Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: Gastric carcinoma TIMP3 promoter methylation and protein expression

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques: Methylation, Expressing

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: TIMP3 protein immunohistochemisty (SP 400×): a, normal gastric tissue; b, early gastric cancer; c, advanced gastric cancer; d, transfer of lymph node.

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques:

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: Relationship between advanced gastric cancer pathology TIMP3 methylation and its protein expression level

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques: Methylation, Expressing

Journal: Journal of Cancer

Article Title: LASS2 impairs proliferation of glioma stem cells and migration and invasion of glioma cells mainly via inhibition of EMT and apoptosis promotion

doi: 10.7150/jca.71256

Figure Lengend Snippet: Effect of LASS2 on cell migration, invasion and apoptosis. (A) Wound healing assay of pLV-vector or pLV-LASS2-transfected glioma/glioblastoma cells at 0 h, 12 h and 24 h after scratch. The images were taken from an inverted microscope under 10× magnification (*P < 0.05 and **P < 0.01, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (B) Colony formation assay in pLV-vector or pLV-LASS2-transfected U251 and U-87 MG cells. Images were acquired at 4× magnification (*P <0.05, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). (C) Transwell assay demonstrated that LASS2 inhibits the migration of U251 and U-87 MG cells compared with the pLV control (*P <0.05; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (D) The immunofluorescence staining of MMP9 and SPHK1 was shown in both U251 and U-87 MG cells. Scale bar = 200 µm. (E) RNA-Seq shows that LASS2 influenced cell migration/invasion, apoptosis, epithelial- mesenchymal transition (EMT) conversion and cellular life activity. (F) Overexpression of LASS2 reduced the protein levels of MMP2, MMP9, and SPHK1 while increasing that of TIMP2 in both U251 and U-87 MG cells. (G) LASS2 overexpression increased the levels of Bax, cleaved Caspase-3, TNF-α, and p53 while reducing that of Bcl-2 in both U251 and U-87 MG cells. (H) Overexpression of LASS2 reduced the protein levels of Vimentin and N-cadherin while increasing that of E-cadherin in both U251 and U-87 MG cells (F, G, and H, *P <0.05 and **P < 0.01, vs. pLV control in each cell line; unpaired two-tailed Student's t-test; n = 3).

Article Snippet: Then, 5% BSA diluted in Tris-buffered saline buffer containing 0.05% Tween 20 (TBST, pH 7.4) was used to block nonspecific protein binding sites at room temperature for 1 h. The membranes were incubated with antibodies specific for Bcl-2 (C-2; 1:1000; sc-7382), Bax (P-19; 1:1000; sc-526), MMP2 (K-20; 1:1000; sc-8835), MMP9 (C-20; 1:1000; sc-6840), and GAPDH (2E3-2E10; 1:1000; sc-293335) (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA) and other antibodies specific for both pro- and cleaved Caspase-3 (1:500; BM3257, Boster; Wuhan, China), TIMP2 (1:500; BM5024, Boster), Notch1 (1:1000; 20687-1-AP, Proteintech), TNF-α (1:1000; 60291-1-Ig, Proteintech), P53 (1:1000; 60283-2-Ig, Proteintech), Vimentin (1:1000; 60330-1-Ig, Proteintech), N-cadherin (1:1000; 66219-1-Ig, Proteintech), E-cadherin (1:1000; 60335-1-Ig, Proteintech) CD133 (1:1000; 18470-1-AP, Proteintech), Nestin (1:1000; 66259-1-Ig, Proteintech), Sox2 (1:1000; 11064-1-AP; Proteintech), LASS2 (1:1000; 20344-1-AP; Proteintech), and SPHK1 antibody (1:100; Proteintech) overnight at 4 °C.

Techniques: Migration, Wound Healing Assay, Plasmid Preparation, Transfection, Inverted Microscopy, Two Tailed Test, Colony Assay, Transwell Assay, Immunofluorescence, Staining, RNA Sequencing Assay, Activity Assay, Over Expression

Journal: Journal of Cancer

Article Title: LASS2 impairs proliferation of glioma stem cells and migration and invasion of glioma cells mainly via inhibition of EMT and apoptosis promotion

doi: 10.7150/jca.71256

Figure Lengend Snippet: LASS2 inhibited tumor growth in a pLV-LASS2-U-87 MG glioblastoma xenograft nude mouse model. (A) Representative photographs showing the gross pLV-LASS2-U-87 MG and empty scramble control glioblastoma xenografts from the nude mouse. (B) The tumor volume was evaluated between the scrambled control and pLV-LASS2 groups (**P < 0.05 and **P < 0.01 vs. pLV control group; unpaired two-tailed Student's t-test; n = 5 animals). (C) The final tumor weight was measured after dissection. The average final weight of tumors derived from pLV-LASS2-transcfected U-87 MG cells was significantly lower than those derived from the scrambled control (**P < 0.01, vs. pLV control group; unpaired two-tailed Student's t-test; n = 5 animals). (D) Representative images for H&E staining from either group were shown. (E) IHC staining of LASS2, TIMP2, MMP9, and SPHK1 in xenografted tumors derived from U-87 MG cells transfected with either pLV-LASS2 or scrambled control. Scale bar = 20 µm. (F) Western blot analysis of LASS2, SPHK1, TIMP2, MMP2, and MMP9 in xenografted tumors derived from U-87 MG cells transfected with either pLV or pLV-LASS2. (G) Western blot analysis of Bax, Bcl-2, pro-Caspase-3, cleaved Caspase-3, TNF-α and p53 in xenografted tumors derived from U-87 MG cells transfected with either pLV or pLV-LASS2. (H) Western blot analysis of EMT conversion-related proteins Vimentin, E-cadherin, and N-cadherin in xenografted tumors derived from U-87 MG cells transfected with either pLV or pLV-LASS2 ( F, G, and H, *P <0.05, **P < 0.01, and ***P < 0.001, vs. pLV control; unpaired two-tailed Student's t-test; n = 3).

Article Snippet: Then, 5% BSA diluted in Tris-buffered saline buffer containing 0.05% Tween 20 (TBST, pH 7.4) was used to block nonspecific protein binding sites at room temperature for 1 h. The membranes were incubated with antibodies specific for Bcl-2 (C-2; 1:1000; sc-7382), Bax (P-19; 1:1000; sc-526), MMP2 (K-20; 1:1000; sc-8835), MMP9 (C-20; 1:1000; sc-6840), and GAPDH (2E3-2E10; 1:1000; sc-293335) (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA) and other antibodies specific for both pro- and cleaved Caspase-3 (1:500; BM3257, Boster; Wuhan, China), TIMP2 (1:500; BM5024, Boster), Notch1 (1:1000; 20687-1-AP, Proteintech), TNF-α (1:1000; 60291-1-Ig, Proteintech), P53 (1:1000; 60283-2-Ig, Proteintech), Vimentin (1:1000; 60330-1-Ig, Proteintech), N-cadherin (1:1000; 66219-1-Ig, Proteintech), E-cadherin (1:1000; 60335-1-Ig, Proteintech) CD133 (1:1000; 18470-1-AP, Proteintech), Nestin (1:1000; 66259-1-Ig, Proteintech), Sox2 (1:1000; 11064-1-AP; Proteintech), LASS2 (1:1000; 20344-1-AP; Proteintech), and SPHK1 antibody (1:100; Proteintech) overnight at 4 °C.

Techniques: Two Tailed Test, Dissection, Derivative Assay, Staining, Immunohistochemistry, Transfection, Western Blot