Figure 1.
XIAP promoted HCT116 cell migration and invasion.
(A), Knockout of XIAP in HCT116 cells was verified by Western Blotting assay. (B and C), Cell migration behavior was evaluated during performance of a wound-healing assay, and images were taken at different time points. Scale bar was 300 µm. The wound area was quantified using Cell Migration Analysis software, and the quantitative data were shown as indicated (error bar represent S.D, n = 3). The asterisk (*) indicates a significant difference in wound area percentage between the indicated cell lines (p<0.05). (D and E), Invasion of WT(Vector), XIAP−/−(Vector), and XIAP−/−(HA-XIAP) HCT116 cells was determined, quantified and expressed as percentage of invasion. Results were represented by the mean ± S.D. of the data from three-independent experiments with duplicate wells for each experiment. The asterisk (*) indicates a significant decrease in invasion percentage compared with that in WT(vector) and XIAP−/−(HA-XIAP) cells (p<0.01). (F), The proliferate rates of the indicated cell lines were assessed by a CellTiter-Glo® Luminescent Cell Viability Assay kit. Results were represented by the mean ± S.D. of the triplicate wells. (G–I), The indicated cells were treated with or without EGF and F-actin induction was analyzed by spectrophotometer (G), or observed under confocal microscope (H), respectively. The fluorescence of cells was quantified by the software of ImageJ (I). The quantitative data was shown as indicated (error bar represent S.D, n = 3). The asterisk (*) indicates a significant decrease compared with that in WT cells (p<0.01).
Figure 2.
The requirement of XIAP for cell motility was confirmed by knocking down approach.
(A), Knockdown of XIAP in HCT116 cells were verified by Western Blotting assay. (B and C), Cell migration behavior was evaluated during performance of a wound-healing assay, and images were taken at different time points. Scale bar was 300 µm. The wound area was quantified using Cell Migration Analysis software, and the quantitative data were shown as indicated (error bar represent S.D, n = 3). The asterisk (*) indicates a significant difference in wound area percentage between the indicated cell lines (p<0.05).
Figure 3.
Various XIAP domains were reconstitutivly expressed into XIAP−/− cells.
(A), Schematic representation of XIAP protein and identified function of each domain. (B and C), Identification of the stable transfectants harboring XIAP and its various deletion plasmids in XIAP−/− HCT116 cells. The numbers under the bands indicated the densitometric analysis of relative ratios of β-Actin levels to loading controls (GAPDH) evaluated by software of ImageQuant Version 5.2 (Molecular Dynamics, Sunnyvale, CA). Results were representative of at least three independent experiments.
Figure 4.
Different XIAP domains involved in cell migration disparately.
(A), Cell migration behavior was evaluated with a wound-healing assay, and images were taken at different time points. Scale bar was 300 µm. (B), The wound area left un-closed on the 4th day was quantified using Cell Migration Analysis software, and the quantitative data was shown as indicated (error bar represent S.D, n = 2). The asterisk (*) indicates a significant difference in percentage of wound area compared with that in WT (Vector) cells (p<0.05).
Figure 5.
F-actin induction by EGF was regulated differently by various XIAP domains.
(A), The indicated cells were treated with or without EGF and F-Actin induction was analyzed by flow cytometry. (B), The quantitative data was shown as indicated (error bar represent S.D, n = 2). The asterisk (*) indicates a significant difference in F-actin induction compared with that in WT(Vector) cells (p<0.05).
Figure 6.
RhoGDI was involved in XIAP regulation of cell migration and actin polymerization.
(A), Lysates from WT and XIAP−/− HCT116 cells were Co-immunoprecipitated with anti-XIAP (mouse) antibody or normal mouse IgG, and immunoprecipitates were then subjected to immunoblotting with anti-RhoGDI (rabbit) or anti-XIAP (rabbit) antibodies. Five percent of lysates was used as input. (B), XIAP−/−(HA-XIAP) cells were transiently transfected with the GFP-RhoGDI or empty vector, GFTP-Vector. Co-immunoprecipitation was performed with anti-GFP antibody-conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting using antibodies as indicated. (C). Stable transfectants of shRNA-RhoGDI in WT and XIAP−/− cells were identified. Cell migration was determined by wound healing assays at the indicated times between Non-silencing and shRNA-RhoGDI transfectants in WT and XIAP−/− cells respectively. The wound area was quantified using Cell Migration Analysis software, and the quantitative data was shown as indicated (error bar represent S.D, n = 3). The asterisk (*) indicates a significant difference between the indicated cell lines (p<0.05). Scale bar was 300 µm. (D), The indicated cells were treated with EGF for 1 min for determination of F-Actin induction by flow cytometry. (E), Constitutive expression of GFP-RhoGDI-Re in XIAP−/−(Si-RhoGDI) was verified by Western Blotting. (F and G), Relative induction of F-Actin in the presence of EGF was determined by spectrophotometer (F), and levels of filamentous Actin were observed under confocal microscopy (G) in the indicated transfectants. The asterisk (*) indicates a significant increase in comparison to those in XIAP−/−(Si-Control) (p<0.05), and the (♣) indicates a significant decrease in comparison to those in XIAP−/−(Si-RhoGDI) cells (p<0.001, n = 3).
Figure 7.
XIAP RING Domain was Responsible for RhoGDI Interaction Independent on its E3 Ligase Activity.
(A), XIAP−/− cells were transfected with GFP-RhoGDI, along with HA-XIAP, HA-XIAP H467A, HA-XIAP ΔRING, or HA-XIAP ΔBIR. Co-immunoprecipitation was performed with anti-GFP antibody-conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting for detection of XIAP using HA antibody. (B). WT(Vector), XIAP−/−(Vector) and XIAP−/−(HA-XIAP) HCT116 cells were transfected with constructs of GFP-RhoGDI in combination with Ubiquitin-WT, Ubiquitin-K48R, Ubiquitin-K63R or Ubiquitin-K48R/K63R (KKRR). Forty-eight hours after transfection, cells were lysed and co-immunoprecipitated with anti-GFP antibody, and then immunoblotted with anti-Ub and anti-GFP antibodies. (C), 293T cells were transfected with various constructs as indicated for detection of RhoGDI ubiquitination by anti-Ub and anti-GFP antibodies. (D), A model for XIAP-regulated modulation of cell motility: XIAP binds to RhoGDI through its RING domain and inhibits RhoGDI SUMOylation which results in down-regulation of RhoGDI's function and promotes actin polymerization and cell motility. Or E3 ligase activity of XIAP RING domain could regulate some un-verified factors which subsequently control cell migration independent of RhoGDI binding.