Journal: Angiogenesis

Article Title: TFEB controls integrin-mediated endothelial cell adhesion by the regulation of cholesterol metabolism

doi: 10.1007/s10456-022-09840-x

Figure Lengend Snippet: TFEB silencing increases the integrin protein levels. A qPCR of ITGB1, ITGB3, ITGA5 and ITGAV expression in scr-shRNA and sh-TFEB-ECs. Data are expressed as relative fold change in sh-TFEB-ECs compared with the expression in scr-shRNA-ECs after normalization to the housekeeping gene TBP ( n = 3 independent experiments, values as mean ± SEM; p = ns sh-TFEB-ECs versus scr-shRNA-ECs by Student’s t test). B Representative western blot of active and total β1 integrins immunoprecipitated from scr-shRNA- or sh-TFEB-ECs lysates. After immunoprecipitation (IP) with anti-active β1 integrin (9EG7) and anti-total β1 integrin antibodies, proteins were blotted (IB) with anti-total β1 integrin antibody. In the same lysates used for immunoprecipitation, the endogenous expression of total β1 integrin and β-tubulin were blotted with the specific antibodies and presented as “input”. C , D Confocal microscopy analysis of plasma membrane active-β1 and active- α5β1 integrin expression and localization in living scr-shRNA- and sh-TFEB-ECs following incubation with anti-active β1 integrin (9EG7), active-α5β1 (SNAKA-51) and phalloidin-647 (scale bar: 25 µm). The bar graphs show the quantification of the mean intensity of active-β1 integrin and active- α5β1 ( n = 10 cells per condition pooled from three independent experiments; values as mean ± SEM; *** p < 0.0001 sh-TFEB-ECs versus scr-shRNA-ECs, as determined by Student’s t test)

Article Snippet: The following TaqMan assays were used: TFEB (Hs00292981_m1), ITGB1 s01127536_m1), ITGB3 (Hs01001469_m1), ITGA5 (Hs01547673_m1), ITGAV (Hs00233808_m1), CAV-1 (Hs00971716_m1), SCAP (Hs00378725_m1), SREBF-2 (Hs01081784_m1), HMGCR (Hs00168352_m1), and TBP (Hs00427620).

Techniques: Expressing, shRNA, Western Blot, Immunoprecipitation, Confocal Microscopy, Clinical Proteomics, Membrane, Incubation

Journal: Oncotarget

Article Title: Tumor secreted ANGPTL2 facilitates recruitment of neutrophils to the lung to promote lung pre-metastatic niche formation and targeting ANGPTL2 signaling affects metastatic disease

doi: 10.18632/oncotarget.27433

Figure Lengend Snippet: ( A ) To induce tissue specific knockout of integrin 5α in Type II alveolar cells, tamoxifen was administrated. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells were purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. Protein lysate from the purified alveolar type II epithelial cells analyzed by western blot with antibodies as shown. ( B ) Osteosarcoma cells were injected into the tibia of either Itga5-flox (herein, wild-type, WT) or Itga5-floxed mice ( n = 40, 10 mice for each group). After primary tumors reached around 800 mm 3 (range from 4 to 5 weeks), primary tumor containing leg was amputated. Animals reaching endpoints were terminated, and lungs were harvested, insufflated, fixed, sectioned, and stained. The number of lung sections with metastatic nodules were compared with ordinary Two-way Anova analysis, Itga5-floxed vs wt-Itga5 (wild-type, WT). **** P < 0.0001. ( C–D ). Prolonged survival of mice following deletion of integrin α5β1. *** P < 0.001, WT vs Itga5-floxed injected with LM9 and WT vs Itga5-floxed injected with K7M2 by Log-rank (Mantel-Cox) test. ( E–F ) Deletion of integrin α5β1 in the alveolar type II (AT-II) cells does not impact on the primary tumor growth in the tibia. Tumor growth was monitored by caliper measurements for four weeks.

Article Snippet: Next, to evaluate the role of ANGPTL2’s receptor integrin α5β1 in the metastatic process, we crossed Itga5 (integrin5α) conditional knockout mice (Taconic) with Sftpc-CreER T2 (Jackson Laboratory) to induce time- and tissue-specific knockout of integrin α5 gene in Type II alveolar cells (herein, Itga5-floxed, after tamoxifen administration).

Techniques: Knock-Out, Purification, Selection, Western Blot, Injection, Staining

Journal: Oncotarget

Article Title: Tumor secreted ANGPTL2 facilitates recruitment of neutrophils to the lung to promote lung pre-metastatic niche formation and targeting ANGPTL2 signaling affects metastatic disease

doi: 10.18632/oncotarget.27433

Figure Lengend Snippet: ( A–B ) The proportions (A) and absolute numbers (B) of neutrophils in the lung were detected by flow cytometry in Itga5-floxed mice or WT littermates after with LM9 and K7M2 cells inoculation into tibia (Gating strategy for neutrophils is shown in Supplementary Figure 4). ( C ) Absolute numbers of neutrophils in the lungs were detected by flow cytometry in WT littermates after intratibial inoculation of LM9-shCtr, LM9-shANGPTL2, K7M2-shCtr and K7M2-shANGPTL2 cells at indicated weeks. ( D ) Serum was isolated from tumor bearing animals one week after intratibial injection of tumor cells and analyzed by ELISA according to the manufacturer’s instructions (R&D Systems). Similar results were obtained in BALF (Supplementary Figure 6A–6B) ( E ) CXCL1, CXCL2, CXCL5, and CXCL12 transcripts were significantly lower in Itga5-floxed AT-II cells compared to WT AT-II cells. AT-II cells were isolated from tumor bearing animals after a week of intratibial injection with indicated cells. Subsequently, total RNA was extracted and used for the gene expression analysis of chemokines. Results were normalized to GAPDH. Unpaired Student’s t -tests, * p < 0.05, ** p < 0.01, *** p < 0.001. ( F ) Depletion of neutrophils results in a dramatic reduction of lung metastases. The numbers of lung sections with metastatic nodules were compared with ordinary Two-way Anova analysis analysis, IgG2a vs anti-Ly6G treated. *** P < 0.001.

Article Snippet: Next, to evaluate the role of ANGPTL2’s receptor integrin α5β1 in the metastatic process, we crossed Itga5 (integrin5α) conditional knockout mice (Taconic) with Sftpc-CreER T2 (Jackson Laboratory) to induce time- and tissue-specific knockout of integrin α5 gene in Type II alveolar cells (herein, Itga5-floxed, after tamoxifen administration).

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

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. Genes and assays included in the low-density array for the study of the expression of angiogenic factors by quantitative RT-PCR.

Article Snippet: ITGA5-Hs00233743_m1 , 0.007016879 , 0.75174636 , 2.4165378 , 0.00529407 , 0.98316264 , 0.003165355 , .

Techniques: Expressing, Amplification

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. 2- DDCt values corresponding to the Nu335 series.

Article Snippet: ITGA5-Hs00233743_m1 , 0.007016879 , 0.75174636 , 2.4165378 , 0.00529407 , 0.98316264 , 0.003165355 , .

Techniques:

Journal: ecancermedicalscience

Article Title: High-risk gastrointestinal stromal tumour (GIST) and synovial sarcoma display similar angiogenic profiles: a nude mice xenograft study

doi: 10.3332/ecancer.2017.726

Figure Lengend Snippet: Supplementary. 2- DDCt values corresponding to the Nu407 series.

Article Snippet: ITGA5-Hs00233743_m1 , 0.007016879 , 0.75174636 , 2.4165378 , 0.00529407 , 0.98316264 , 0.003165355 , .

Techniques: