Figure 5.

SOX9 is a critical mediator involved in Gal-1-induced upregulation of β-catenin activity and CIC features. (A) Ingenuity Pathway Analysis (IPA) for the prediction of candidate mediators within the LGALS1/CTNNB1/Twist1 axis. IPA database revealed the several major pathways which might be involved in tumor development and metastasis. According to the IPA results and literature review, SOX9 was selected and confirmed whether it is the downstream gene of Gal-1 by Western blot. (B) Western blot for the analysis of SOX9 protein levels in KM12C after culturing in MRC-5- or WS1-CM; KM12C-CM was used as the control. Internal control: GAPDH. (C) Western blot for SOX9 levels in whole cell lysate (top panel) and nuclear fraction (bottom panel) of KM12C with addition of increasing doses of rhGal-1 for 48 hours; for nuclear protein blot, histone H1 is used as the positive control and α-Tubulin as the negative control. (D) Sphere formation capacity of shC-KM and shSOX9-II-KMC12 (shSOX9-KM) after treating with rhGal-1 (100 ng/ml) for 72 hours. (E) Drug resistance capacity of shC- and shSOX9-KM to cisplatin (25 µM) after pretreatment with rhGal-1 (100 ng/ml) for 24 hours. Cell viability was assessed 48 hours after drug treatment. (F) qPCR analysis for the gene expression of Twist1 in shC- and shSOX9-KM after treatment with Gal-1 (100 ng/ml) and XAV (10 µM). (G) Invasion capacity of shC- and shSOX9-KM with addition of rhGal-1 (100 ng/ml) and XAV (10 µM). All results are shown as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01, and ***p < 0.005 compared to the control. N.S., not significant.