Fig 1: BMP9 promotes EpCAM+ CSC properties in HCC cells. (A, B) Flow cytometry analysis of the percentage of EpCAM-positive cells in Huh7 cells and the percentage of CD90-positive cells in HLE cells. Cells were treated with dimethyl sulfoxide or BMP9 (2.5 ng·mL-1, 5 ng·mL-1) for 10 days. (C) Relative gene expression levels of EpCAM, Endoglin, SMAD1, ID1, TGF-ß1, and SNAI2 in Huh7 cells treated with BMP9 (2.5 ng·mL-1) for 5 days. (D) Western blot analysis of EpCAM and ID1 expression in Huh7 cells treated with different concentrations of BMP9 for 5 days. (E) Optical density at 450 nm (OD450) in cell proliferation assay of Huh7 cells treated with BMP9 (5 ng·mL-1) for 48 h. (F) Representative spheroid assay images of Huh7 cells treated with or without BMP9 (5 ng·mL-1) for 14 days. BMP9 was added to the medium twice a week. Scale bar = 500 µm. (G) Spheroid diameter of (F). (H) Number of spheroids measuring > 200 µm of (F). (I) Representative invasion assay images of Huh7 cells treated with BMP9 (5 ng·mL-1) for 24 h. Scale bar = 200 µm. (J) Relative cell numbers from invasion assay. (K) Representative migration assay images of Huh7 cells treated with BMP9 (5 ng·mL-1) for 24 h. Scale bar = 200 µm. (L) Relative cell numbers from migration assay. Error bars represent the SD from at least three independent biological replicates. Student's t-test was used to calculate P values represented as *P < 0.05; **P < 0.01; ***P < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com]
Fig 2: MACC1-AS1 is induced by mesenchymal stem cell (MSC)-derived TGF-ß1 and contributes to stemness and chemoresistance. a, b Expression of MACC1-AS1 in gastric cancer (GC) cells (a) and spheres of GC cells (b) after co-culture with MSCs. c Expression of MACC1-AS1 in the indicated subcutaneous tumor of nude mice, formed by MKN45 cells with or without MSCs. d The score of MACC1-AS1 in CD29(-)CD90(-) and CD29(+)CD90(+) GC tissues. e, f Expression levels of stemness-associating genes was increased in the indicated AGS and MKN45 cells after MACC1-AS1 overexpression by quantitative real-time polymerase chain reaction (e) and western blotting (e) (V vector, M MACC1-AS1 overexpression). g Representative images of sphere-formation assay in AGS and MKN45 cell after overexpressing MACC1-AS1. Scale bar = 500 µm. h Colony-formation assay and the quantitative graph of AGS and MKN45 cells treated with 5-florouracil (1 µg/mL) and oxaliplatin (3 µg/mL) after transfected with MACC1-AS1 compared to vector. i Expression of MACC1-AS1 in AGS and MKN45 incubated with GC medium, <3 kD MSC-CM or >3 kD MSC-CM. j Transforming growth factor (TGF)-ß1 concentration in AGS and MKN45 medium, >3 kD MSC-CM and <3 kD MSC-CM measured by enzyme-linked immunosorbent assay. k Expression levels of TGF-ß receptors and SMAD family in AGS and MKN45 cells after co-culture with MSCs. l Expression of MACC1-AS1 in AGS and MKN45 cells treated with TGF-ß1 (20 µg/mL) for 24 h. m, n Expression of MACC1-AS1 in AGS and MKN45 cells incubated with >3 kD MSC-CM treated with TGF-ß1 inhibitor disitertide (m) and TGFßR-I inhibitor LY-364947 (n) for 24 h. *P < 0.05; **P < 0.01; ***P < 0.001
Fig 3: UCCs are heterogeneous for cytokeratin expression and proportions of differentiation states. a Differentiation state model of UC according to Volkmer et al. [10]. Relative mRNA expression of epithelial markers E-cadherin and miR-200 and mesenchymal markers Vimentin and ZEB1 (b) and CK14, CK5, and CK20 (c) measured by qRT-PCR in a panel of 11 human UCCs. UCC expression levels were quantified relative to an internal standard. TBP was used as reference gene. d Mean percentages of CD90, CD44, and CD49f positive cells in 11 UCCs as measured by flow cytometry. UCCs were categorized into epithelial and mesenchymal phenotype. Values are expressed as the mean ± SD of triplicates
Fig 4: Sphere-forming ability of CD90+ and CD90- cells. Spheroid formation assay. CD90- and CD90+ cells were cultured on 6-well plates or as cell spheres. The cells were imaged under a light microscope (magnification, ×200) following 20 days of incubation. Each experiment was repeated three times. Scale bar, 100 µm.
Fig 5: The identification of the freshly isolated spermatogonia, pachytene spermatocytes, and round spermatidsImmunocytochemistry showed the protein expression of UCHL1 a, GFRA1 b, THY1 c, and GPR125 d in the freshly isolated mouse spermatogonia. Immunocytochemistry demonstrated the protein expression of PRM1 e and PNA f in the isolated mouse round spermatids. Meiosis spread assays by triple immunostaining revealed the expression of CREST (blue fluorescence), MLH1 (green fluorescence) and SCP3 (red fluorescence) in the freshly isolated mouse pachytene spermatocytes g. The data shown in a–g were representatives from three independent experiments. Replacement of primary antibodies with isotype IgGs in male germ cells served as a negative control h. Scale bars in a–h = 10 µm
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