max his tagged  (Sino Biological)

Journal: Oncotarget

Article Title: Tumor regression is mediated via the induction of HER263-71- specific CD8+ CTL activity in a 4T1.2/HER2 tumor model: no involvement of CD80 in tumor control

doi: 10.18632/oncotarget.15816

Figure Lengend Snippet: IFN-γ and the expression level of co-stimulatory molecules in TC-1 and MC32 tumor models ( A ) Each group of C57BL/6 mice ( n = 3/group) was challenged s.c. with TC-1 and MC32 cells (5 × 10 5 cells/mouse). When tumor sizes reached 7–8 mm in mean diameter (9, 9 and 4 mm for TC-1 tumor; 11, 8 and 5,5 mm for MC32 tumor), the mice were bled and sera were collected to measure systemic IFN-γ levels using ELISA. ( B ) The mice were sacrificed to measure their spleen weights. ( C ) The splenocytes were isolated and reacted for 2 days with class I CTL peptides (control CEA vs. E7 peptides for TC-1 tumor; control E7 vs. CEA peptides for MC32 tumor). The collected cell supernatants were used to measure IFN-γ levels using ELISA. ( D ) Five × 10 5 TC-1 and MC32 tumor cells were treated with PE-labeled anti-CD80, anti-CD86 and anti-CD40 (thick line), as well as control Abs (thin line) to measure the expression of CD80, CD86 and CD40 molecules using a flow cytometer. The values and bars show IFN-γ levels or spleen weights, and SDs, respectively.

Article Snippet: To generate CD80 expression vectors, CD80 genes were amplified using a pair of primers (forward primer, 5′-CGGAATTCATGGCTTGC AATTGTCAGTTG-3′ and reverse primer, 5′-CCGCTC GAGCTAAAGGAAGACGGTCTGTT-3′).

Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Labeling, Flow Cytometry

Journal: Oncotarget

Article Title: Tumor regression is mediated via the induction of HER263-71- specific CD8+ CTL activity in a 4T1.2/HER2 tumor model: no involvement of CD80 in tumor control

doi: 10.18632/oncotarget.15816

Figure Lengend Snippet: 4T1.2/HER2 and CT26/HER2 cell tumor growth patterns with/without CD80 expression and CT26/HER2 cell tumor growth patterns in IFN-γ deficient mice ( A ) 4T1.2/HER2 cells were stably transfected with CD80 cDNA and then selected against hygromycin B. The selected cells tested positive for CD80 based on a FACS analysis. Thin line, control Ab; bold line, anti-CD80 Ab. ( B ) BALB/c mice ( n = 5/group) were challenged s.c. with wild type or CD80-expressing 4T1.2/HER2 cells (2 × 10 5 cells/mouse). Tumor sizes were measured over time. ( C ) CD80 expression levels on wild type CT26/HER2 and CT26/HER2-1 cells, as determined via FACS analysis. Thin line, control Ab; bold line, anti-CD80 Ab. ( D ) BALB/c mice ( n = 5/group) were challenged s.c. with CT26/HER2 and CT26/HER2-1 cells (5 × 10 5 cells/mouse). ( E ) BALB/c IFN-γ knockout and wild type mice ( n = 5/group) were challenged s.c. with CT26/HER2 cells (5 × 10 5 cells/mouse). Tumor sizes were measured over time. The values and bars represent tumor sizes and SDs, respectively.

Article Snippet: To generate CD80 expression vectors, CD80 genes were amplified using a pair of primers (forward primer, 5′-CGGAATTCATGGCTTGC AATTGTCAGTTG-3′ and reverse primer, 5′-CCGCTC GAGCTAAAGGAAGACGGTCTGTT-3′).

Techniques: Expressing, Mouse Assay, Stable Transfection, Transfection, FACS, Knock-Out

Journal: Oncotarget

Article Title: Tumor regression is mediated via the induction of HER263-71- specific CD8+ CTL activity in a 4T1.2/HER2 tumor model: no involvement of CD80 in tumor control

doi: 10.18632/oncotarget.15816

Figure Lengend Snippet: MHC-I, CD80, CD86, CD40 and HER2 antigen expression levels on the surface of 4T1.2/HER2 vs. CT26/HER2 cells. Five × 10 5 tumor cells were reacted with FITC-labeled anti-MHC-I (thick line) and isotype control (thin line) Abs and with PE-labeled anti-CD80, anti-CD86, anti-CD40 Abs (thick line) and isotype control Abs (thin line) For HER2 expression, the cells were treated with anti-HER2 mouse serum (thick line) or control serum (thin line), followed by treatment with FITC-labeled anti-mouse IgG.

