Figure 1.
Figure 1. Activation of Notch pathway in myeloma cells following ligation to Jagged-1. (A) Expression of Notch family proteins and their ligand Jagged-1 was analyzed in 8 different human myeloma cell lines by Western blotting. (B) Expression of Notch family proteins and their ligands was evaluated by Western blotting in BMS cells obtained from 5 different BM donors. (C) Interaction between BMS and H929 cells resulted in activation of the Notch pathway in myeloma cells. H929 cells transfected with reporter or control constructs were incubated on BMS for 48 hours or kept in suspension (control). Luciferase activity (LA) was measured according to the manufacturer's protocol and normalized to 1 μg total protein. Data are presented as fold increase of the specific LA (H929 cells transfected with p6 × CBF-1-pKA9 plasmid) over control LA activity (cells transfected with pKA9 plasmid). Experiments were performed in triplicate and repeated twice with similar results. (D) Jagged-1 ligation activates Notch pathway in myeloma cells. H929, U266, RPMI-8226, or MM1S human myeloma cells and Burkitt lymphoma HS-Sultan cells were starved for one hour in serum-free RPMI-1640 medium and then incubated on the monolayer of irradiated 3T3-Jagged (J) or control 3T3-MSCV (Con) cells for 8 hours. Cells were then collected and nuclear extract was prepared. EMSA with CBF-1-specific probe was performed. “Cold inh” indicates cold inhibition control. (E-F) Notch activation in myeloma cells results in an increase of HES-1 mRNA espression. The pcDNA3.1-Notch-1IC or pcDNA3.1 plasmids were introduced in U266 cells by electroporation and cultured for 24 hours and 48 hours (E). H929, RPMI-8226, or U266 myeloma cells were cultured on 3T3-Jagged or 3T3-MSCV cells for 48 hours (F). Real-time PCR was performed as described in “Materials and methods.” The mRNA expression for each sample was normalized to 18S mRNA expression. Data shown are fold increase of HES-1 expression in myeloma cells cultured on 3T3-Jagged over cells cultured on 3T3-MSCV (F) or U266 cells transfected with Notch-1IC-carrying plasmid over control plasmid (E). Differences were statistically significant for all cell lines studied. Error bars (C, E, F) indicate standard deviation.

Activation of Notch pathway in myeloma cells following ligation to Jagged-1. (A) Expression of Notch family proteins and their ligand Jagged-1 was analyzed in 8 different human myeloma cell lines by Western blotting. (B) Expression of Notch family proteins and their ligands was evaluated by Western blotting in BMS cells obtained from 5 different BM donors. (C) Interaction between BMS and H929 cells resulted in activation of the Notch pathway in myeloma cells. H929 cells transfected with reporter or control constructs were incubated on BMS for 48 hours or kept in suspension (control). Luciferase activity (LA) was measured according to the manufacturer's protocol and normalized to 1 μg total protein. Data are presented as fold increase of the specific LA (H929 cells transfected with p6 × CBF-1-pKA9 plasmid) over control LA activity (cells transfected with pKA9 plasmid). Experiments were performed in triplicate and repeated twice with similar results. (D) Jagged-1 ligation activates Notch pathway in myeloma cells. H929, U266, RPMI-8226, or MM1S human myeloma cells and Burkitt lymphoma HS-Sultan cells were starved for one hour in serum-free RPMI-1640 medium and then incubated on the monolayer of irradiated 3T3-Jagged (J) or control 3T3-MSCV (Con) cells for 8 hours. Cells were then collected and nuclear extract was prepared. EMSA with CBF-1-specific probe was performed. “Cold inh” indicates cold inhibition control. (E-F) Notch activation in myeloma cells results in an increase of HES-1 mRNA espression. The pcDNA3.1-Notch-1IC or pcDNA3.1 plasmids were introduced in U266 cells by electroporation and cultured for 24 hours and 48 hours (E). H929, RPMI-8226, or U266 myeloma cells were cultured on 3T3-Jagged or 3T3-MSCV cells for 48 hours (F). Real-time PCR was performed as described in “Materials and methods.” The mRNA expression for each sample was normalized to 18S mRNA expression. Data shown are fold increase of HES-1 expression in myeloma cells cultured on 3T3-Jagged over cells cultured on 3T3-MSCV (F) or U266 cells transfected with Notch-1IC-carrying plasmid over control plasmid (E). Differences were statistically significant for all cell lines studied. Error bars (C, E, F) indicate standard deviation.

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