Figure 3.

Involvement of Src in MUC2 mucin induction by P. aeruginosa. (A and B) Inhibition of P. aeruginosa-induced MUC2 up-regulation by coexpressing a dominant-negative mutant form of c-Src (A) and Src inhibitor PP1 (B). p-2864luc was transfected into HM3 cells with (A) or without (B) a dominant-negative mutant form of c-Src. Forty hours later, HM3 cells were pretreated with (B) or without PP1 (A) (5, 14, and 28 μM) (Calbiochem) for 30 min and then exposed to PAO1 culture supernatant (CS) for 6 hr before harvesting. Luciferase activity was measured as described in the text. (C) Effect of coexpressing p-2864luc and v-Src. p-2864luc (15 μg) was cotransfected with v-Src (0.3, 1, 3, and 5 μg) into HM3 cells. Forty-eight hours after being transfected, HM3 cells were lysed for assessing luciferase activity. (D) Effect of PD98059, CAPE, and coexpressing a dominant-negative mutant form of Ras and MEK1/2 on v-Src-induced MUC2 up-regulation. p-2864luc (15 μg) was cotransfected with v-Src (3 μg) and a dominant-negative mutant form of either Ras or MEK1/2 into HM3 cells. Forty hours after being transfected, the cells transfected with only p-2864luc were pretreated with either PD98059 (37 μM, 30 min) or CAPE (15 μg/ml, 1 hr). All the cells were then exposed to P. aeruginosa for 6 hr. Forty hours after being transfected, HM3 cells were either exposed or not exposed to PAO1 culture supernatant (CS) 6 hr before harvesting for assessing luciferase activity. Similar results were observed in another MUC2-expressing epithelial cell line, NCIH292. In all the experiments described above, all transfections were carried out in triplicate. Values are the means ± SD; n = 3.