Western blot analysis of SHH and Gli-1 in TOV-112D and Caov3 cells stably transfected with lentivirus. (F) Schematic depicting of indirectly co-cultured OC cells and CAFs from the top to bottom. (G) Western blot analysis of Gli-1 expression in CAFs that were indirectly co-cultured with SKOV3, TOV-112D or CAOV3 cells. CAFs cultured alone were used as the control. (H) Western blot analysis of Gli-1 expression in CAFs that were indirectly co-cultured with TOV-112D and Caov3 cells stably transfected with lentivirus. Data are expressed as mean ± s.e.m. (*P < 0.05).

Article Snippet: Human EOC cell lines SKOV3, TOV-112D, TOV-21G, A2780, OV-90, OVCAR3 and Caov3 were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, shRNA, Cell Culture

Journal: Oncotarget

Article Title: Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis

doi: 10.18632/oncotarget.18621

Figure Lengend Snippet: (A) and (B) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and NOFs after 48 h treatment with rSHH (1 μg/ml). (C) ELISA of VEGF-C secreted by CAFs and NOFs after 48 h treatment with rSHH (1 μg/ml). (D) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (E) ELISA of VEGF-C secreted by CAFs co-cultured with SKOV3, TOV-112D or Caov3 cells. (F) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (G) ELISA of VEGF-C secreted by CAFs co-cultured with TOV-112D cells stably transfected with either shNC or SHH-shRNA lentivirus. (H) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (I) ELISA of VEGF-C secreted by CAFs co-cultured with Caov3 cells stably transfected with either NC or SHH-OE lentivirus. (J) Western blot analysis of Gli-1 expression in CAFs stably transfected with NC or Gli-shRNA lentivirus in the presence of rSHH (1 μg/ml) for 48 h. (K) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (L) ELISA of VEGF-C secreted by CAFs stably transfected with NC or Gli-shRNA lentivirus in the presence of rSHH (1 μg/ml) for 48 h. The data are expressed as the mean ± s.e.m. (*P<0.05).

Article Snippet: Human EOC cell lines SKOV3, TOV-112D, TOV-21G, A2780, OV-90, OVCAR3 and Caov3 were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Stable Transfection, Transfection, shRNA, Western Blot, Expressing

Journal: Oncotarget

Article Title: Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis

doi: 10.18632/oncotarget.18621

Figure Lengend Snippet: (A) Western blot analysis of SHH and Gli-1 expression in OC cells. GAPDH served as the loading control. (B) ELISA of SHH ligand secreted by OC cells. (C) ELISA of SHH ligand secreted by TOV-112D cells stably transfected with either shNC or SHH-shRNA lentivirus. (D) ELISA of SHH ligand secreted by Caov3 cells stably transfected with either NC or SHH-OE lentivirus. (E) Western blot analysis of SHH and Gli-1 in TOV-112D and Caov3 cells stably transfected with lentivirus. (F) Schematic depicting of indirectly co-cultured OC cells and CAFs from the top to bottom. (G) Western blot analysis of Gli-1 expression in CAFs that were indirectly co-cultured with SKOV3, TOV-112D or CAOV3 cells. CAFs cultured alone were used as the control. (H) Western blot analysis of Gli-1 expression in CAFs that were indirectly co-cultured with TOV-112D and Caov3 cells stably transfected with lentivirus. Data are expressed as mean ± s.e.m. (*P < 0.05).

Article Snippet: Human EOC cell lines SKOV3, TOV-112D, TOV-21G, A2780, OV-90, OVCAR3 and Caov3 were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, shRNA, Cell Culture

Journal: Oncotarget

Article Title: Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis

doi: 10.18632/oncotarget.18621

Figure Lengend Snippet: (A) and (B) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and NOFs after 48 h treatment with rSHH (1 μg/ml). (C) ELISA of VEGF-C secreted by CAFs and NOFs after 48 h treatment with rSHH (1 μg/ml). (D) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (E) ELISA of VEGF-C secreted by CAFs co-cultured with SKOV3, TOV-112D or Caov3 cells. (F) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (G) ELISA of VEGF-C secreted by CAFs co-cultured with TOV-112D cells stably transfected with either shNC or SHH-shRNA lentivirus. (H) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (I) ELISA of VEGF-C secreted by CAFs co-cultured with Caov3 cells stably transfected with either NC or SHH-OE lentivirus. (J) Western blot analysis of Gli-1 expression in CAFs stably transfected with NC or Gli-shRNA lentivirus in the presence of rSHH (1 μg/ml) for 48 h. (K) qRT-PCR analysis of Gli-1 and VEGF-C mRNA in CAFs and (L) ELISA of VEGF-C secreted by CAFs stably transfected with NC or Gli-shRNA lentivirus in the presence of rSHH (1 μg/ml) for 48 h. The data are expressed as the mean ± s.e.m. (*P<0.05).

