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. 2011 Mar 29;30(9):1753–1765. doi: 10.1038/emboj.2011.95

Figure 5.

Figure 5

Selective induction of the TSC22D1.2 transcript is linked to OIS. (A) Scheme indicating the two transcripts, TSC22D1.1 and TSC22D1.2, expressed from the TSC22D1 locus. The known protein-coding transcripts have four exons, from which only the third and fourth are used in both transcripts. Our data suggest that the TSC22D1.1 transcript encodes the TSC22D1.1 protein and that the TSC22D1.2 transcript encodes three proteins, TSC22D1.2 (144, 134 and 86 aa), by differential use of translation initiation sites. All TSC22D1 protein variants, but TSC22D1.286aa, harbour the TSC box, a region that is highly conserved in all TSC family members (indicated in fair blue), and a leucin zipper (indicated in dark grey). (B) qRT–PCR of Tig3(et)-16i HDF with and without BRAFE600. Samples were taken 10 days after infection and the transcript levels of TSC22D1.2 and TSC22D1.1 determined using specific primers. Transcript levels were standardized to those of proliferating control cells. Mean and s.d. of three independent experiments are shown. (C) Immunoblot with the indicated antibodies on lysates from the cells described in (B). Samples were taken 6 and 10 days after transduction with BRAFE600. Arrowheads point to the TSC22D1 protein variants. β-Actin serves as a loading control. (D) HDF (Tig3(et)-16i) senescent and proliferating control cells were treated 9 days after transduction with the indicated constructs with the proteasome inhibitor MG132 (16 h at 10 μM). TSC22D1.1 and BRAF levels were determined by immunoblotting. β-Actin serves as loading control. (E) Scheme showing the TSC22D1 sequences targeted by shRNAs. Whereas sh-TSC22(1) and (2) target all TSC22D1 transcripts, sh-TSC22(3) specifically targets TSC22D1.2 and sh-TSC22(4) TSC22D1.1 only. (F) HDF were transduced with the hairpins described in (E) and subsequently transduced with BRAFE600-encoding or empty vector (control). Protein levels were assessed 8 days after oncogene exposure by immunoblotting. β-Actin serves as loading control. (G) Cell proliferation assay of the experiment described in (E). Cells were stained 10 days after exposure to BRAFE600.