Figure 6.

Breast tumor kinase and signal transducer and activator of transcription 5b roles in DNA synthesis. Knockdown of breast tumor kinase (Brk) and/or signal transducer and activator of transcription 5b (STAT5b) in SKBr3 cells (a, b) or BT-20 cells (c, d) was performed using the Dharmacon siGenome SMARTpool duplex for each protein as per the manufacturer's instructions. (a, c) Seventy-two hours after transfection of the siRNA, cells were lysed. Total lysates were analyzed via immunoblotting with anti-STAT5b (top), anti-Brk (middle), or anti-β-actin (bottom) antibodies. (b, d) Sixty-six hours after transfection of the siRNA, bromodeoxyuridine (BrdU) was added to the medium for 6 hours. The cells were fixed and stained with a fluorescent antibody against BrdU. Cells were scored for BrdU incorporation and graphed. Between 160 and 200 cells were counted for each treatment group. (b) Average percentage of BrdU incorporation ± standard error from three independent experiments performed in triplicate for the SKBr3 cells: media (21.54 ± 0.16), siLuc (24.98 ± 2.78), siBrk (12.37 ± 1.65), siSTAT5b (12.37 ± 0.51), siBrk/siSTAT5b (12.39 ± 0.21). Student's t test was utilized to determine statistical significance between media and siBrk, siSTAT5b, or siBrk/siSTAT5b, and between siLuc and siBrk, siSTAT5b, or siBrk/siSTAT5b. *P ≤ 0.0050, • P < 0.0025. (d) Average percentage of BrdU incorporation ± standard error from three independent experiments performed in triplicate for the BT-20 cells: media (25.28 ± 0.55), siLuc (26.87 ± 0.52), siBrk (18.97 ± 0.32), siSTAT5b (18.87 ± 0.03), siBrk/siSTAT5b (17.37 ± 0.88). Student's t test was utilized to determine statistical significance between media and siBrk, siSTAT5b, or siBrk/siSTAT5b, and between siLuc and siBrk, siSTAT5b, or siBrk/siSTAT5b. *P ≤ 0.0016, • P < 0.0007.