Figure 6

shRNA based variable knockdown of E4BP4 correlates with corresponding repression of Bim expression: CEM C7-14 cells were transfected with three different E4BP4 shRNA plasmidsID1, ID2 and ID4 with a puromycin selection marker. Transfected cells were selected in the presence of 1 μg/ml puromycin, and surviving cells were analyzed for extent of E4BP4 knockdown and Bim expression in comparison to untransfected cells. Cells were treated with 0.1% ethanol or 1 μM Dex for 24h and total RNA was extracted in TriZol. Seven microgram RNA was subjected to reverse transcription reaction and real-time qPCR using primers specific for E4BP4 or Bim as listed in Table 1. Fold change in expression in knockdown cells (ID1, ID2 or ID4), compared to CEM C7-14 cells, for ethanol treated (Panel A) and Dex treated (Panel B) cells was calculated by the Pfaffl formula: (E_target)^ΔCTtarget/(E_ref)^ΔCTref where ΔCTtarget = (CT_{CEM C7-14}-CT_ID) for E4BP4 or Bim and ΔCTref = (CT_{CEM C7-14}-CT_ID) for β-actin. Induction of E4BP4 or Bim expression in response to 1 μM Dex was calculated for CEM C7-14, ID1, ID2, and ID4, by the Pfaffl method, whereΔCTtarget = (CT_ethanol-CT_Dex) for E4BP4 or Bim and ΔCTref = (CT_ethanol-CT_Dex) for β-actin (Panel C). Efficiency for each reaction was calculated using the software LinRegPCR. Data represent averages ± S.D. from three independent experiments. Correlation between E4BP4 and Bim expression was determined by calculating the correlation coefficients (r) which are indicated on each panel. Panel D: CEM C7-14 and mass cultures of cells transfected with ID1, ID2 and ID4 shRNA plasmids against E4BP4, and selected with puromycin were seeded at a density of 1 × 10⁵cells/ml and treated for 96 h with either 0.1% ethanol (EtOH) or 1 μM Dex. Aliquots were taken at 24 h intervals and viable cell number was determined by trypan blue dye exclusion assay. Data represent average ± S.D. of three independent experiments with two replicates each.