The Survival Effect of Prolactin on PC3 Prostate Cancer Cells Alain Ruffion, Kaltoom A. Al-Sakkaf, Barry L. Brown, Colby L. Eaton, Freddie C. Hamdy, Pauline R.M. Dobson  European Urology  Volume 43, Issue 3, Pages 301-308 (March 2003) DOI: 10.1016/S0302-2838(03)00038-1

Fig. 1 Expression of the prolactin receptor (PRL-R) in the three prostate cancer cell lines. Positive controls are Nb2 and MCF7 cells. Cells were grown for 2 (PC3, DU145) or 3 days (LNCaP) before protein extraction. They were then harvested, lysed in the designated buffer and expression of the three forms of the PRL-R was investigated by Western blot analysis as indicated in Section 2. Both the long (A) and short (C) forms of the receptor are expressed in the three prostate cell lines, as in the MCF7 cell line. Note that the Nb2 cells do not express the long form of the PRL receptor, and is the only cell line to express an intermediate form (B) of the receptor. European Urology 2003 43, 301-308DOI: (10.1016/S0302-2838(03)00038-1)

Fig. 2 The survival effect of PRL on PC3 cells using Timelapse microscopy. Images were collected at 0 (left side) and 24hours (right side) of living cells grown in medium with 1% charcoal-treated foetal calf serum and 10mM HEPES: (A and B) control; (C and D) 2hours control pre-incubation followed by TRAIL (25ng/ml); (E and F) 2hours PRL (10ng/ml) pre-incubation followed by TRAIL (25ng/ml). All experiments were repeated three times. European Urology 2003 43, 301-308DOI: (10.1016/S0302-2838(03)00038-1)

Fig. 3 Time and dose response of TRAIL-induced apoptosis in PC3 cells. Cells were treated with several concentrations of TRAIL (10–100ng/ml) for various times. Apoptosis was measured using the Annexin assay (described in Section 2). Each point represents mean values of three replicates in one of three similar experiments which gave similar results. Error bars represent the standard error. European Urology 2003 43, 301-308DOI: (10.1016/S0302-2838(03)00038-1)

Fig. 4 Apoptotic and dead cells, after a 2hour pre-incubation with various concentrations (0ng/ml, 10ng/ml and 100ng/ml) of PRL, followed by TRAIL stimulation (24hours). Apoptosis was measured using the Annexin assay (described in Section 2). Each point represents mean values of three replicates in one of three experiments, which gave similar results. Error bars represent the standard error. PRL significantly ((∗) p<0.05) inhibited TRAIL-induced apoptosis. European Urology 2003 43, 301-308DOI: (10.1016/S0302-2838(03)00038-1)

Fig. 5 Akt/PKB phosphorylation by PRL in PC3 cells. Cells were grown in Petri dishes for 2 days, then washed twice with PBS. PRL (10ng/ml) was then added for various time points (0, 5, 15, 30 and 60minutes). Western blotting (see Section 2) was performed to assess the existence of phosphorylated Akt/PKB (A). The membrane was then stripped and reprobed to assess the equal presence of the protein at each time point (B). European Urology 2003 43, 301-308DOI: (10.1016/S0302-2838(03)00038-1)