Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulation of SPINT 2/ HAI ‐2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion

doi: 10.1111/jcmm.14066

Figure Lengend Snippet: SPINT 2/ HAI ‐2 expression in bone marrow mesenchymal stromal cells (BMMSC) from patients after Azacytidine treatment. (A) SPINT 2/ HAI ‐2 mRNA and protein expressions in BMMSC from 1 healthy donor treated with Aza for 48 hours. (B) SPINT 2/ HAI ‐2 mRNA and protein expressions in BMMSC from 1 low‐risk myelodysplastic syndromes ( MDS ) patient treated with Aza for 48 hours. (C) SPINT 2/ HAI ‐2 mRNA and protein expressions in BMMSC from 1 high‐risk MDS patient treated with Aza for 48 hours. (D) SPINT 2/ HAI ‐2m RNA and protein expressions in BMMSC from 1 de novo AML patient treated with Aza for 48 hours. mRNA expression levels of SPINT 2 were normalized by HPRT endogenous control, as indicated. Results were analysed using 2 −ΔΔ CT . Experiments were performed in triplicate. Western blotting was done using protein extracted from total cell. The membrane was blotted with antibodies against SPINT 2/ HAI ‐2 (34 KD a), DMNT 3a (130 KD a), or GAPDH (37 kDa), as a control for equal sample loading, and developed with the ECL Western Blotting Analysis System. P values are indicated and correspond to the difference between BMMSC treated with vehicle ( DMSO ) and BMMSC treated with Aza for each sample

Article Snippet: HS‐5 stromal cells were transduced with lentivirus‐mediated shRNA nonspecific control (sc‐108080, Santa Cruz Biotechnology) or lentivirus‐mediated shRNA targeting SPINT2 (sc‐39556‐V, Santa Cruz Biotechnology) and namely shControl and shSPINT2 cells, respectively.

Techniques: Expressing, Control, Western Blot, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulation of SPINT 2/ HAI ‐2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion

doi: 10.1111/jcmm.14066

Figure Lengend Snippet: SPINT 2 silencing induces HGF secretion, hematopoietic cells adhesion onto HS ‐5 stromal cells and CD 34 + cells survival. (A) Quantitative expression of SPINT 2 mRNA in sh SPINT 2 cells relative to the shControl cells in HS ‐5 stromal cells. Lentivirus‐mediated SPINT 2 sh RNA effectively silenced SPINT 2 in HS ‐5 stromal cells. mRNA expression levels of SPINT 2 were normalized by HPRT endogenous control, as indicated. Results were analysed using 2 −ΔΔ CT . Experiments were performed in triplicate. P values are indicated. (B) Western blot analysis of shControl and sh SPINT 2 total cell extracts of HS ‐5 stromal cells. The membrane was blotted with antibodies against SPINT 2/ HAI ‐2 (34 kDa) or actin (42 kDa), as a control for equal sample loading, and developed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). (C) Analysis of HGF secreted by shControl and sh SPINT 2 HS ‐5 after 48 hours of culture. (D) CD 34 + cells from de novo AML patients were added to a monolayer of nontransduced and shControl and sh SPINT 2 HS ‐5 stromal cells and allowed to adhere for 24 hours. After 24 hours, nonadherent cells were removed by gentle aspiration, and the CD 34 + cells that adhered to HS ‐5 stromal cells were collected by gentle pipetting cold phosphate‐buffered saline. The percentage of CD 34 + adherent cells were measured by flow cytometry using CD 34‐ APC and analysed as percentage of total cells. Percentages of cells expressing the marker were determined out of a total 10,000 events. (E) CD 34 + cells from de novo AML total bone marrow were added to a monolayer of nontransduced and shControl and sh SPINT 2 stromal cells and cultured for 48 hours. After 48 hours, nonadherent cells were carefully collected by gentle aspiration, labelled with CD 34‐ APC antibodies and measured by flow cytometry. Values are means ± standard deviation of three independent experiments. Statistical analysis: Mann‐Whitney test. P values are indicated

Article Snippet: HS‐5 stromal cells were transduced with lentivirus‐mediated shRNA nonspecific control (sc‐108080, Santa Cruz Biotechnology) or lentivirus‐mediated shRNA targeting SPINT2 (sc‐39556‐V, Santa Cruz Biotechnology) and namely shControl and shSPINT2 cells, respectively.

Techniques: Expressing, Control, Western Blot, Membrane, Gentle, Saline, Flow Cytometry, Marker, Cell Culture, Standard Deviation, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulation of SPINT 2/ HAI ‐2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion

doi: 10.1111/jcmm.14066

Figure Lengend Snippet: Schematic pathway hypothesis of the biological effects of SPINT 2/ HAI ‐2 in haematological neoplasms (Myelodysplastic Syndrome and Acute Myeloid Leukemia, AML). Methylation results in the inhibition of SPINT 2/ HAI ‐2 expression in bone marrow mesenchymal stromal cells from myelodysplastic syndromes (MDS) and de novo AML patients, resulting in increased secretion of HGF with consequent increase in cell adhesion and in survival/growth of hematopoietic cells, mainly of the abnormal MDS and de novo AML cells, and contributing with the functional abnormalities of microenvironment niche and with cancer progression. This figure was created using Servier Medical Art tools ( http://www.servier.com )

Article Snippet: HS‐5 stromal cells were transduced with lentivirus‐mediated shRNA nonspecific control (sc‐108080, Santa Cruz Biotechnology) or lentivirus‐mediated shRNA targeting SPINT2 (sc‐39556‐V, Santa Cruz Biotechnology) and namely shControl and shSPINT2 cells, respectively.

