Fig. 6

ACLY promoted colon cancer metastasis via promoting CTNNB1 translocation to nucleus. a HEK293T extracts transfected with Flag-ACLY for 48 h were immunoprecipitated with anti-Flag or mouse IgG and immunoblotted by anti-CTNNB1. b HEK293T extracts were immunoprecipitated with anti-ACLY or rabbit IgG and immunoblotted by anti-CTNNB1. c After 48 h of transfection, the colocation (yellow) of exogenous Flag-ACLY (green) and HA-CTNNB1 (red) in HEK293T cells were analyzed using a fluorescence microscope (magnification, 400×). Cell nucleus was stained by DAPI (blue). d HCT116 cells were transfected with siRNA-NC or siRNA-ACLY for 48 h, followed by 0, 1 and 2 h cycloheximide (CHX; 100 μg/ml) treatment or DMSO. Cell lysates were immunoblotted with anti-ACLY or anti-CTNNB1. Actin was the loading control. e HCT116 cells were transfected with siRNA-NC or siRNA-ACLY for 48 h. MG132 (100 mmol/l) was added for 2, 4 h or DMSO. Cell lysates were immunoblotted with anti-ACLY or anti-CTNNB1. Actin was the loading control. f, g mRNA levels of the indicated genes [TCF4, Slug, CCND1, c-MYC, Survivin, PYGO1, PYGO2] in ACLY stably silenced HCT116 cells and RKO cells were analyzed by QPCR. (H) Luciferase reporter assay using Top-flash and Fop-flash vectors was used to study CTNNB1 TCF promoter activity. 293 T cells were transfected with siACLY-1 or siACLY-2 (or siRNA-NC) for 48 h before luciferase reporter assay. i HCT116 cells transfected with siRNA-NC, siRNA-ACLY or Flag-ACLY for 48 h. Part of the cells was used to extract nuclear and cytosolic fractions. Cell lysates were immunoprecipitated with anti-ACLY or anti-CTNNB1. CTNNB1 band intensity was normalized to actin. Actin was the loading control. j-m HCT116 cells were cotransfected with Flag-ACLY or empty vector (as control) plus siRNA-NC or siRNA-CTNNB1 for 48 h. Efficiency of knockdown CTNNB1or overexpression of ACLY was assayed by western blot (Additional file 3: Figure S6B). Transwell migration assay (j, k) and invasion assay (l, m) were performed in HCT116 cells cotransfected with Flag-ACLY (or vector as control) and siCTNNB1 (or siNC as control). The quantitative analysis of cells across the transwell membrane to access the migration and invasion abilities (k and m)