Article Snippet: To generate CD80 expression vectors, CD80 genes were amplified using a pair of primers (forward primer, 5′-CGGAATTCATGGCTTGC AATTGTCAGTTG-3′ and reverse primer, 5′-CCGCTC GAGCTAAAGGAAGACGGTCTGTT-3′).

Techniques: Expressing, Labeling

Journal: Oncotarget

Article Title: Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

doi: 10.18632/oncotarget.20834

Figure Lengend Snippet: IMD-4482 suppressed proliferation and induced G0/G1 cell cycle arrest and apoptosis in PAI-1-positive ovarian cancer cells Effect of IMD-4482 on cell proliferation (A) . SKOV3ip1, HeyA8, and OVCAR3 cells were plated onto 96-well plates and cultured in DMEM containing 2% FBS with or without IMD-4482. Effect of IMD-4482 on cell cycle distribution (B and C) . Cells treated with or without IMD-4482 for 24 hours were stained with propidium iodine and analyzed by flow cytometry. Representative flow histograms (B), and percentages of cells in G0/G1, S, and G2/M phase (C) are shown. Western blot (D) . Cells were incubated with or without IMD-4482 for 24 hours. Cell lysates were immunoblotted with antibodies against PARP, CDK2, cyclin D3, and p27kip1. β-actin was used as a loading control.

Article Snippet: Human SERPINE1 expression plasmid (HG10296-UT) and control vector (pCMV) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Radial Immuno Diffusion, Cell Culture, Staining, Flow Cytometry, Western Blot, Incubation

Journal: Oncotarget

Article Title: Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

doi: 10.18632/oncotarget.20834

Figure Lengend Snippet: IMD-4482 inhibits PAI-1 activation of PAI-1-positive ovarian cancer cells PAI-1 expression in 6 serous ovarian cell lines and 2 different primary cultures of ovarian surface epithelium (OSE) cells was analyzed by western blot (A) . β-actin was used as a loading control. Real-time RT-PCR (B) . Total RNAs from the six cell lines and one OSE were collected and subjected to RT-PCR. The relative abundance of PAI-1 with respect to GAPDH expression was calculated. Molecular formula of IMD-4482 (C) . Western blot (D) . SKOV3ip1, HeyA8, and OVCAR3 cells were incubated with or without IMD-4482 in serum-free medium for 24 hours. Cell lysates were immunoblotted with antibodies against PAI-1 and uPAR. β-actin was used as a loading control. Experiments were repeated three times and are expressed as mean ± SD. * ; P

Article Snippet: Human SERPINE1 expression plasmid (HG10296-UT) and control vector (pCMV) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Radial Immuno Diffusion, Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: Oncotarget

Article Title: Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

doi: 10.18632/oncotarget.20834

Figure Lengend Snippet: IMD-4482 inhibits adhesion and invasion of PAI-1-positive ovarian cancer cells (SKOV3ip1 and HeyA8 cells) but not PAI-1-negative cells (OVCAR3) In vitro adhesion assay (A) . A total of 1 × 10 5 ovarian cancer cells (left, SKOV3ip1; middle, HeyA8; right, OVCAR3) were plated onto vitronectin-, fibronectin-, collagen type I-, and laminin-coated 96-well plates. After incubation for 50 minutes at 37°C, plates were washed to discard non-adherent cells, and the relative number of attached cells was measured, Data represents mean ± SD, n = 5 from triplicate independent experiments. (B) Representative images of in vitro adhesion assay of SKOV3ip1 cells (B). Bar, 200 μm. In vitro invasion assay (C) . A total of 4 × 10 4 SKOV3ip1 (left) and 8 × 10 3 HeyA8 (right) cells were plated on the top chamber in serum-free medium with IMD-4482, and allowed to invade for 48 hours. Invading cells on the underside of the filter were counted. Representative images are shown. Bar, 200 μm. Data represents mean ± SD, n = 5 from triplicate independent experiments. * ; P

Article Snippet: Human SERPINE1 expression plasmid (HG10296-UT) and control vector (pCMV) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Radial Immuno Diffusion, In Vitro, Cell Adhesion Assay, Incubation, Invasion Assay

