Fig 2: Correlation between expression of FOXC2 and Ang-2 in HCC tissues. a The correlation between FOXC2 and Ang-2 expression levels (R2 = 0.709, p < 0.0001) in terms of bivariate analysis. b The Ang-2 expression was detected through western blot assay on MHCC-97H and Huh-7 cells when FOXC2 was knocked-down and overexpressed; the membrane was probed with antibody to ß-actin. c pGL3-FOXC2 was transfected into MHCC-97H cells that had been previously transfected with Ang-2 promoter-WT, Ang-2 promoter-Mut1, or Ang-2 promoter-Mut2 construct. d The activities of three Ang-2 promoters were determined through luciferase assay. The SD was obtained by conducting three experiments independently

Fig 3: Knockdown of Ang-2 in MHCC-97H and Huh-7 cells with FOXC2 overexpression. Ang-2 knockdown led to impairment of cell proliferation of both MHCC-97H (a) and Huh-7 (b) cells with FOXC2 overexpression. Decreased Ang-2 contributed to impairment of cell migration of both cells with FOXC2 overexpression (c–d). The FOXC2 overexpressing cells with Ang-2 knockdown exhibited decreased cell invasion (e–f). g Western blot assessing Ang-2 levels in MHCC-97H and Huh-7 cells with FOXC2 overexpression and Ang-2 knockdown (KD). Significance at p < 0.01 is denoted by ‘*’ relative to control by student’s t-test

Fig 4: FOXC2 facilitates in vivo and in vitro proliferation of HCC through Ang-2. a Representative images of NOD/SCID mice injected with FOXC2 OE and FOXC2 OE Ang-2 KD Huh-7 cells at day7, 9, 11 and 13. b Quantification analysis of bioluminescent signal of tumor-bearing mice injected with FOXC2 OE or FOXC2 OE Ang-2 KD Huh-7 cells at specified time points. Ang-2 downregulation impeded proliferation of FOXC2 OE Huh-7 cells c Tumor volume of FOXC2 OE Huh-7 cells were decreased with Ang-2 downregulation. d Tumor weight of FOXC2 OE Huh-7 cells were lightened with Ang-2 knockdown. e Ang-2 and FOXC2 expression in FOXC2 OE and FOXC2 OE Ang-2 KD tumors were analyzed through western blot. Values are presented as mean ± SEM. Significance at p < 0.01 is denoted by ‘*’ relative to control by one-way ANOVA

Fig 5: Schematic of the proposed mechanisms of action of GT3. From various assays and methods of detection determining the differential expression of various target proteins from Ang2-Tie2 pathway affecting the downstream pathways AKT and P38 pathways resulting an ultimate effect on cell survival, angiogenesis and vasculature of the lung tissue.