Techniques: Methylation, Inhibition, Expressing, Functional Assay

Journal: Cancer science

Article Title: Hepatocyte growth factor activator inhibitor type 1 suppresses metastatic pulmonary colonization of pancreatic carcinoma cells.

doi: 10.1111/j.1349-7006.2010.01808.x

Figure Lengend Snippet: Fig. 1. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) ⁄ serine protease inhibitor Kunitz type 1 (SPINT1) knockdown in SUIT-2 cells and application of cytokeratin 19 (CK19) mRNA measurement by one-step nucleic acid amplification (OSNA) to detect SUIT-2 and its sublines. (A) Effect of HAI-1 ⁄ SPINT1 knockdown on E-cadherin expression. The knockdown cells (SUIT-2-KD) showed significantly reduced membranous E- cadherin immunoreactivity compared with the control cells (SUIT-2-sc). An immunoblot analysis of cellular HAI-1 ⁄ SPINT1 is also shown. (B) Correlation between CK19 mRNA copy numbers measured by OSNA and actual cell numbers. (C) CK19 mRNA levels in carcinoma cell lines derived from the pancreas (MIA Paca2, PANC-1, SUIT-4) and the biliary tract (TGBC1TKB, TGBC2TKB). (D) Comparison of CK19 mRNA copy numbers between parent SUIT-2, SUIT-2-sc and SUIT-2-KD. Mean ± standard error of eight independent experiments. (E) Effect of murine lung tissue extracts on the measurement of CK19 mRNA from metastatic SUIT-2 cells. Macroscopically metastasis-positive lung tissues (four cases) were obtained after tail vein injection of SUIT-2 cells. Human CK19 mRNA levels were directly measured by OSNA using each lung homogenate at different dilutions.

Article Snippet: The following primary antibodies were used in the present study: anti-human HAI1 ⁄ SPINT1 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA), anti-E-cadherin mouse monoclonal antibody (Takara Bio, Shiga, Japan) and anti-b-actin mouse monoclonal antibody (AC-74; Sigma, St Louis, MO, USA).

Techniques: Protease Inhibitor, Knockdown, Expressing, Control, Western Blot, Derivative Assay, Comparison, Injection

Journal: Cancer science

Article Title: Hepatocyte growth factor activator inhibitor type 1 suppresses metastatic pulmonary colonization of pancreatic carcinoma cells.

doi: 10.1111/j.1349-7006.2010.01808.x

Figure Lengend Snippet: Fig. 2. Effect of hepatocyte growth factor activator inhibitor type 1 (HAI-1) ⁄ serine protease inhibitor Kunitz type 1 (SPINT1) knockdown on experimental pulmonary metastasis. (A) Analysis of cytokeratin 19 (CK19) mRNA copy numbers in lung tissues 2 and 4 weeks after injection of tumor cells. Lung tissue homogenate diluted 10-fold was used for analysis and a logarithmic plot of CK19 mRNA copy numbers ⁄ microlitre of diluted sample is shown. Histologically confirmed cases are indicated as closed circles. (B) Histology (HE stain) and E-cadherin immunoreactivity of metastatic tumors (4 weeks after injection). Bar, 100 lm.

Article Snippet: The following primary antibodies were used in the present study: anti-human HAI1 ⁄ SPINT1 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA), anti-E-cadherin mouse monoclonal antibody (Takara Bio, Shiga, Japan) and anti-b-actin mouse monoclonal antibody (AC-74; Sigma, St Louis, MO, USA).

Techniques: Protease Inhibitor, Knockdown, Injection, H&E Stain

Journal: Cancer science

Article Title: Hepatocyte growth factor activator inhibitor type 1 suppresses metastatic pulmonary colonization of pancreatic carcinoma cells.

doi: 10.1111/j.1349-7006.2010.01808.x

Figure Lengend Snippet: Fig. 3. Effect of sHAI-1 ⁄ KD1 on SUIT-2 sublines in vitro. (A) Schematic representation of the molecular structure of hepatocyte growth factor activator inhibitor type 1 (HAI-1) ⁄ serine protease inhibitor Kunitz type 1 (SPINT1) and recombinant sHAI-1 ⁄ KD1. Amino acid number of the truncated position is shown above the structure. (B) RT-PCR analysis for the effect of sHAI-1 ⁄ KD1 on SIP1 ⁄ ZEB2, Snail, Slug, Twist and MMP-9 expression. (C) Restoration of E-cadherin expression by addition of sHAI-1 ⁄ KD1 (100 lg ⁄ mL) to SUIT-2-KD cells. Immunoblot analysis (upper panel) and immunocytochemical analysis are shown. Red, E-cadherin; blue, DAPI (nuclei). (D) Effect of sHAI-1 ⁄ KD1 (100 lg ⁄ mL) on Matrigel invasion in vitro. Representative images (chemoattractant, fibronectin) and a summary of the data are shown. Fibronectin (FN, 10 lg ⁄ mL; left graph figure) or FBS (5%; right graph figure) was used as a chemoattractant. Values are mean ± standard deviation. *P < 0.05; **P < 0.01 (Mann–Whitney U-test).

Article Snippet: The following primary antibodies were used in the present study: anti-human HAI1 ⁄ SPINT1 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA), anti-E-cadherin mouse monoclonal antibody (Takara Bio, Shiga, Japan) and anti-b-actin mouse monoclonal antibody (AC-74; Sigma, St Louis, MO, USA).

Techniques: In Vitro, Protease Inhibitor, Recombinant, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation, MANN-WHITNEY