Article Snippet: Human EOC cell lines SKOV3, TOV-112D, TOV-21G, A2780, OV-90, OVCAR3 and Caov3 were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Stable Transfection, Transfection, shRNA, Western Blot, Expressing

Journal: Oncotarget

Article Title: Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis

doi: 10.18632/oncotarget.18621

Figure Lengend Snippet: (A) Growth properties of subcutaneous tumours in mice derived from Caov3 SHH-OE or Caov3 NC cells alone or co-injected with CAFs. Kaplan-Meier plot of time to tumour progression, as evidenced by survival rates over time (n=5). (B) Bright field images of subcutaneous tumours from each group (n=5) 20 days after tumour implantation. (C) Graphical representation of the weights of subcutaneous tumours from each group 20 days after tumour implantation. (D) Immunohistochemical staining for Ki-67, α-SMA, Gli-1 and LYVE1 in tumour sections from mice in each group. (E) Graphical representation of the number of LYVE1+ vessels counted from IHC images in panel D. (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: Human EOC cell lines SKOV3, TOV-112D, TOV-21G, A2780, OV-90, OVCAR3 and Caov3 were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Derivative Assay, Injection, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis

doi: 10.18632/oncotarget.18621

Figure Lengend Snippet: (A) Representative images and statistical analyses of migration of LECs in a co-culture invasion system with CAFs stably transfected with shNC or Gli-shRNA lentivirus in the presence of rSHH (1 μg/ml). (B) Representative images and statistical analyses of capillary tube formation of LECs treated with supernatants from cultures of CAFs stably transfected with shNC or Gli-shRNA lentivirus in the presence of rSHH (1 μg/ml). (C) Bright field images and graphical representation of the weights of tumours of subcutaneous tumours from mice (n=5) bearing Caov3 SHH-OE cells co-injected with CAFs stably transfected with shNC or Gli-shRNA lentivirus 20 days after tumour implantation. (D) Growth properties of subcutaneous tumours in mice derived from Caov3 SHH-OE cells co-injected with CAFs stably transfected with shNC or Gli-shRNA lentivirus. Kaplan-Meier plot of time to tumour progression, as evidenced by survival rates over time. (E) IHC staining for Ki-67, α-SMA, Gli-1 and LYVE1 in tumour sections from mice in the two groups. (F) Graphical representation of the number of LYVE1+ vessels counted from IHC images in panel E. (**P<0.01, ***P<0.001).

Article Snippet: Human EOC cell lines SKOV3, TOV-112D, TOV-21G, A2780, OV-90, OVCAR3 and Caov3 were purchased from American Type Culture Collection (Rockville, MD, USA).

Techniques: Migration, Co-Culture Assay, Stable Transfection, Transfection, shRNA, Injection, Derivative Assay, Immunohistochemistry

Journal: Cell host & microbe

Article Title: Human Cytomegalovirus miRNAs Regulate TGF-β to Mediate Myelosuppression while Maintaining Viral Latency in CD34 + Hematopoietic Progenitor Cells

doi: 10.1016/j.chom.2019.11.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: Kasumi-3 , ATCC , Cat# CRL-2725.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, TaqMan Assay, Binding Assay, Reporter Assay, Negative Control, Software

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD158b/j costimulates expression of IFN-γ in CD4 + CD28 null T cells. CD4 + CD28 null CD158b/j + T cell clones were stimulated using immobilized mouse IgG and anti-CD3 mAb alone or in combination with anti-CD158b/j mAb (shown as μg/ml). The number of IFN-γ transcripts was quantified using real-time PCR. Anti-MHC mAb is included as a negative control (A). Cells were stimulated as above and IFN-γ cytokine production was quantified by ELISA (B).

Article Snippet: Clones were maintained on 1.0 × 10 6 irradiated heterologous PBMCs/ml, 2.0 × 10 5 irradiated Epstein Barr virus–transformed B cells/ml, 30 ng/ml anti-CD3 mAb (OKT3, CRL 8001; American Type Culture Collection), and 50 U/ml recombinant human IL-2 (Proleukin; Chiron Corp.).

Techniques: Expressing, Clone Assay, Real-time Polymerase Chain Reaction, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Stimulation of CD158b/j in CD4 + CD28 null T cells leads to JNK, but not ERK, phosphorylation. CD4 + CD28 null CD158j + T cell clones shown in Figure 1 (clones #1, #2, and #4) were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels), after which the blots were stripped and reprobed with Abs against total JNK (right panels) (A). After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of ERK (left panels), after which the blots were stripped and re-probed with Abs against total ERK (right panels) (B).

Article Snippet: Clones were maintained on 1.0 × 10 6 irradiated heterologous PBMCs/ml, 2.0 × 10 5 irradiated Epstein Barr virus–transformed B cells/ml, 30 ng/ml anti-CD3 mAb (OKT3, CRL 8001; American Type Culture Collection), and 50 U/ml recombinant human IL-2 (Proleukin; Chiron Corp.).

