Fig 1: Isoliquiritin apioside does not affect the adhesion ability and expression of EMT-related proteins and integrins of HT1080 cells. (A) The wells of 96-well culture plates were coated overnight at room temperature with 5 µg/mL fibronectin (Sigma), 0.3% type I collagen solution (Cellmatrix type I-A; Nittazerachin Co., Osaka, Japan), and 10 µg/mL vitronectin (Sigma) in a volume of 50 µL. After blocking with 200 µL RPMI containing 3% BSA, ISLA-treated or -untreated HT1080 cells suspended in serum-free RPMI (1 × 105/200 µL) were added to ECM-coated wells and then allowed to adhere for 1 h at 37°C. Unbound cells were washed with PBS three times, and the attached cells were fixed and stained with 0.2% crystal violet/20% methanol (w/v) solution. After washing with distilled water, dye was lysed with 1% SDS and measured absorbance at 560 nm using SpectraMaxi3 Multi-mode reader. Data are expressed as means ± SD (n = 3). (B,C) Cells were treated with or without ISLA for 24 h and the whole cell lysates were extracted. The levels of EMT-related proteins and integrins were measured by western blotting using EMT Antibody Sampler Kit (Cell Signaling Technology, cat. no. 9782) and Integrin Antibody Sampler Kit (Cell Signaling Technology, cat. no. 4749).
Fig 2: SMOC2 binds to integrin ß3.a ACHN and b 786-O cells were transfected with either a SMOC2-Myc or empty-Myc vector, then protein harvested after 24 h. Cell extracts were immunoprecipitated for Myc. Western blot analysis was performed on whole cell extracts (2.5% Input), supernatant and Myc-immunoprecipitated samples for Myc, integrin ß3 (ITGB3) and GAPDH. Western blot images are representative of repeated experiments.
Fig 3: SMOC2 interacts with integrin ß3 to mediate EMT.a ACHN and b 786-O cells were transfected with either scrambled siRNA (ssiRNA) or integrin ß3 siRNA (siITB3) 24 h prior to 5 ng/mL SMOC2 recombinant protein treatment, then protein harvested after 24 and 48 h. Western blot was performed on whole cell extracts for integrin ß3 (ITGB3), and EMT markers fibronectin and aSMA. GAPDH immunoblotting served as a loading control. Images are representative of n = 3; *P < 0.05 determined by t-test.
Fig 4: Cell adhesion properties and integrin expression of AGR-2-silenced PC3 cells. A. Adhesion properties of PC3control and PC3AGR2sh cells were evaluated following growth in culture dishes coated with various ECM proteins. First, cells were seeded in duplicate onto the coated substrates and allowed to adhere. Next, unbound cells were washed away, and the adherent cells were fixed and stained. Finally, the stain was extracted and quantified colorimetrically. Significant reduction in cellular adhesion to fibronectin (***p<0.001), collagen I (**p<0.01), collagen IV (***p<0.001), laminin (*p<0.05) and fibrinogen (*p<0.05) were observed in case of PC3AGR2sh cells when compared to PC3control cells. BSA coated wells were used as the reagent control (p>0.1). Data presented here are mean ± SEM. B. Significant down-regulation of a4, a5, aV, ß3 and ß4 integrins was observed in PC3AGR2sh cells as compared to PC3control cells. No difference in ß1 integrin levels was observed between the two cell types. Beta-actin was used as the loading control. Multiple protein bands present in the blots include integrin precursor and mature proteins as well as cleaved products is expected molecular mass according to manufacturer’s information (Cell Signaling Technology, Cat# 4749S).
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