Fig 1: NUSAP1 enhanced the proliferation of BLCA cells. (A and B). NUSAP1 expression in transfected 5637 cells determined by PCR and Western blotting. **P<0.01 vs NC. (C and D). NUSAP1 expression in transfected T24 cells by real-time PCR and Western blotting. **P<0.01 vs NC. (E and F). Cell proliferation was determined by a Cell Counting Kit-8 assay. **P<0.01, ***P<0.001 vs NC (5637) or NC (T24). (G, H and I). Cell proliferation was determined by an EdU assay (100× magnification). **P<0.01 vs NC (5637) or NC (T24).
Fig 2: NUSAP1 downregulation induced cell cycle arrest and apoptosis in BLCA cells. (A). GSEA suggests that NUSAP1 is closely related to the G2/M cell cycle phase. (B and C). Cell cycle analysis via flow cytometry. **P<0.01 vs NC. (D). Western blotting analysis of CDK1 and cyclin B1 protein expression. (E and F). Apoptosis was analyzed by flow cytometry. **P<0.01 vs NC (5637), *P<0.05 vs NC (T24). (G). Western blotting analysis detected the protein expression levels of Bax, Bcl-2 and cleaved-caspase3.
Fig 3: Individual knockdowns of ANKLE2, BANF1, VPS18, EIF1AD, or NUSAP1 do not induce spontaneous tau aggregation.(A) FACS plots of knockdown and control cells (non-targeting sgRNA) without adding exogenous tau seeds. Quadrants (Q2 shaded in yellow) in which FRET-positive cells were detected revealed the absence of a FRET signal in both control (NonT) and knockdown cells for ANKLE2, BANF1, VPS18, EIF1AD, and NUSAP1, indicating that no spontaneous tau aggregation was initiated. (B) However, these cells (bottom panels) showed FRET-positive cells only after tau seeds (400 ng of sarkosyl tau) were added, implying the requirement for an exogenous tau seed to trigger the aggregation of endogenous tau. Percentages of FRET-positive cells in Q2 are shown (n = 3, average ± SEM, 40,000 cells/experiment were analyzed).
Fig 4: NUSAP1 expression in BLCA tissues and cell lines and survival analysis. (A). NUSAP1 mRNA expression levels in 46 BLCA tumors and 14 normal tissues extracted from GSE3167. ***P<0.001 vs Normal. (B). Nineteen paired BLCA tumors and normal tissues extracted from the TCGA data portal. ***P<0.001 vs Normal. (C). NUSAP1 mRNA expression levels in 25 BLCA tumors and 23 normal tissues obtained from the Department of Urology of the First Affiliated Hospital of Chongqing Medical University. ***P<0.001 vs Normal. (D and E). NUSAP1 protein expression in BLCA and normal tissues as detected by immunohistochemistry (100× and 400× magnification). ***P<0.001 vs Normal. (F). NUSAP1 protein expression in 6 paired BLCA tissues. (G). Survival analysis of patients with BLCA obtained from PrognoScan (GSE13507). (H and I). The expression of NUSAP1 in BLCA cell lines was detected by real-time PCR and Western blotting. **P<0.01, ***P<0.001 vs SV-HUC-1 cells.
Fig 5: NUSAP1 downregulation in BLCA cells enhanced GEM chemosensitivity and suppressed cell migration, invasion, and EMT. (A). The chemosensitivity of BLCA cells to GEM was determined by a Cell Counting Kit-8 assay. *P<0.05, **P<0.01, ***P<0.001 vs NC (5637) or NC (T24). (B and C). A wound healing assay was performed to detect the migration of cells (100× magnification). **P<0.01, vs NC (5637) or NC (T24). (D and E). Transwell assays were performed to evaluate the migration and invasion of cells (100× magnification). **P<0.01, vs NC (5637) or NC (T24). (F). Western blotting analysis detected the protein expression levels of E-cadherin, vimentin and N-cadherin.
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