Fig. 2. Morphological and molecular changes associated with LIF deficiency in involuting mammary glands. (A) Delayed involution in Lif^-;/-; mammary glands compared with controls. Haematoxylin and Eosin staining of mammary gland sections from Lif^-;/-; and Lif^+/-; mice at day 2 involution. (B) Absence of pSTAT3 in involuting glands from LIF-deficient animals. Immunohistochemistry on sections of mammary glands from animals at 2 day involution using antibodies against pSTAT3. (C) LIF deficiency at 2 day involution is associated with changes in pSTAT3 and cleaved caspase 3 levels. Levels of pSTAT3, STAT3, cleaved caspase3 and the epithelial marker protein cytokeratin 18 (Ker18) were assessed by western blot analysis. Graphs indicate densitometry analysis for three independent mice, mean±s.e.m.; Lif^-;/-;, black bars; Lif^+/-;, white bars. (D) Regulation of C/EBP transcription in glands from LIF-deficient mice at day 2 of involution. RT-PCR analysis of C/EBP expression in glands from wild-type mice (lane 1-3), Stat3^-;/-; mice (lane 4), Lif^+/-; control mice (lane 5) and Lif^-;/-; mice (lane 6). The glands were harvested at 5 day lactation (lane 1), 24 hours involution (lane 2) and 48 hours involution (lane 4-6). Cyclophilin (Cyclo.) was used as an internal control. Graph indicates densitometry analysis normalised to cyclophilin, mean±s.e.m., n=3.