Fig. 7
CCL5 essential for PCa hormone resistance induction by MSCs. a ELISA kit (R&D Systems, Minneapolis, MN, USA) applied to examine concentration of CCL5 in coculture medium of MSCs and PCa cells in AD. b ELISA performed to examine CCL5 secretion of MSCs in AD. c RT-PCR analysis of CCL5 expression in MSCs under AD. d CCL5 small hairpin RNA (shRNA) oligonucleotides (Neuron Bio, Shanghai, China) with stem-loop structure containing CCL5-target sequence (5′-GTGTGTGCCAACCCAGAGA-3′) used for knockdown analysis. MSCs (1–3 × 106 cells) growing to 50–60% confluence in 10-cm Petri dishes transfected with CCL5-targeting shRNA or their corresponding nontargeting shRNA using lentiviral vectors for 48 h. Effective knockdown effect of CCL5 in MSCs confirmed by RT-PCR measurement. e Influence of MSCs on sphere formation assay of PCa cells with CCL5 knockdown in AD. f mRNA expression of stem markers in LNCaP cells tested when cocultured with CCL5-knocked MSCs in AD. g LNCaP tumor volume observed again to evaluate effect of CCL5 on tumor castration-resistant growth. h Expression of stem markers also examined after tumor removed at 6th week after CCL5-knocked MSC treatment. *p < 0.05, **p < 0.01. AD androgen deprivation, CCL-5 chemokine ligand 5, MSC mesenchymal stem cell