Single-agent Δ1–9-G129R-hPrl inhibits colony formation of cell lines and primary ductal carcinoma in situ samples. (a) MCF-7 cells treated for 2 hours in monolayer culture with control medium, Δ1–9-G129R-hPrl (Δ1–9) (1,000 ng/ml), doxorubicin (1 μM) or the combination were subsequently plated into soft agar (see Materials and methods) and colonies were counted at 14 days. * p < 0.05 compared to untreated control and Δ1–9 alone, † p < 0.01 compared to each of the other three conditions (both by Student t-test). (b) MCF-7 cells plated into soft agar with Δ1–9 incorporated into the agar at the concentrations shown. (c) Effects of bovine pituitary extract (BPE) and prolactin on the ductal carcinoma in situ (DCIS) mammosphere forming efficiency. DCIS mammospheres were counted on day 3 in complete culture medium (complete medium), complete medium without BPE (-BPE) and with 500 ng/ml human prolactin substituted for BPE (-BPE + PRL). Grey-filled and empty bars represent the means of duplicate observations from two independent samples; error bars, standard error of the mean. (d) Table of the six DCIS samples detailing their mammosphere forming efficiency (MFE), the maximum reduction in MFE with Δ1–9 and the concentration of Δ1–9 at which maximum inhibition was achieved. (e) Photomicrograph of a DCIS mammosphere following embedding in paraffin and immunohistochemical analysis for the PRLR using a Fluorescein isothiocyanate (FITC)-labelled secondary antibody (green fluorescence). Cell nuclei counterstained using 4',6-diamidino-2-phenylindole (blue). Scale bar = 50 μm.