Figure 6.

Role of PIN1 in AP-1 activity and breast tumorigenesis induced by IL-34. MCF7 cells were seeded and co-transfected with the luciferase reporters, c-Fos-luc (A), c-Jun-luc (B), and AP-1-luc (C) along with the pRL-TK vector. At 24 h after transfection, the cells were serum starved for 24 h and then treated with the indicated doses of IL-34 for 24 h before luciferase assay was performed. (D) MCF7 cells were seeded and co-transfected with the luciferase reporter AP-1-luc and siRNA-control or AP-1-luc and siRNA-PIN1. At 24 h after transfection, the cells were serum starved for 24 h, treated with 10 ng/mL IL-34 for 24 h or left untreated, before a luciferase assay was performed. (E) MCF7 cells were transfected with siRNA-control and siRNA-PIN1. After 24 h, the cells were treated with 10 ng/mL IL-34 in a soft agar matrix or left untreated and incubated at 37 °C in a 5% CO₂ atmosphere. After 14 days, colonies from three separate experiments were photographed (left), followed by calculation of the average colony numbers and sizes (diameter > 200 μm, right). (F) 4T1 cells were injected into the mammary gland of BALB/c mice in the presence or absence of 100 ng/mL IL-34 and 100 μM juglone, and allowed to grow until tumors were formed. Shown are representative pictures of tumor (left), measured volumes and weights of tumors (right). Error bars indicate the mean ± S.D. of triplicate measurements from two independent experiments. Statistical analyses were conducted using one-way ANOVA (*p < 0.05, ** p < 0.01, *** p < 0.001, compared to control group or only IL-34-treated group, respectively).