Figure 5.

Galectin-1 and FAK1 mutually activated each other in hypoxic lung epithelial cells. (a) Galectin-1 protein levels were increased following hypoxia treatment (1% O₂; 72 h) as determined by LC-MS/MS in H441 cells. *P<0.05 (Mann–Whitney U-test). Data are shown as mean±S.D. (n=3 independent experiments). (b) Loss of FAK1 H441 reduced galectin-1 mRNA expression in hypoxic H441 cells. FAK-null (FAK−) H441 cells were exposed to hypoxia for 24 h followed by quantitation of mRNA levels of galectin-1 via qRT-PCR. *P<0.05 (Student’s t-test). Data are shown as mean±S.E. (n=3 independent experiments). (c) SMAD3 inhibition reduced galectin-1 mRNA expression in hypoxic H441 cells. Cells were pretreated with SMAD3 inhibitor SIS3 (3 μM; 2 h) or vehicle followed by exposure to hypoxia (1% O₂; 24 h) and mRNA levels of galectin-1 were determined by qRT-PCR. *P<0.05 (Student’s t-test). Data are shown as mean±S.E. (n=3 independent experiments). (d) Galectin-1 treatment increased pFAK (Y397) levels in H441 cells. H441 cells were serum starved for 24 h and then treated with recombinant galectin-1 (50 ng/ml; 15 min). P<0.05 (Student’s t-test). Data are shown as mean (n=3 independent experiments). (e) Co-IP experiment detected interaction between galectin-1 and FAK1. HEK-293T cells were transfected with plasmid vectors expressing either V5-galectin-1 (lane 2) or GFP-FAK1 (lane 3) or both (lane 4). Cells transfected with empty vector (pCDNA, lane 1) were used as control. IP with V5 antibody and immunoblotting (IB) with GFP antibody revealed interaction between galectin-1 and FAK1 (lane 4). (f) PLA performed in H441 cells demonstrated physical interaction of endogenous galectin-1 and FAK1 proteins. Each red dot indicates interaction between the two proteins (scale bar=10 μm). No primary was used as a negative control, which displays minimal background staining. Images are representative of two independent experiments.