Hui-Ling Guo et al . npg 1251 Figure 3 Knockdown of Axin, TNKS or KIF3A impaired GLUT4 translocation. 3T3-L1 cells were induced to differentiate into adipocytes and on day 6 were infected with adenovirus vector (pAdEasy-1) expressing different siRNAs as indicated. (A) Knockdown of Axin, TNKS or KIF3A blocks insulin-dependent GLUT4 translocation. After 48 h of infection with the indicated siRNA-delivering adenovirus, adipocytes were serum starved for 4 h, followed by incubation with or without 200 nM insulin for 30 min. Cells were then fixed and analyzed by immunofluorescence staining using the GLUT4 antibody and a Texas Red- coupled secondary antibody. Arrow heads point to the PM. The outlines of adipocytes (gray) were displayed by GFP co-ex- pressed from adenovirus. Scale bars represent 20 µm. The siRNA effects on GLUT4 translocation were re-assessed by Rim intensity/TOTAL fluorescence (Right), which were collected and analyzed using Leica TCS SP2 confocal laser scanning soft- ware. The results show that knockdown of each component of the Axin complex attenuates insulin-stimulated GLUT4 trans- location to the cell membrane (right, **P < 0.01; ***P < 0.001). (B, C) Knockdown of Axin (B) or TNKS (double knockdown of TNKS1 and TNKS2) (C) attenuates insulin-stimulated GLUT4 translocation to PM. Adipocytes were infected with adenovirus on day 6 and 48 h later PM fractions were prepared as described in Materials and Methods after insulin (200 nM for 30 min) stimulation. Equal amounts (15 µg) of protein of the PM fractions (membrane pellet) and the Post-PM (representing cell su- pernatant) fractions, along with the total cell lysates (TCL), were subjected to immunoblotting using antibodies indicated. The immunoblots of PM GLUT4 were quantified by densitometry on the right. (Data are mean ± SEM of three independent experi- ments. ***P < 0.001.) tant role in mediating insulin-stimulated glucose uptake in vivo. Insulin stabilizes Axin/TNKS/KIF3A complex by sup- pressing TNKS PARsylase activity We next assessed whether the Axin and TNKS pro- teins are dynamically modulated before and after insulin treatment. As shown in Figure 6A, insulin treatment in- creased cellular levels of Axin and TNKS, which is not due to altered mRNA abundance as indicated by quan- titative RT-PCR analysis (Supplementary information, Figure S7A and S7B). Since TNKS1/2 PARsylate them- Figure 4 Knockdown of components of the Axin complex attenuated insulin-stimulated glucose uptake. Single knockdown of Axin (siA) (A), KIF3A (siK) (B) or TNKS2 (siT2, **P < 0.01; ***P < 0.001) (C), and double knockdown of TNKS1 and TNKS2 (D) or KIF3A and TNKS2 (E) were carried out, respectively. 3T3-L1 cells were induced to differentiate into adipocytes and on day 6 were infected with adenovirus vector (pAdEasy-1) expressing different siRNAs as indicated. Experiments were performed 48 h after infection according to the protocol described in Materials and Methods. www.cell-research.com | Cell Research