Fig. 2.

CSK mediates hormone-independent breast cancer cell growth. (A) Top positively selected genes in both T47D and MCF7 cells cultured in hormone-depleted medium plus 10% dextran-charcoal–treated FBS (DCC-FBS) treated with Veh (EtOH) conditions, compared with E2 (10 nM) conditions. The RRA scores obtained by comparing Veh versus E2 conditions from MAGeCK are shown. (B, Left) Effects of cell growth by knocking out CSK in T47D and MCF7 cells using three different gRNAs against CSK and one AAVS1_gRNA as a control. The CSK function was rescued by the expression of three gRNA-resistant CSK cDNAs in these CSK-null cells. Cell growth was measured by crystal violet staining assays, and the cells were cultured in hormone-depleted medium plus 10% DCC-FBS. (B, Right) Immunoblot analysis for indicated proteins of control (gAAVS1), CSK-null, and rescued CSK-null cells. GAPDH was used as a loading control. (C, Left and Center) Estrogen-independent growth of ZR75-1 and HCC1428 cells harboring two different gRNAs against CSK and one gAAVS1 as a control. All of the cells were cultured in hormone-depleted medium plus 10% DCC-FBS. The data are represented as mean ± SD (n = 3; two-tailed Student’s t test: *P < 0.05, **P < 0.005). (C, Right) Immunoblot analysis for indicated proteins of control (gAAVS1) and CSK-null cells. GAPDH was used as a loading control. (D) Estrogen-independent growth of MCF7 xenografts. MCF7 cells harboring either gAAVS1 or gCSK were injected into the ovariectomized nude mice in the presence of estrogen. Mice were assigned randomly (day 7), in groups of eight, to continued estrogen supplementation (E2, 0.1 mg⋅kg⁻¹⋅wk⁻¹) or estrogen withdrawal (−E2). Luminescence values were plotted as an average of percentage of the first measurement (% relative bioluminescence) for each mouse in each respective group. The measurements were performed in intact male mice at days 10, 17, 25, 31, and 37 after tumor cell injection (two-tailed Student’s t test: *P < 0.05, **P < 0.005).