Fig 1: Knockdown of ESM1 on the migration and invasion of PC3 and DU145 cell lines(A) The abilities of migration and invasion of shLuc or shESM1-PC3 and shESM1-DU145 cells were determined by using a migration assay and Matrigel-invasion assay. Cells in the lower surface of the Borden chamber were stained and photographed under a light microscope at ×400 magnification. Quantification of migrated cells was shown as a histogram chart. (B) Western blots analysis on ESM1, MMP-9 and TIMP-1 expression in shLuc or shESM1-PC3 and shESM1-DU145 cells. ß-actin was used as internal control for protein equal loading. (C) The immunofluorescence staining of ESM1, MMP-9, and TIMP-1 expression. DAPI staining of nucleus in each cell line. Data are presented as the mean ± SE of at least three independent experiments. **p < 0.01, compared with shLuc cells. Scale bars = 100 mm.
Fig 2: The expression of ESM1 in TCGA pancancer gene expression analysis and GSE161533 GEO database. (a) ESM1 is significantly highly expressed in 12 of 20 types of human cancer, including ESCA. Red and blue represent tumor (T) and normal (N) tissues, respectively. (b) ESM1 is distinctly highly expressed in ESCA (T = 162, N = 11). (c) ESM1 is highly expressed in tumors compared with the paired normal tissue (T = 11, N = 11). (d–f) ESM1 was remarkably highly expressed in ESCA tumors compared with normal and/or the paired tissues (T = 28, N = 28, and para-T = 28). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig 3: Expression of ESM1 in prostate cancer cells and knockdown ESM1 on the expression of ESM1 of PC3 and DU145 cells(A) Total lysate from PC3, DU145, and 22Rv1, LNCap cells were isolated and analyzed by western blotting (B) Total RNAs were isolated and then qRT-PCR assay was applied to detect ESM1 mRNA expression. (C) PC3 and DU145 cells were infected with shLuc or shESM1 and then purpomylin (2 or 10 mg/ml) for 5 days. Then, total lysates were isolated and analyzed by western blotting. (D) qRT-PCR assay was applied to detect ESM1. β-actin was used as internal control for protein equal loading. Values are expressed as the mean ± SE of three independent experiments. **p < 0.01.
Fig 4: Rh-TIMP-1 attenuates knockdown ESM1 induces migration and invasion in PC3 cellsThe shLuc and shESM1-PC3 cells were treated with 100 ng/mL RhTIMP-1 or cotreated with 100 ng/ml and 1mg TIMP-1 antibody for 24 h. (A, C) Protein expression of ESM1, MMP-9 and TIMP-1, as measured with western blotting. (B, D) The abilities of migration and invasion of cells were determined by using migration and invasion assay. Quantification of migrated cells was shown as a histogram chart. **p < 0.01, compared with shLuc cells. #p < 0.01, compared with Rh-TIMP-1 treated cells.
Fig 5: ESM1 is regulated by TNFα through RelB subunit of NF-κB. (a) qRT-PCR of ESM1 levels in MDA-MB-231-S tumor cells treated with various concentrations of TNFα (ng/mL). 0 ng/mL TNFα treated group as control, ** p < 0.01; *** p < 0.001, by t test. (b) Dot blotting of ESM1 in MDA-MB-231-S tumor cells supernatant under various concentrations of TNFα (ng/mL) treatment. (c) RelA or RelB was overexpressed in MDA-MB-231-S cells and detected by western blotting (Figure S2). (d) Dot blotting of ESM1 in MDA-MB-231-S tumor cells supernatant after overexpressed RelA or RelB, dot of PBS as control. (e–g) Design of luciferase reporter assays in HEK293T cells transfected with the indicated plasmids for 24 h, luciferase activity was determined, *** p < 0.001; **** p < 0.0001, by t test.
Supplier Page from Abcam for Anti-ESM1 antibody