Figure 5: SUMOylation of TARBP2 may suppress tumour progression.

(a) The mutation E54K abolishes SUMOylation of TARBP2. Myc-TARBP2-WT, or -K52R or E54K with Flag-Ubc9 and His-SUMO1 were co-transfected into 293T cells. The SUMOylation assay by Ni²⁺-NTA resin pull down was conducted as before. For full scans of western blot analysis, see Supplementary Fig. 8. (b) SUMOylation of TARBP2 suppresses anchorage-independent growth. Each of A549^luc stable cell line expressing the control vector, or Flag-TARBP2-WT, or Flag-TARBP2-K52R (right panels), were seeded at a density of 2,000 cells per well in 1.5 ml of medium containing 10% FBS with 0.35% soft agarose and were layered on 0.6% solidified agarose. Photographs were taken 12 days after seeding, and the number of colonies was scored. Each value represents the mean±s.e.m. of three independent experiments with triplicates (left panels). (c) SUMOylation of TARBP2 suppresses xenograft tumour growth in vivo. Each of stable A549^luc cell line expressing the control vector, or Flag-TARBP2-WT, or Flag-TARBP2-K52R (2.5 × 10⁶ cells/each) was injected subcutaneously into male BALB/c nude mice (n=4) individually. Mice were killed 6 weeks later, and tumours were dissected (left panels) and assessed by weight (right panels). (d) SUMOylation of TARBP2 inhibits tumour cell migration. The migration assay of stable A549^luc cell lines was conducted with xCELLigence RTCA-DP and the kinetic curves were real-time-recorded. Differences between individual groups as indicated were analysed using the t-test (two-tailed and unpaired), and P values of <0.05 (*) or <0.01 (**) or <0.001 (***) are considered significant.