Fig. 8

Fibronectin up-regulated WISP3 expression by activating HIF-1α in NSCLC cells. (A) IHC assay was performed to detect the WISP3 expression in NSCLC cancer tissues with high or low fibronectin expression, and representative data were shown. (B) Luciferase experiments were used to determine the role of fibronectin on the transcriptional activity of WISP3 promoter. (C) The potential binding site for HIF-1α to WISP3 promoter was predicted by JASPAR dataset and details were shown in the schematic diagram. (D) Western blot assay was used to detect the proteins of HIF-1α in H1299 cells treated with or without dish-coated fibronectin (10 µg/mL) for 24 h. β-actin was used as a loading control. Quantitative analysis of proteins expression were shown in the histogram. (E) H1299 and H460 cells were transfected with siRNA against HIF-1α or negative control (NC) for 24 h, followed by treating with or without dish-coated fibronectin (10 µg/mL) for 24 h. Then, qPCR was performed to examine the expression of WISP3 mRNA. (F) Western blot assay was used to detect the WISP3 proteins levels expressed in H1299 and H460 cells treated as (E). β-actin was used as a loading control. Quantitative analysis of proteins expression were shown in the histogram. (G) H1299 and H460 cells were treated with or without dish-coated fibronectin (10 µg/mL) for 24 h, CHIP assay was performed using HIF-1α antibody to determine the binding site of HIF-1α on WISP3 promoter. (H) Luciferase experiment was used to identify the role of fibronectin on the wildtype WISP3 promoter or the mutant one, in which the binding site1 to HIF-1α was mutant. Data were presented by mean ± SD from three independent experiments. *P < 0.05; **P < 0.01 vs. control