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. 2010 Aug 11;5(8):e12107. doi: 10.1371/journal.pone.0012107

Figure 4. ARG1 and ARG2 are metabolically active.

Figure 4

LNCaP cells were transfected with either a siCTRL or a cocktail of three siRNA against the ARG1 or ARG2. Post-transfection (24 hours), cells were plated in charcoal-stripped serum supplemented media for 72 hours and then stimulated for 72 hours with 10 nM R1881. A) siRNA inhibition of ARG1 and ARG2 expression was evaluated by Western Blot. Representative experiment is shown, (n = 4). B) Decreased arginase activity following transfection with siARG1 or siARG2 in LNCaP cells. The corresponding Western blot is shown in the bottom panel. Representative experiment is shown, (n = 3). C) Decreased metabolism of l-arginine in the absence of arginase expression. Conditioned media of LNCaP cells transfected with siCTRL, siARG1 or siARG2 were analyzed by HPLC for l-arginine concentration. The conditioned media analyzed by HPLC were from the LNCaP cells presented in Figure 4A. *Statistically significant difference (p<0.05, Mann-U). D) Decreased proliferation of LNCaP cells in the absence of arginase expression. LNCaP cells were transfected as previously described. Proliferation was measured by cell count 96 hours post-transfection. *Statistically significant difference (p<0.05, Mann-U), (n = 3). E) Inhibition of ARG2 expression causes increased PBMC proliferation and activation. PBMCs from normal donors were activated with anti-CD3 (OKT3, 1 µg/ml) with or without IL-2 in the presence of fresh media or conditioned media of LNCaP cells transfected with either control, siCTRL, siARG1 or siARG2 as previously described. Control PBMCs were incubated with IgG isotype control (1 µg/ml) and with PBS instead of IL-2. Left panel: PBMC proliferation was quantified by BrdU incorporation following 120 hours of OKT3 and IL-2 stimulation. Mean absorbance (n = 4) is shown with standard error (error bars). Right panel: PBMC secretion of IFN-γ quantified by ELISA. Same experiment as previously described, but the PBMCs were activated for 24 hours without IL-2. Representative expression is shown (n = 4) with standard error of the mean (error bars).