Fig 1: GINS1 physically interacts with TOP2A, while GINS1 induces glioma cell proliferation and migration in a TOP2A-dependent manner(A) Immunoaffinity purification and mass spectrometry analysis of GINS1-containing protein n complexes. Cellular extracts from U251 cells stably expressing FLAG (Vector) or FLAG-GINS1 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptide. The eluates were resolved by SDS–PAGE and silver-stained. The protein bands were retrieved and analyzed by mass spectrometry. Detailed results from the mass spectrometric analysis are provided in Table S4.(B) IP of whole-cell lysates from U251 cells followed by IB with antibodies against the indicated proteins.(C) GST pull-down assays with GST-fused GINS1 and in vitro transcribed/translated TOP2A, VCP, EBP1, and NPM as indicated.(D) GST pull-down assays with the indicated GST-fused proteins and in vitro transcribed/translated GINS1.(E) WB analysis of TOP2A and USP15 expression in GINS1-silenced U251 cells and GINS1-overexpressing A172 cells.(F) Relative TOP2A mRNA expression in GINS1-silenced U251 cells and GINS1-overexpressing A172 cells measured by real-time PCR. Bar graph data are presented as the mean ± SD.(G) Western blot analysis of exogenous TOP2A in HEK293T cells co-expressing GINS1 and TOP2A.(H) Western blot analysis of the protein levels of GINS1 and TOP2A in clinical glioma specimens.(I) Western blot analysis of the protein levels of GINS1 and TOP2A in normal brain cells (HEB) and glioma cell lines (T98G, U251, U373, and A172).(J) Western blot analysis of TOP2A levels in GINS1-silenced U251 cells treated with MG132 and CQ.(K and L) CHX chase analysis of TOP2A protein half-life in GINS1-overexpressing A172 cells and GINS1-silenced U251 cells.(M and N) Ubiquitination assays of endogenous TOP2A in lysates from A172 cells transfected with Flag-GINS1 (M) or U251 cells stably expressing GINS1 shRNA (N).(O and P) Stable GINS1 overexpression (A172-GINS1) cells were transfected with TOP2A shRNA, and stable GINS1 knockdown (U251-shRNA) cells were transfected with TOP2A cDNA. The role of TOP2A in GINS1-induced proliferation was examined by MTT assays. Bar graph data are presented as the mean ± SD; ∗, p < 0.05.(Q and R) Transwell assays detected the effect of TOP2A on GINS1-induced migration. Representative pictures are shown on the left, and the statistical data are counted on the right. Experiments were performed independently thrice. Bar graph data are presented as the mean ± SD; ∗, p < 0.05. Scale bars, 200 μm.(S) Western blot analysis of the expression level of MMP-9 protein and EMT-related proteins in co-transfected U251 and A172 cells.