Fig 1: Effect of cleaved GLI2 fragment on liver metastasis in CRC.(A–D) Effect of LA on liver metastasis was examined in the orthotopic colon cancer models using CT26 cells-BALB/c mice and HT29 cells-nude mice. In GLI2 knockdown (KD) group, cells were knocked downed GLI2 before inoculation. In LA or KD+LA groups, mice were fed with 15% LA diet for the first week. (A, B) Growth of the primary tumors in the cecum. (Insert) Plasma LA concentration. (C, D) Number of the liver metastasis foci. Error bars indicate standard deviations of 5 mice. Statistical differences were calculated by chi-square test. (Insert) Protein levels of GLI2 and the cleaved GLI2 fragment examined by immunoblotting. (E) Protein levels of GLI2, cleaved GLI2 and SOX17 were examined in the 11 CRCs with serosal invasion (pT3) and a few nodal metastasis (pN1) by immunoblotting. Abbreviations: LA: linoleic acid; GLI: glioma-associated oncogene homolog; cGLI2: cleaved GLI2; SOX: SRY-box transcription factor; Cont: control.
Fig 2: Effect of LA on generation of cleaved GLI2 in CT26 cells.(A) Nuclear protein levels of GLI2 and cleaved GLI2 in CT26 cells. (B) Nuclear protein levels of determined using an anti-GLI2 N-terminus antibody. GAPDH was examined as a cytosolic marker. (C) Nuclear protein levels of GLI2 and cleaved GLI2 in Colo320 and HT29 cells (D) Effect of siGLI2 on nuclear GLI2 protein levels in LA-treated CT26 cells. (E) Effect of Gli2 knockdown on SOX17 expression in CT26 cells treated with LA. (F) Sphere formation assay. The bar graph shows the quantification of total sphere numbers of 3 cell lines. Production of SOX17 in untreated control, control siRNA-treated, and siGLI2-treated CRC cells. CRC cells were treated with 35 μM of LA, which was equivalent to inhibitory concentration 25. Abbreviations: LA: linoleic acid; GLI: glioma-associated oncogene homolog; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; siGLI2: short interference RNA for GLI2; siC: short interference RNA; SOX: SRY-box transcription factor; Cont: control.
Fig 3: Effect of LA on the ubiquitination of GLI2 in CT26 cells.(A) Effect of LA on the ubiquitination of GLI2 in nuclear fraction of LA-treated CT26 cells. (B) The same nuclear extract of A was subjected to western blotting with anti-GLI2 antibody. (C) Effect of acylation inhibitor hydroxylamine (NH2OH, 1.75 mM) on LA-induced inhibition of GLI2 ubiquitination in nuclear fraction of CT26 cells. (D) The same nuclear extract of C was subjected to western blotting with anti-GLI2 antibody. (A, C) Nuclear fraction from LA- or LA+hydroxylamine-treated CT26 cells were immunoprecipitated using an anti-ubiquitin antibody (UB ppt). The electrophoresed UB ppt samples were detected by immunoblotting with an anti-GLI2 antibody. (E) LA-binding to recombinant GLI2. Mouse recombinant GLI2 (rGLI2) was incubated with ADAM-labeled LA (ADAM-LA). rGLI2 was transported to CT26 cells by ChariotTM. After 12 h culture, whole cell lysate was extracted from CT26 cells to detect GLI2 and cleaved GLI2 by western blot. In right panel, the membrane was also observed by excitation wavelength 365 nm, fluorescence wavelength 412 nm. (F) Using the whole cell lysate in G, SOX17 was detected. Error bars indicate standard deviations based on three independent experiments. Statistical differences were calculated by ordinary analysis of variance. Abbreviations: C: control; LA: linoleic acid; GLI: glioma-associated oncogene homolog; Cl-GLI2: cleaved GLI2; ADAM: 9-anthryldiazomethane.
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