Fig. 3

c-Jun overexpression inhibited SH-SY5Y cell proliferation and migration. (A). EdU proliferation assay of negative control (NC) and c-Jun OE SH-SY5Y cells with representative images shown. (B). Calculated ratio of number of EdU + cells to total number of cells. Data were represented as mean ± SEM of three independent experiments with mean number collected from five areas of each well. (C). qPCR assay to detect the mRNA level of proliferation related genes in NC and c-Jun OE SH-SY5Y cells. (D). Viability of NC and c-Jun OE cells measured with CCK-8 assay. (E). Cell scratch experiment to evaluate the effect of c-Jun overexpression on wound healing ability of SH-SY5Y cells. Migration rate was statistically analyzed with the ratio of migrated area to original scratched area. (F-G). Transwell assay to evaluate the effect of c-Jun overexpression on cell migration and invasion. Migrated cell number were calculated and statistically analyzed by unpaired t-test. (H). Protein immunoblotting assay to detect changes of EMT and migration-related proteins (N-cadherin, E-cadherin, vimentin, MMP9, MMP2 and TIMP2). Results were presented as mean ± SEM (n = 3). (I). qPCR to examine mRNA changes of migration-related genes (MMP9, MMP2 and TIMP2) in c-Jun OE cells. qPCR results were calculated using 2^−ΔΔCT and analyzed by Graphpad software. The results were presented as mean ± SEM (n = 3). (J). Immunoblot assay to detect phosphorylation level of GSK3β at ser9 and expression level of β-catenin. (K). Relative protein levels of c-Jun, pGSK3β and β-catenin were determined by the ratio to GAPDH. (L). Changes of β-catenin in nucleus as detected by nucleocytoplasmic separation experiment. All data were statistically analyzed with unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001. OE represents SH-SY5Y cells infected with c-Jun overexpression lentivirus, whereas NC represents SH-SY5Y cells infected with control virus. Scale bar = 100 μm