Fig. 6.

Effect of ZFAS1 knockdown on cell phenotype. a Four different shRNA constructs (BC1-4) designed to target ZFAS1 were transfected into MDA-MB-468 cells. The efficiency of shRNA knockdown was analysed by qPCR in relation to a scrambled shRNA control. ZFAS1 transcripts were knocked down to the greatest extent by shRNA BC1 and BC2, these were used for further analysis. b (i) Western blot analysis of phosphorylated RPS6 and total RPS6 in ZFAS1 knockdown and scrambled control cells in three biological replicates. Actin was used as an internal control. (ii) Semiquantitatve analysis of western blot results using densitometry comparison to actin. shRNA BC1–BC3 transfected cells and scrambled shRNA were used. Error bars are SEM of three biological replicates, p values were calculated using Student’s t-test. c Cells transfected with scrambled shRNA and ZFAS1-shRNA cells were maintained for 48 h in medium containing low serum (SS), and then refed (RF) for 48 h in medium containing serum at 10 %. qPCR was then performed to measure the expression of 45S rRNA at different time points. Expression is relative to three housekeeping genes (3HK). Error bars are SEM of three biological replicates, p values were calculated using Student’s t test