Fig. 1: Inverse tumorigenicity of ECM1 subtypes.

a Detection of ECM1 mRNA levels in CLs of ovarian cancer cell lines (Hey, HeyA8, SKOV3, SKOV3ip1, OVCA433, OVCA429, and A2780) and a normal cell line (HOSE) by qRT-PCR. ACTB (encoding β-actin) was used to normalize the expression of ECM1. Data are presented as mean ± SD. n = 3 biologically independent repeats. Representative images of ECM1 staining performed by IHC in normal ovary (b), normal fallopian tube (c), and ovarian carcinoma (OC) (d) tissues. The arrows indicate typical tissues (normal or cancer epithelium) stained with ECM1. Bars with 200 or 100 μm indicate the magnification of images. Silencing efficiency of ECM1 with specific shRNAs detected by WB (e), 3D culture image showing spheroid formation (f, bars = 400 µm) and numbers (g, data are presented as mean ± SD, n = 3 biologically independent repeats, two-tailed t-test), and xenograft tumor growth (h, data are presented as mean ± SD, n = 8v8 mice, two-tailed t-test) in eight mice generated from cells expressing ECM1 shRNA or control shRNA. Functional examination of ECM1 subtypes in HeyA8-ECM1 shRNA cells (A8i) after transfection of ECM1a (A8i-A), ECM1b (A8i-B), or empty vector (A8i-V), as confirmed by WB analysis (i); 3D culture images (j, bars = 400 µm) and spheroid number (k, data are presented as mean ± SD, n = 3 biologically independent repeats, two-tailed t-test); and assessment of xenograft tumor growth (l, data are presented as mean ± SD, n = 8v8 mice, two-tailed t-test) and tumor tissues (m) in eight mice injected with cells expressing ECM1a or ECM1b. n Alterations in signaling molecules associated with AKT/FAK/Paxillin/Rac after silencing of ECM1 and overexpression of ECM1a and ECM1b. Detection of the expression of phosphorylated proteins in cells treated or not treated with an HA antibody (o, A8i-A cells) or treated/not treated with bioactive ECM1 (ECM1a) (p, OVCA429 cells). PBS and mouse IgG were used as controls. β-actin was used as a loading control for WB analysis.