Figure 7.

Higher PTCH1b mRNA relative translation efficiency during hypoxia in HEK 293T cells overexpressing GLI1. HEK 293T cells were transfected with an empty vector or a construct overexpressing GLI1 and then cultured in normoxia or hypoxia for 16 hours (48 hours post transfection). (A). Total RNA levels of the indicated mRNAs, quantified by qPCR as described in the method section and presented as relative expression compared to the reference genes (ΔCq). c-MYC mRNA was included as an example of mRNA whose 5'UTR is considered to possess IRES activity, while PCNA was included as negative control for IRES function. Error bars plot the average and standard deviations of 3 replicates (B). Cytoplasmic lysates of HEK 293T cells transfected and cultured as for panel A, were separated by sucrose-gradient equilibrium density; subpolysomal (SUB) and polysome-associated (POL) mRNAs were identified and collected by UVC scanning and the indicated transcripts were quantified by qPCR, as for panel A. (C). The results from panel B, are also plotted as ratio between polysome-associated and subpolysomal mRNAs as a function of the different treatment. This ratio is considered an estimate of relative mRNA translation efficiency. (D). Global relative mRNA translation rates in HEK 293T cells cultures in normoxia or hypoxia for 16 hours were assessed by incorporation of an immune-detectable methionine analog, as described in Materials and Methods. (E). Western blot analysis of PTC1-L protein and PCNA. Transfection of the GLI1 overexpression plasmid and treatment are indicated. Beta-tubulin was used as reference protein.