Fig. 4

Androgen receptor (AR) protein is upregulated and transcriptionally activated in letrozole-resistant (LetR) cells treated with recombinant prosaposin (PSAP); the effect of PSAP can be diminished by co-treatment with the anti-AR drug enzalutamide (Enza). a (i) Western blot analysis was used to quantify AR protein levels in LetR cells treated with recombinant human PSAP (rhPSAP) (10 ng/ml) versus control (Tris–HCl). (ii) Densitometry values from separate experiments. Error bars are representative of mean ± standard error of the mean (SEM) of three separate experiments. b (i) Modified TransAM assay was used to evaluate AR recruitment to a direct AR binding sequence 5′ – TGTTCT – 3′ when LetR cells are cultured in the presence of rhPSAP. Results are representative of two separate experiments (ii) In LetR cells the AR binds the direct AR consensus sequence in the presence of either R1881 or rhPSAP c. Nuclear translocation assays were used to observe the trafficking of AR in aromatase-inhibitor (AI)-resistant LetR cells, as visualised by the detection of immunofluorescently labelled AR within the nucleus. c (i) rhPSAP treatment significantly increased AR nuclear translocation in LetR cells (p <0.0001). rhPSAP and Enza combination treatment significantly decreased AR nuclear translocation in LetR cells (p <0.05). Error bars are representative of mean ± SEM of three separate experiments. Anti-AR drug treatment (Enza) significantly decreased AR nuclear translocation in LetR cells (p <0.05). (ii) There was no change in AR nuclear translocation in MCF7 cells following treatment with either rhPSAP or Enza individually or with a combination of both rhPSAP and Enza. Error bars are representative of mean ± SEM of three separate experiments. (iii) Representative images of AR nuclear translocation in LetR cells following individual treatments with rhPSAP and Enza and combination treatments: *p <0.05, **p <0.001, ***p <0.0001. HRE hormone response element, Veh vehicle, DAPI 4′, 6-diamidino-2-phenylindole