Figure 3.

TSC22D1 is crucial for the induction of OIS. (A) HDF (Tig3(et)-16i) expressing two independent hairpins against TSC22D1 (sh-TSC22(1)/(2)) were exposed to BRAF^E600 for 8 days. Amount of TSC22D1.2 transcript was analysed by qRT–PCR. Transcript levels from three independent experiments were normalized to those found in non-senescent control cells and are represented as mean with s.d. (B) The samples described in (A) were analysed by immunoblotting for the amount of TSC22D1 protein. CDK4 serves as loading control, arrowheads indicate the three TSC22D1.2 proteins (see Figures 1C and 2A). (C) Cell proliferation assay of HDF described in (A). Cells were fixed and stained 10 days after exposure to BRAF^E600 (upper row). Representative images of senescence-associated (SA) β-galactosidase staining (middle row) and phase contrast (lower row) are shown for the cells described in (A). Quantification was performed for three independent experiments, with s.d. (D) Nine days after exposure to BRAF^E600 BrdU incorporation was measured in the samples described in (A), after a 3-h BrdU pulse (measured by FACS). Levels are represented as mean from at least three independent experiments. Error bars represent s.d. (E) Cell proliferation assay of FM186 human melanocytes. Cells were lentivirally transduced with the indicated constructs, fixed and stained 8 days after BRAF^E600 expression. The lower row represents phase contrast images of the cells described in (E).