Journal: Oncogene

Article Title: Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells

doi: 10.1038/onc.2011.122

Figure Lengend Snippet: (A) Univariate survival analyses by log-rank were conducted to assess the contribution of the Notch ligands to disease prognosis in 779 patients using GEO database (GSE7390, GSE2034, and NKI-295). (B) Metastasis free- and overall- survival rates over a period of 5 years were analyzed in 295 patients (NKI-295) with breast cancer, in relation to Jagged2 expression. Solid line, Jagged2 negative patients; dotted line, patients with increased expression of Jagged2. (C) Multivariate Cox regression analysis of GSE7390 was conducted to assess the contribution of the indicated variables to disease prognosis. b, the estimation for the regression coefficient β; CI, confidence interval.

Article Snippet: Western blot analysis was performed as described previously using antibodies against cleaved Notch1 (1:200; Val1744; Cell Signaling Technology), Jagged2 (1:200; R&D Systems Inc.), α -tubulin (1:1,000 Cell Signaling Technology), HIF1α (1:200; BD Bioscience), phospho-Akt1 (1:500; Ser473; Cell Signaling Technology), and Akt1 (1:500; Cell Signaling Technology) ( ).

Techniques: Expressing

Journal: Oncogene

Article Title: Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells

doi: 10.1038/onc.2011.122

Figure Lengend Snippet: (A) Representative photos of breast tumor specimens at various stages after IHC with anti-NICD antibody. (B) Staining intensity of NICD at different stages was quantified. (C) Representative photos of two consecutive sections stained with anti-NICD and anti-Jagged2 antibodies at invasive front and inside of tumor. (D) Representative photos of IHC using antibodies to NICD, CA9 and Glut1 at invasive front and inside of tumor.

Article Snippet: Western blot analysis was performed as described previously using antibodies against cleaved Notch1 (1:200; Val1744; Cell Signaling Technology), Jagged2 (1:200; R&D Systems Inc.), α -tubulin (1:1,000 Cell Signaling Technology), HIF1α (1:200; BD Bioscience), phospho-Akt1 (1:500; Ser473; Cell Signaling Technology), and Akt1 (1:500; Cell Signaling Technology) ( ).

Techniques: Staining

Journal: Oncogene

Article Title: Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells

doi: 10.1038/onc.2011.122

Figure Lengend Snippet: (A) Breast cancer cell lines, MCF7 and MDA-MB231, were cultured in two sets of 24-well plates under normoxic (21%O 2 ) or hypoxic (1%O 2 ) conditions for 24 hrs. One set of cells (in triplicate) was collected, and RNA was prepared. The samples were then subjected to qRT-PCR using primers for the Jagged2 and β-actin genes. Another set of cells was collected, and the cell lysates were subjected to Western blot analysis using anti-Jagged2 and anti-tubulin antibodies (inserted fig). For Jagged2 reporter assay, MCF7 cells were transfected with the Jagged2 reporter plasmid and they were incubated under normoxia or hypoxia. Luciferase activity was measured after 24hrs incubation. (B) MCF7 cells were transfected with Notch reporter plasmid (4XCBF-Luc) and they were incubated under normoxia or hypoxia for 24 hrs followed by luciferase activity assay. Western blot was performed to measure the protein level of NICD and HIF1α in MCF7 cells under normoxia or hypoxia (inserted figure). (C) MDA-MB231 were cultured under 1%O 2 and NICD immunocytochemistry was performed. Values are means ± SD of triplicate measurements. *, P<0.01.

Article Snippet: Western blot analysis was performed as described previously using antibodies against cleaved Notch1 (1:200; Val1744; Cell Signaling Technology), Jagged2 (1:200; R&D Systems Inc.), α -tubulin (1:1,000 Cell Signaling Technology), HIF1α (1:200; BD Bioscience), phospho-Akt1 (1:500; Ser473; Cell Signaling Technology), and Akt1 (1:500; Cell Signaling Technology) ( ).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Reporter Assay, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Immunocytochemistry

Journal: Oncogene

Article Title: Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells

doi: 10.1038/onc.2011.122

Figure Lengend Snippet: (A) mRNA and whole cell lysates were prepared from the non-tumorigenic cell line, MCF10A and three human breast cancer cell lines, MCF7, MDA231 and MDA231LM, and quantitative RT-PCR and Western blot were performed to evaluate the amount of Jagged2 expression. (B) MDA231, MDA231LM and MDA231LM-DNMAML were cultured in 96-well plates under hypoxic condition (1%O 2 ). Cell viability was measured at different time point by MTT assay. (C) MDA231 and MDA231LM cells were infected with sh-Jagged2 and pSin-Jagged2 lentivirus, respectively. Cells were also infected with control lentiviruses. These cells were incubated for 12 hours under normoxic condition and they were transferred to hypoxic condition (1%O 2 ). After 72 hrs, cell viability was measured by MTT assay. Knock-down and ectopic expression of Jagged2 were also examined by Western blot (inserted photos). (D) MDA231LM cells were also cultured in the presence or absence of DAPT under hypoxia for 48 hrs and the expression of phospho-Akt was examined by Western blot. (E) MDA231LM cells were seeded in Matrigel invasion chambers in the presence or absence of DAPT (20 µM) under hypoxia. After 24hrs incubation, the number of invading cells was counted. (F) mRNA were prepared from MCF7, MDA231 and MDA231LM, and qRT-PCR was performed to evaluate the snail expression.(G) MDA231LM cells were cultured in the presence or absence of DAPT under normoxia or hypoxia for 48 hrs and cell morphology was observed under microscope. (H) mRNA levels of E-cadherin expression were examined in MDA231LM (left panel) and MCF7 cells (right panel) that were cultured under normoxia or hypoxia with or without the treatment of DAPT (20 µm). (I) MDA231LM cells were infected with sh-Jagged2 or control lentivirus for 12hrs under normoxia, and they were transferred to hypoxic condition (1%O 2 ). After 48 hrs of incubation, mRNA levels of Hes1 and snail were measured by qRT-PCR. Values are means ± SD of triplicate measurements. *, P<0.01.

