Fig. 8

CDC16 overexpression abolished the inhibitory effect of c-Jun on SH-SY5Y cell proliferation and cell migration. (A). Proliferation of NC, c-Jun OE and c-Jun OE + CDC16 OE cells measured using EdU proliferation assay. (B). Proliferation of NC, c-Jun OE and c-Jun OE + CDC16 knockdown cells using EdU proliferation assay. Ratio of the number of EdU⁺ cells to the total number of cells was calculated from five areas in each well. The result was represented as mean ± SEM of three independent experiments. (C-E). Transwell assay to detect the effect of CDC16 on the inhibition of cell migration and invasion mediated by c-Jun overexpression. Numbers of migrated and invaded cells were calculated and analyzed statistically with ordinary one-way ANOVA combined with Sidak’s multiple comparisons test. (F-G). Protein immunoblotting assay to detect protein expression level of EMT and migration-related genes (N-cadherin, E-cadherin, vimentin, MMP9, MMP2 and TIMP2) in c-Jun OE + CDC16 OE cells (F) and c-Jun OE + CDC16 knockdown cells (G). Relative protein level was present as the ratio to GAPDH (mean ± SEM, n = 3). (H). qPCR to detect mRNA change of migration-related genes (MMP2 and TIMP2). (I-J). Immunoblot assay to detect phosphorylation level of GSK3β at ser9 and expression level of β-catenin in c-Jun OE cells (I, J), c-Jun + CDC16 OE cells (I) and c-Jun OE + CDC16 knockdown cells (J). Relative protein level was present as ratio to GAPDH. Data were statistically analyzed with ordinary one-way ANOVA combined with Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar = 100 μm