Figure 1: HGAL enhances intracelular BCR signalling.

Raji and VAL lymphoma cells were transfected with short interfering RNA (siRNA) for HGAL or scrambled control siRNA and HBL-1 cells (a) and normal peripheral blood B-lymphocytes (b) transfected with pcDNA3.1-HGAL or pcDNA3.1-mock plasmids for 48 h. After 1 min stimulation with goat F(ab')2 anti-human IgM, western blot of BCR receptor effectors was performed. HGAL expression and equal loading were analysed by immunoblotting with HGAL and actin antibodies. (c) Kinetic of calcium mobilization in Raji cells transfected with siRNA for HGAL or scrambled control siRNA. Arrow indicated the time point of goat F(ab')2 anti-human IgM stimulation. (d) Raji lymphoma cells were transfected with firefly luciferase reporter plasmid pNFkB-Luc or pNFAT-Luc and renilla luciferase plasmid pRL-TK and with either siRNA for HGAL or scrambled control siRNA. Forty-eight hours after transfection, the cells were stimulated for 10 min with goat F(ab')2 anti-human IgM and luciferase activities were detected with the dual luciferase assay kit. Numbers refer to luciferase activities representing three independent experiments, each performed in triplicate. * Indicate statistically significant difference (NFκB P=0.0001 and nuclear factor of activated T cells P=0.001 by two-tailed Student’s t-test). Data are presented as mean±s.d. of the mean. (e) BJAB lymphoma cells were transfected with siRNA for HGAL or scrambled control siRNA for 48 h. After stimulation with goat F(ab')2 anti-human IgM, cell lysates were used for western blot of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway effectors at indicated time points. HGAL knockdown or expression and equal loading were confirmed by immunoblotting with HGAL and actin antibodies. Normalized densitometry measurements are shown below the corresponding blots. Results in (a–e) are representative of three independent experiments. NFκB, nuclear factor κB.