Constructs used in this study.

A. Oligonucleotides used for the construction of the shRNA expressing lentiviral vectors. In red the target sequence, in blue the antisense (guide) strand, in green the sequence corresponding to the miR-30 'head'. The sequence of the Ambion siRNA on which a given shRNA construct was based is indicated in italics, underlined is the base that has been modified in order to introduce a bulge into the miRNA produced. B. Scheme of the pInducer lentiviral vectors used [15]. Expression of the red fluorescent protein (RFP) and the coupled miR30 construct containing the inserted shRNA sequence (pInducer-10) or of the inserted CDS (pInducer-20) is driven by the synthetic TRE2 promoter regulated in a doxycycline-dependent manner by the rtTA3 transcription factor expressed constitutively by the Ubc promoter. The vectors express resistance markers, allowing selection of infected cells by puromycin or G418. C. Modifications introduced in the sequences targeted by the shRNA's in the coding sequence of PIT-1. The CDS with these modifications have been used to construct the pInducer-20 based expression vectors. The wt sequence is given in the upper lines, the modified sequence with the introduced changes indicated in capital letter in the middle lines. The lower line indicated the amino acids corresponding to the codons. The numbers in parentheses indicate the ordinal number of the first codon.