Journal: Oncotarget

Article Title: Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

doi: 10.18632/oncotarget.20834

Figure Lengend Snippet: IMD-4482 inhibits the phosphorylation of FAK and ERK followed by the dissociation of PAI-1 and uPAR from αVβ3 integrin and FAK Western blot (A) . Cells were incubated with IMD-4482 for 24 hours. Cell lysates were immunoblotted with an antibody against integrin αV and integrin β3. β-actin was used as a loading control. Western blot (B) . Cells were incubated with or without IMD-4482 for 24 hours. Cell lysates were immunoblotted with an antibody against PARP, p-FAK (Tyr-397), FAK, p-ERK, and ERK. β-actin was used as a loading control. (C and D) Immunoprecipitation. Ovarian cancer cells were incubated with IMD-4482 for 24 hours, and lysed in buffer containing 1% Triton X-100. Cell lysates were subjected to immunoprecipitation with anti-PAI-1 antibody (C) or anti-uPAR antibody (D) , followed by immunoblotting to detect integrin αV, integrin β3, and FAK. Representative blots from three independent experiments are shown. Arrow indicates heavy chain of IgG band. Schema (E) . PAI-1 inhibition with IMD-4482 dissociates the interaction between PAI-1/uPAR and αVβ3 integrin, which leads to the inhibition of FAK phosphorylation.

Article Snippet: Human SERPINE1 expression plasmid (HG10296-UT) and control vector (pCMV) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Radial Immuno Diffusion, Western Blot, Incubation, Immunoprecipitation, Inhibition

Journal: Oncotarget

Article Title: Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

doi: 10.18632/oncotarget.20834

Figure Lengend Snippet: IMD-4482 inhibits peritoneal dissemination of ovarian cancer cells through inhibition of FAK phosphorylation and intratumoral vessel formation Experimental protocol (A) . A total of 1 × 10 6 HeyA8 cells were injected intraperitoneally into female BALB/c nu/nu mice. Three days after the injection, IMD-4482 (50 mg/kg body weight) or an equal amount of 0.5% CMC-Na (control) was injected intraperitoneally daily for 11 days. Effect of IMD-4482 on intraperitoneal tumor weight (B) and number of metastases (C) . Results are expressed as mean ± SD, each n = 5. Representative tumor areas were stained with H E, and immunostained with antibodies against PAI-1 and p-FAK (D) . Bar, 50 mm. Number of microvessels per field by CD31 staining (×200) (E, right). Results are expressed as mean ± SD, n = 5, each. The representative tumor areas immunostained with an antibody against mouse CD31 are shown (E, left). Bar, 50 μm. * , P

Article Snippet: Human SERPINE1 expression plasmid (HG10296-UT) and control vector (pCMV) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Radial Immuno Diffusion, Inhibition, Injection, Mouse Assay, Staining

Journal: Oncotarget

Article Title: Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

doi: 10.18632/oncotarget.20834

Figure Lengend Snippet: PAI-1 expression correlates with poor prognosis in patients with ovarian cancer Immunohistochemical staining of PAI-1 in different malignant ovarian tissue sections (A). Representative areas of three different ovarian cancers scored as 1, 2, and 3, and one normal ovarian tissue sample scored as 0. Sections from breast cancer are shown as positive control for PAI-1 staining, and as negative control stained with nonimmune sera. Bar, 100 μm. Kaplan-Meier plot showing progression-free survival (PFS) (B) and overall survival (OS) (C) of patients with ovarian cancer treated at Gifu University Hospital and Osaka University Hospital (n = 154) stratified by PAI-1 expression level. Kaplan-Meier plot showing PFS (D) and OS (E) of patients with serous adenocarcinoma (n = 54) stratified by PAI-1 expression level. Kaplan-Meier plot showing PFS of patients with ovarian cancer (n = 1307) (F) and PFS of patients with serous ovarian cancer (n = 1144) (G) stratified by PAI-1 gene expression. Patients are split by the threshold of lower tertile according to the microarray expression data for the probe representing SERPINE 1 (202628_s_at). Kaplan-Meier plot showing PFS (H) (n = 395) and OS (I) (n = 485) of patients with stage II-IV high-grade serous ovarian cancer in TCGA database stratified by PAI-1 mRNA expression.

Article Snippet: Human SERPINE1 expression plasmid (HG10296-UT) and control vector (pCMV) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Immunohistochemistry, Staining, Positive Control, Negative Control, Microarray