Fig 2: A) GD3 synthase gene expression patterns in human breast cancer cells. Total mRNA was isolated from breast cancer and SK-Mel II melanoma cells. The bar graphs represent the relative band intensity obtained from RT-PCR results by densitometry. For the expression levels of GD3 synthase mRNA, RT-PCR (B) and real-time PCR (C) analyses were performed. The protein expression was analyzed by Western blot (D). The pc3-GD3s cells were cultured with 1 mg/ml G-418. All experiments have been performed at least three times and we examined mean differences between groups by using the error bar graph procedure.

Fig 3: Differential regulation of ICAM-1 expression in pc3-GD3s-trasnfected SK-BR3 (ER-) and MCF-7 (ER+) cells. Expression levels of GM3 synthase, GD3 synthase and ICAM-1 mRNAs in the pc3-GD3s-transfected SK-BR3 (ER-) were checked by RT-PCR (A) and relative band intensities were compared with each ß-actin control (B). ß-Actin was included as an internal control. For MCF-7 (ER+) cells, ICAM-1 mRNA level in the pc3-GD3s-transfected MCF-7 cells was examined by Western blot analysis (C) and relative band intensity was also compared with GAPDH control (D). All experiments performed at least three times. We examined mean differences between groups by using the error bar graph procedure.

Fig 4: Accumulation of the GD2 ganglioside in pc3-GD3s cells. (A) The total ganglioside on a HPTLC plate. (B) Comparison of ganglioside contents in pc3 and pc3-GD3s cells. Percentage of each ganglioside was measured by densitometry with total gangliosides of each lane as 100%. (C) Total mRNA was isolated from pc3 and pc3-GD3s cells. GD2 synthase (ß4-GalNc T) was detected by RT-PCR (30 cycles). The bar graphs represent the relative band intensity obtained from the RT-PCR results by densitometry.

Fig 5: Downregulation of ICAM-1 expression (A, B, C, D, E) in pc3-GD3s cells. Expression levels of ICAM-1 mRNA were checked by RT-PCR (A) and real-time PCR (B). Expression of ICAM-1 confirmed by Western blot (C) and FACS analysis (D). A schematic representation of DNA constructions containing two different forms of the ICAM-1 promters linked to the luciferase reporter gene is shown. The values represent the mean ±S.D. for three independent experiments with triplicate measurements (E). Inhibition of AKT phosphorylation in pc3-GD3s cells (F). Total protein extracts from pc3 and pc3-GD3s cells were examined by Western blot analysis with anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38 and anti-p38 antibodies. The nonspecific IgG antiserum has been used to block the cell reaction. GAPDH was included as an internal control. All experiments performed at least three times and we examined mean differences between groups by using the error bar graph procedure.