Fig 1: CLU interacted with MEF2A to upregulate COL15a1(A) Jaspar database was used to estimate the potential TFs that could bind to the promoter of COL15a1, and ChIP assay was performed to prove this in oeCLU and NC groups. (B) Luciferase reporter assay was performed in shMEF2A cells to detect the luciferase activity. The mRNA expression of COL15a1 was then explored with qRT-PCR in shMEF2A, oeCLU, and oeCLU + shMEF2A groups. (C) The mutated promoter of COL15a1 was constructed. The luciferase activity of MEF2A was then detected in WT and mutant groups with cells transfected by shCLU, shMEF2A, oeCLU + shMEF2A, and NC. (D) Immunoprecipitation assay was performed with total, nucleus, and cytoplasm protein respectively to study the directly interaction between CLU and MEF2A. (E) Co-localized IF staining displayed the distribution of CLU and MEF2A. (F) The analysis of COL15a1 expression in seminoma based on UALCAN database. (G) The mechanism of CLU in inhibiting testicular seminoma metastasis.
Fig 2: ColXV induced ECM remodeling dependent on MMPs. All mice were pretreated with either Ad-GFP or Ad-Col15a1. (A) Relative mRNA expressions of Col1a1, Col4a1, Col6a1 and Fibronectin in mice inguinal white adipose tissue (iWAT) with or without GM6001 (n = 6). (B–D) Relative protein expressions of Fibronectin, Col I, Col VI, MMP-2, MMP-9, TIMP-1, TIMP-2 and Cleaved Caspase-3 in mice iWAT with gradients concentration of GM6001 (n = 6). (E-F) Relative protein expressions of Col I, Col VI, MMP-2, TIMP-1 and TIMP-2 in the Control group, APMA group and co-treatment of Ad-Col15a1 and APMA group in mice iWAT (n = 6), each group has two technical repeats (E). (G–J) Representative images of Col I and MMP-9 in adipocytes by immunofluorescent staining in different groups (n = 4). (K) Representative images of collagen bundles microstructure on adipocytes surface by scanning electron microscope. Scale bar, 30 µm (top) and 10 µm (bottom) (n = 4). Arrowheads indicate disrupted collagen fibers and exposed adipocytes surface (middle). Asterisks indicate adipocytes. Values are presented as mean ± SEM. *p < 0.05, ** p < 0.01, ns, not significant.
Fig 3: ColXV raised excessive deposition of ECM composition in WAT. All mice were pretreated with either Ad-GFP or Ad-Col15a1. (A) Relative mRNA expressions of Col15a1, Col1a1, Col6a1, Fibronectin, MMP-2, MMP-9 and MMP-14 in mice inguinal white adipose tissue (iWAT) (n = 6). (B–D) Relative protein expressions of Fibronectin, Col I, Col VI, MMP-2, MMP-9, TIMP-1, TIMP-2 and Cleaved Caspase-3 in mice iWAT (n = 6), each group has two technical repeats. (E–F) Representative images of Masson’s trichrome staining (collagen is shown as blue, nuclei as black and cytoplasm as red) in frozen-section iWAT samples. Scale bar, 100 µm (top) and 30 µm (bottom) (n = 4). Arrowheads indicate thinner collagen fibers (left) and thicker collagen fibers (right). (G) Representative images of collagen bundles microstructure on adipocytes surface by scanning electron microscope. Scale bar, 30 µm (top) and 10 µm (bottom) (n = 4). Arrowheads indicate thinner collagen bundles (left) and thicker collagen bundles (right). Asterisks indicate adipocytes. Values are presented as mean ± SEM. *p < 0.05, ** p < 0.01.
Fig 4: 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.
Fig 5: ColXV exacerbated apoptosis in unfavorable state adipocytes and involved a mitochondrial pathway. (A–C) Relative mRNA and protein expressions of Caspase-3/Cleaved Caspase-3, Caspase-9/Cleaved Caspase-9, Bax, Bcl-2, Bid, and Bad in mice adipocytes in the Control group, sh-Col15a1 group and Ad-Col15a1 group with or without FBS (Fetal Bovine Serum) treatment (n = 4). (D–F) Relative mRNA and protein expressions of Caspase-3/Cleaved Caspase-3, Caspase-9/Cleaved Caspase-9, Bax, Bcl-2, Bid, and Bad in mice adipocytes in the Control group, sh-Col15a1 group and Ad-Col15a1 group with or without PA (palmitate) treatment (n = 4). (G–H) JC-1 staining of mitochondrial membrane potential in adipocytes. Scale bar, 100 µm (n = 4). (I) Representative images and enlarged image (bottom right) of Mito-tracker staining (Red) and Cytc (Cytochrome C) immunofluorescent staining (Green) in adipocytes in different groups. Scale bar, 100 µm (n = 4). Arrowheads indicate Cytc released from mitochondria to the cytoplasm. Scale bar, 20 µm. Values are presented as mean ± SEM. *p < 0.05, ** p < 0.01.
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