Rm 9116 s1

Monoclonal anti-CCL5 antibodies and the recombinant protein IgG were purchased from R&D systems (MAB678, MAB002, Minneapolis, MN, USA), and 500 μg/mL stock was reconstituted in phosphate buffered saline (PBS). For anti-CCL5 treatment, stocks were adjusted to a final concentration of 6 μg/mL. Ficoll-Paque was purchased from Amersham Pharmacia Biotech (17144002, Piscataway, NJ, USA). Collagenase D was purchased from Roche Diagnostic (100 mg, 11088858001 Indianapolis, IN, USA) and was adjusted to a final concentration of 1 mg/mL to use. Antibodies used for flow cytometry included PE-Cy 7-conjugated mouse anti-human CD3 antibody (341091), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD4 antibody (340133), phycoerythrin (PE)-conjugated mouse anti-human CD8 antibody (340046), PE-Cy 7-conjugated mouse IgG1, κ isotype (555872), FITC-conjugated mouse IgG1 κ isotype (555909), or PE-conjugated mouse IgG1κ isotype (554680). All these FACs antibodies were purchased from BD Biosciences (NJ, USA). Antibodies used for immunohistochemistry included Rabbit anti-CD4(+) (dilution 1 : 50, ab133616, Abcam, Cambridge, UK), anti-CD8(+) (dilution 1 : 50, RM-9116-S1, Thermo Fisher Scientific, Cheshire, UK), and the rabbit anti-CCL5 (+) (2 μg/mL, ab9679, Abcam, Cambridge, UK).

Antibodies used for immunohistochemistry and western blot included rabbit anti-CD8(+) (RM-9116-S1, Thermo Fisher Scientific, Cheshire, UK), rabbit anti-CCL5 (ab9679, Abcam, Cambridge, UK), mouse anti-PCNA (2586, Cell Signaling Technology, MA, USA), rabbit anti-GAPDH (2118, Cell Signaling Technology, MA, USA), mouse anti-β-ACTIN (3700, Cell Signaling Technology, MA, USA), rabbit anti-STAT5 (9363, Cell Signaling Technology, MA, USA), rabbit anti-phospho-STAT5 (9314, Cell Signaling Technology, MA, USA) and rabbit anti-CCND1 (2978, Cell Signaling Technology, MA, USA). The reagents used in the in vitro experiment included recombinant human CCL5 (rhCCL5), anti-CCL5 neutralizing antibody and Pimozide were purchased from R&D systems (278-RN, MAB678, 0937 Minneapolis, MN, USA). rhCCL5 was reconstituted in phosphate buffered saline (PBS) and adjusted to a final concentration of 2, 20, 100 ng/ml. For blocking the proliferation effect of rhCCL5 and conditioned media, the anti-CCL5 neutralizing antibody was reconstituted in PBS and adjusted to a final concentration of 2 μg/ml. The STAT5 inhibitor Pimozide was used for interruption assay at a final concentration of 10 μM (dissolved in DMSO). All reagents were used in a low androgen condition to evaluate the proliferation effect and the mechanism dissection of CD8+ T cells or rhCCL5 on BECs.