Fig 2: LRG1 Overexpression Promoted Proliferation, Migration, and Invasion in NSCLC Cells(A and B) The expression levels of LRG1 in A549 cells transfected with LRG1 overexpression plasmid (LRG1oe) or empty vector (Vec) were confirmed using qRT-PCR (A) and western blotting (B). (C) MTT assays showed LRG1 overexpression significantly enhanced proliferation of A549 cells. (D) Wound-healing assays showed the enhancement of cell migration of A549 due to overexpression of LRG1. (E) Transwell assays showed the increase of cell invasion of A549 due to overexpression of LRG1. **p < 0.01; ***p < 0.001 (two-way ANOVA for C, Student’s t test for others).

Fig 3: The overexpression of leucine-rich alpha-2-glycoprotein (LRG) in Panc1 cells promoted invasion with transforming growth factor (TGF)-ß1 treatment. A 48-h invasion assay revealed that Panc1/LRG cells had a stronger invasion ability with TGF-ß1 exposure than parental Panc1 and Panc1/pcDNA cells (P < .05). The upper panel shows a representative picture (upper: low magnification, middle: high magnification). Cells were stained for 48 h after plating. The lower panel shows the number of invading cells. The number of invading Panc1/LRG cells was approximately 180% that of the number of invading parental Panc1 cells

Fig 4: Cytokines induced leucine-rich alpha-2-glycoprotein (LRG) production in Panc1 cells. A, The LRG protein level in the supernatant of pancreatic ductal adenocarcinoma cells and hepatocellular carcinoma cells. In contrast with hepatocellular carcinoma cells, the pancreatic ductal adenocarcinoma cell lines did not produce LRG. B, The phosphorylation of NF-?B and STAT3 in Hep G2 and Panc1 cells, stimulated by interleukin (IL)-1ß or tumor necrosis factor (TNF)-a/IL-6 or IL-22. Panc1 and HepG2 cells were pre-treated with the indicated cytokines for 30 min. C, The LRG protein level in culture supernatants of Panc1 cells stimulated for 72 h with increasing doses of IL-1ß, TNF-a, IL-6 or IL-22 (1-100 ng/mL). D, The relative LRG mRNA expression in Panc1 cells following IL-1ß (1-100 ng/mL) or IL-6 (1-100 ng/mL) exposure for 24 h. *P < .05 in comparison with control

Fig 5: Promoter methylation level of LRG1 gene in ccRCC and subgroups. (a) Promoter methylation level of LRG1 gene is significantly downregulated compared with normal controls. (b) Methylation level of LRG1 gene in male and female patients is decreased compared with normal patients (p < 0.0001). (c) Methylation level of LRG1 gene in different ages has significant differences compared with normal controls (p < 0.0001). (d) Methylation level of LRG1 gene in different races has significant differences compared with normal controls (p < 0.001). (e) Methylation level of all of the stages are downregulated than normal controls (p < 0.001). (f) Methylation level of all of the grads are downregulated than normal controls (p < 0.0001). (g) Methylation level of metastatic ccRCC is lower than nonmetastatic, but both of them are significantly downregulated than normal controls (p < 0.001).