Figure 4. Induction of MMP10 by HRG is mediated by P-Rex1.

(A) T-47D cells were transfected with two different P-Rex1 RNAi duplexes (P-Rex #1 and P-Rex #2), or a non-target control RNAi duplex (NTC). After 16 h, cells were serum starved for 48 h and stimulated with HRG (20 ng/ml) or vehicle for 6 h. MMP10 mRNA levels were determined by qPCR. Expression was normalized to the housekeeping gene B2M. Data was expressed as fold-change relative to NTC, +HRG. The experiment was performed in triplicate samples. Similar results were obtained in two additional independent experiments. *p < 0.05; **p < 0.01 vs. NTC, +HRG. ND, non detectable. (B) Effect of β2-chimaerin on HRG-induced activation of Rac1. T-47D cells were serum starved for 48 h and then infected with AdVs for either β2-chimaerin or LacZ (control) using different m.o.i.'s (1–100 pfu/cell). After 16 h, cells were stimulated with either HRG (20 ng/ml) or vehicle for 5 min, and Rac1-GTP levels were determined using a pull-down assay. Upper panel, representative experiment. Lower panel, densitometric analysis of Rac1-GTP levels normalized to total Rac1. Data (mean ± S.E.M., n = 3) are expressed as fold-change relative to cells infected with LacZ AdV (m.o.i. = 1 pfu/cell), + HRG (lower panel). *p < 0.05 vs. LacZ AdV 100 m.o.i., +HRG. (C) MMP10 mRNA levels were determined by qPCR in response to HRG (20 ng/ml) or vehicle treatment (6 h). Data was normalized to the housekeeping gene B2M, and expressed as fold-change relative to cells infected with LacZ AdV (m.o.i. = 1 pfu/cell), +HRG. The experiment was performed in triplicate samples. Similar results were obtained in two additional independent experiments. ***p < 0.005 vs. LacZ AdV 100 m.o.i. + HRG. ND, non detectable.