Fig. 1. LIF activates STAT3 and induces apoptosis in vitro and is differentially regulated in vivo. (A) LIF activates STAT3 in KIM-2 cells. Western blot analysis of KIM-2 cells treated with increasing concentrations of LIF (1-100 ng/ml) for 30 minutes. Two western blots show pSTAT3 and total STAT3. (B) LIF induces apoptosis in KIM-2 cells. Flow cytometry analysis of annexin V stained KIM-2 cells treated with 100 ng/ml LIF in growth medium (MM + LIF) for 24 hours and MM alone. The graph shown represents fold induction of annexin V-positive cells calculated relative to the negative control, mean±s.e.m, n=3. (C) LIF does not activate STAT5 in mammary epithelial cells. Western blot analysis of KIM-2 cells untreated or treated with 100 ng/ml LIF for 24 hours in MM. The blot was probed with antibodies against pSTAT3, total STAT3 and pSTAT5. KIM-2 cells treated with prolactin for 30 minutes were used as positive control for pSTAT5. (D) Distinct expression profiles of LIF, LIF receptor and gp130 during mammary gland developmental cycle. RNA was extracted from mammary glands harvested at different time points during mammary gland development. RT-PCR analysis was performed using the primers indicated and Cyclophilin (Cyclo.) was used as an internal control. Each gel is representative of three independent mammary gland time courses. wc, water control; d Preg, day pregnancy; d Lac, day lactation; hrs Inv, hours involution. (E) Taqman quantitative PCR data for LIF, performed on a fourth independent set of mammary gland samples, normalised to 18s RNA and displayed as the mean of three PCR reactions.

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