Effect of silencing GP88 expression on cell proliferation and letrozole responsiveness. A: Top panel: Comparison of GP88 expression in MCF-7CA cells (letrozole sensitive) and LTLT cells (letrozole resistant cells). Bottom panel: LTLT-CA cells were treated with either siRNA control or with GP88 siRNA for 48 hours before harvesting the cells to determine GP88 expression. GP88 level was determined by IP-Western analysis and normalized to cell number as described previously (Lu and Serrero, 2001). (B) LTLT-CA cells were transfected with GP88 siRNA or control siRNA according to manufacturer's instructions one day before being plated at 2 × 103 cells/well in 96 well plates in PRF-5%ChX medium and incubated for the time indicated. Cell proliferation was then measured by using the cellTiter-GloR assay using a luminometer (Molecular Devices).(C) LTLT-CA cells were transfected with either GP88 siRNA or Control siRNA as described above. The next day, cells were plated at 2 × 103 cells/well in 96-well plate and incubated in PRF-ChX medium for 24 hrs more. After 24 hrs, the medium was removed and cells were washed with PRF medium twice. Cells were then incubated in serum free fresh medium with the indicated concentration of letrozole for 3 days. After 3 days cells were counted using cellTiter-Glo assay, p < 0.05. D) Increase of GP88 expression in AC1-LetR cells derived from AC1 cells rendered letrozole resistant by long term culture in the presence of 5 nM letrozole. E) Effect of silencing GP88 on proliferation of AC1-LetR cells. AC1-LetR cells were transfected with either control siRNA or GP88 siRNA as described in the method section. Proliferation was measured after 24 and 48 hours exposure by cellTiter-GloR assay, p < 0.05.