Article Snippet: Western blot analysis was performed as described previously using antibodies against cleaved Notch1 (1:200; Val1744; Cell Signaling Technology), Jagged2 (1:200; R&D Systems Inc.), α -tubulin (1:1,000 Cell Signaling Technology), HIF1α (1:200; BD Bioscience), phospho-Akt1 (1:500; Ser473; Cell Signaling Technology), and Akt1 (1:500; Cell Signaling Technology) ( ).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Cell Culture, MTT Assay, Infection, Control, Incubation, Knockdown, Microscopy

Journal: Oncogene

Article Title: Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells

doi: 10.1038/onc.2011.122

Figure Lengend Snippet: (A) Human bone marrow stromal cell line, HS5, was cultured in two sets of 24-well plates under normoxic (21%O 2 ) or hypoxic (1%O 2 ) conditions for up to 48hrs. One set of cells was collected and RNA was prepared. The samples were then subjected to qRT-PCR using primers for the Jagged2 and β-actin genes. Another set of cells was collected, and the cell lysates were subjected to Western blot analysis using anti-Jagged2 and anti-tubulin antibodies (inserted figure). Similar experiments were performed to examine the mRNA and protein levels of Jagged2 in HS5 after DFO treatment by qRT-PCR and Western blot (right panel). (B) MDA231BoM cells which stably expressed the Notch reporter gene were seeded on monolayer of HS5 cells followed by DFO (left panel) or hypoxia (right panel) treatment. Luciferase activity was measured after 48hrs of co-culture. Values are means ± SD of triplicate measurements. *, P<0.01.

Article Snippet: Western blot analysis was performed as described previously using antibodies against cleaved Notch1 (1:200; Val1744; Cell Signaling Technology), Jagged2 (1:200; R&D Systems Inc.), α -tubulin (1:1,000 Cell Signaling Technology), HIF1α (1:200; BD Bioscience), phospho-Akt1 (1:500; Ser473; Cell Signaling Technology), and Akt1 (1:500; Cell Signaling Technology) ( ).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Stable Transfection, Luciferase, Activity Assay, Co-Culture Assay

Journal: Blood Cancer Journal

Article Title: Autocrine insulin-like growth factor 1 and stem cell factor but not interleukin 6 support self-renewal of human myeloma cells

doi: 10.1038/bcj.2013.18

Figure Lengend Snippet: Silencing of IGFR1 , imatinib and wortmannin did not decrease JAG2 expression. ( a ) IGF1R silencing. KMM1 cells were transiently transfected with siControl or siRNA human IGF1R (si IGF1R ) messenger RNA (mRNA) for 48 h and IGF1R expression was assessed by flow cytometry. Overlay histograms represent the IGF1R staining (thick line) over that of control staining (thin line). The specific fluorescence was expressed as the ratio of the specific mean fluorescence intensity divided by the control fluorescence ( r ). A representative experiment out of two is shown. ( b ) IGF1R silencing inhibited the spontaneous colony formation. KMM1 cells were transiently transfected with siCt or si IGF1R mRNA for 48 h, and 660 cells were seeded in two wells containing semisolid collagen. Colonies were counted after 3 weeks. The data represent the means±s.d. of four wells from two experiments. ( c ) IGF1R silencing did not inhibit JAG2 expression. The western blot analysis was performed 48 h after siRNA transfection as described in a . ( d ) JAG2 silencing did not inhibit P-AKT. Stable sh JAG2 and shControl KMM1 cells were incubated for 48 h in serum-free RPMI 1640 medium supplemented with 0.5% bovine serum albumin (BSA). ( e ) Imatinib and wortmannin did not decrease the expression of JAG2. Cells were treated for 48 h with 20 μℳ imatinib or 10 μℳ wortmannin in serum-free RPMI 1640 medium supplemented with 0.5% BSA, and JAG2 expression was assessed by flow cytometry. Overlay histograms represent the JAG2 staining (thick lines) over that of control staining (thin lines). ** P <0.01.

Article Snippet: Allophycocyanin-conjugated control and anti-JAG2 mAbs were purchased from R&D Systems (Lille, France).

Techniques: Expressing, Transfection, Flow Cytometry, Staining, Control, Fluorescence, Western Blot, Incubation