Techniques: Phospho-proteomics, Clone Assay, SDS Page, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Stimulation of CD158b/j in CD4 + CD28 null T cells leads to MKK4 phosphorylation. CD4 + CD28 null CD158j + T cell clones (clones #1, #2, and #4) were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of MKK4 (left panels). The blots were stripped and reprobed with Ab against total MKK4 (right panels).

Article Snippet: Clones were maintained on 1.0 × 10 6 irradiated heterologous PBMCs/ml, 2.0 × 10 5 irradiated Epstein Barr virus–transformed B cells/ml, 30 ng/ml anti-CD3 mAb (OKT3, CRL 8001; American Type Culture Collection), and 50 U/ml recombinant human IL-2 (Proleukin; Chiron Corp.).

Techniques: Phospho-proteomics, Clone Assay, SDS Page, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Phosphorylation of JNK is initiated by stimulation specifically through CD158j. Two CD4 + CD28 null CD158j + T cell clones (top panels) and a CD4 + CD28 null CD158b1 + T cell clone (bottom panels) were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels; A). Jurkat T cells were infected with either wild-type vaccinia virus (WR) or vaccinia virus containing CD158j cDNA and were analyzed for expression of CD158j by flow cytometry (B). Jurkat T cells infected with WR vaccinia virus or CD158j + vaccinia virus were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (top left panels) and MKK4 (bottom left panel). The blots were stripped and reprobed with Abs against β-actin (top right panels) or MKK4 (bottom right panel) (C).

Article Snippet: Clones were maintained on 1.0 × 10 6 irradiated heterologous PBMCs/ml, 2.0 × 10 5 irradiated Epstein Barr virus–transformed B cells/ml, 30 ng/ml anti-CD3 mAb (OKT3, CRL 8001; American Type Culture Collection), and 50 U/ml recombinant human IL-2 (Proleukin; Chiron Corp.).

Techniques: Phospho-proteomics, Clone Assay, SDS Page, Membrane, Infection, Virus, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Stimulation of CD158j in CD4 + CD28 null T cells leads to expression of ATF-2 and c-Jun. CD4 + CD28 null CD158j + T cell clones (clones #2, #3, #4), and Jurkat T cells expressing CD158j were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for expression of ATF-2 and c-Jun. The membranes were stripped and reprobed with Ab against β-actin.

Article Snippet: Clones were maintained on 1.0 × 10 6 irradiated heterologous PBMCs/ml, 2.0 × 10 5 irradiated Epstein Barr virus–transformed B cells/ml, 30 ng/ml anti-CD3 mAb (OKT3, CRL 8001; American Type Culture Collection), and 50 U/ml recombinant human IL-2 (Proleukin; Chiron Corp.).

Techniques: Expressing, Clone Assay, SDS Page, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Mutation of transmembrane lysine residue in CD158j abolishes ability to induce JNK phosphorylation. Jurkat T cells were transiently transfected with constructs containing the CD158j cDNA or the CD158j233I cDNA. Cell-surface expression was confirmed by flow cytometry (A). Jurkat T cells transfected with either CD158j or CD158jK233I were stimulated with anti-CD3 or anti-CD158b/j mAb and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels) (B).

Article Snippet: Clones were maintained on 1.0 × 10 6 irradiated heterologous PBMCs/ml, 2.0 × 10 5 irradiated Epstein Barr virus–transformed B cells/ml, 30 ng/ml anti-CD3 mAb (OKT3, CRL 8001; American Type Culture Collection), and 50 U/ml recombinant human IL-2 (Proleukin; Chiron Corp.).

Techniques: Mutagenesis, Residue, Phospho-proteomics, Transfection, Construct, Expressing, Flow Cytometry, SDS Page, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by streptavidin-HRP (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).

Article Snippet: Clones were maintained on 1.0 × 10 6 irradiated heterologous PBMCs/ml, 2.0 × 10 5 irradiated Epstein Barr virus–transformed B cells/ml, 30 ng/ml anti-CD3 mAb (OKT3, CRL 8001; American Type Culture Collection), and 50 U/ml recombinant human IL-2 (Proleukin; Chiron Corp.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Negative Control, SDS Page, Membrane, Phospho-proteomics, Expressing, Stable Transfection, Transfection, Flow Cytometry, Immunoprecipitation, Western Blot

Journal: Cancer cell

Article Title: β2 adrenergic-neurotrophin feed-forward loop promotes pancreatic cancer

doi: 10.1016/j.ccell.2017.11.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: BxPC-3 cells , ATCC , CRL-1687.

Techniques: Plasmid Preparation, Virus, Recombinant, Blocking Assay, Enzyme-linked Immunosorbent Assay, MTT Cell Proliferation, SYBR Green Assay, Western Blot, Knock-Out, Software