Figure 3

Regulated silencing of EGFP expression in HeLa G/Rcells. (A) Control of shRNA expression, as determined by flow cytometry, based on the presence (yellow) or absence (blue) of Dox. HeLa G/R cells were co-transduced with a lentiviral-based silencing construct at an MOI of 25 and lentiviral vectors encoding the Tet-tTS repressor at MOIs varying from 10 to 50 as indicated. An shRNA corresponding to CXCR4 was used as a control. Results shown were taken from cells harvested 7 days posttransduction. (B) Northern blot analysis of 10 μg of total RNA isolated from HeLa G/R cells 7 days posttransduction. Sample 1: Mock control. Samples 2–6 were transduced with 25 MOIs of the lentiviral silencing constructs and 25 MOIs of the Tet-tTS repressor lentivirus. An shRNA corresponding to CXCR4 was used as a control (sample 6). RNA levels seen in the Northern blot were quantified by imaging of phosphor excitation. Results of quantification are shown in the graph. All levels represent the percentage of EGFP compared to the mock control (sample 1) and are based on the EGFP:GAPDH RNA ratios.