Figure 6

C/EBPβ expression is important for cell survival following anoikis. A). Flow cytometry of MCF10A cells in forced suspension culture at 48 hr and 96 hr. Cell death was analyzed by measuring the sub-G₁ cell cycle fraction. Vector control (Vector) and C/EBPβ knock down (shRNA) cells were treated with Doxycycline (1 μg/ml) for 2 days to activate shRNA expression, followed by one more day of Dox treatment in serum free conditions. IGF-1 (I), (2.6 nM or 39 nM) was then added to the treated cultures at time 0 hour. n = 3B). Flow cytometry of Annexin V positive MCF10A cells, that were transduced to overexpress LIP in forced suspension culture for 24 and 48 hours. n = 3. C). Anoikis assay of MCF10A cells transduced with lenti-LIP (LIP) or vector control (vector) and cultured in suspension for 48 and 96 hours prior to Flow cytometry for the sub G₁ cell cycle fraction. Statistical significance was determined using an unpaired, 2-tailed T-test with a * p value < 0.05. n = 3. D). After 24 hours of suspension culture in serum free media containing IGF-1 (39 nM), vector control or C/EBPβ knock down cells were transferred to standard 6 well cell culture plates and permitted to adhere and expand for two weeks for analysis of clonogenic outgrowth potential followed by staining with crystal violet. E-F). Western blot analysis of MCF10A cells with shRNA knock down for C/EBPβ (E) or transduced to overexpress LIP (F).