Fig 1: The expression of ECM-associated COL15A1 and COL7A1 was significantly changed with CLU knockdown or overexpression(A) The most enriched GO terms based on the RNA-seq data of oeCLU and oe-NC groups. (B) Data downloaded from TCGA were submitted to GSEA and extracellular matrix organization was also significantly enriched in samples with higher CLU expression. (C) 21 and 115 upregulated ECM-associated genes from GO and GSEA terms were overlapped. COL15a1, COL7A1, and VCAN were screened out. (D) The co-expression of CLU with COL15a1, COL7A1, and VCAN based on the database: GEPIA. (E) The verification of co-expression relationship with 50 seminoma tissues. (F and G) mRNA and protein expression of COL15a1, COL7A1, and VCAN in pretreated Tcam-2 cells. Data are represented as mean ± SD. *p < 0.05.
Fig 2: COL15a1 competitively binds to the DDR1 with collagen I to block the phosphorylation of PYK2 and so inhibit the EMT process(A) Tcam-2 cells (EV) and Tcam-2 cells with high level of CLU (oeCLU) were transfected with NC and shCOL15a1 lentivirus. The invasion abilities of pretreated cells were determined by the transwell assay. (B) The expression of indicated proteins was detected by western blot. (C) COL7a1 was also downregulated as above methods, and the transwell assays were followed subsequently. (D) Western blot assays were performed to detect the indicated proteins level. (E) Pretreated cells were coated or non-coated with collagen I. The cell aggregation indicated their scattering ability. (F) The downstream markers of collagen I (pPYK2, pFAK) and collagen XV (pPYK2) were detected by western blot. (G) In vivo imaging of testicular xenografts in situ injected with four groups of pretreated cells. Metastasis occurred in the lung, medial iliac, mediastinal, and inguinal lymph nodes. The number of metastatic foci and the tumor volumes were recorded. (H) Photographs and H&E staining of testicular xenografts in situ. (I) The protein markers of EMT (E-cad, N-cad, and Vimentin) in group 2 primary tumor and group 4 pulmonary metastasis were detected by IHC. Data are represented as mean ± SD. Scale bar: 20 µm.
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