primary neuronal culture sh sy5y human neuroblastoma cells  (ATCC)

Journal: Biology direct

Article Title: c-Jun promotes neuroblastoma cell differentiation by inhibiting APC formation via CDC16 and reduces neuroblastoma malignancy.

doi: 10.1186/s13062-025-00630-1

Figure Lengend Snippet: Fig. 1 c-Jun induced neuroblastoma cell differentiation. (A). c-Jun expression in different neuroblastoma cell lines and glioblastoma cell lines deposited at The Human Protein Atlas. (B). SH-SY5Y cells were induced for differentiation by RA and expression of c-Jun protein during differentiation was detected by Western blot (n = 3). (C). Ch-IP assay by using c-Jun antibody to detect the binding of c-Jun onto the promoters of PDGFRα, RARα, RARβ and RARγ. (D). Immunoblots showing the expression of differentiation markers in control cells and c-Jun OE cells after RA-induced differentiation. (E-G). Relative protein level of TUBB3, GAP43 and c-Jun were analyzed by unpaired t-test (mean ± SEM of triplicate experiments). *p < 0.05, **p < 0.01. (H). Neural differentiation of negative control (NC, cells transfected with Lentiviral vector) and c-Jun OE SH-SY5Y cells. Cells were stained with TUBB3 and DAPI, and images were acquired using a fluorescence microscope (Nikon). (I). Number of cells with neurite outgrowth (> 20 μm) was counted using Image J software. Pictures were taken from 3–5 areas of each well with at least three independent experiments. The mean of each well was used for statistical analysis with unpaired t-test. Means with SEM were shown. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Cell line and culturing conditions Human neuroblastoma cell line SH-SY5Y and human embryonic kidney cell line 293T were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Differentiation, Expressing, Western Blot, Binding Assay, Control, Negative Control, Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Software

Journal: Biology direct

Article Title: c-Jun promotes neuroblastoma cell differentiation by inhibiting APC formation via CDC16 and reduces neuroblastoma malignancy.

doi: 10.1186/s13062-025-00630-1

Figure Lengend Snippet: 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

Article Snippet: Cell line and culturing conditions Human neuroblastoma cell line SH-SY5Y and human embryonic kidney cell line 293T were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Migration, Proliferation Assay, Negative Control, CCK-8 Assay, Transwell Assay, Western Blot, Software, Phospho-proteomics, Expressing, Infection, Control, Virus

Journal: Biology direct

Article Title: c-Jun promotes neuroblastoma cell differentiation by inhibiting APC formation via CDC16 and reduces neuroblastoma malignancy.

doi: 10.1186/s13062-025-00630-1

Figure Lengend Snippet: Fig. 5 c-Jun interacted with CDC16 and inhibited its interaction with CDC20 and CDC27. (A). Proteins interacting with c-Jun were detected by co-immu noprecipitation (Co-IP) and silver staining in c-Jun OE cells. (B). Different protein bands in IgG group (NC) and c-Jun antibody group (IP) were analyzed by mass spectrometry, and the number of different proteins in the two groups were shown by Venn diagram. (C-D). Co-IP assay with c-Jun antibody (C) and CDC16 antibody (D) to confirm the interaction of CDC16 with c-Jun. IgG were used as negative control. (E). Co-IP assay with CDC16 antibody to detect the interaction between CDC16 and c-Jun or the interaction between CDC16 and CDC20/CDC27 in negative control (NC, cells transfected with Lentiviral vec tor) and c-Jun OE SH-SY5Y cells. (F). Relative protein level precipitated by CDC16 antibody. The ratio of IP group to total protein was statistically analyzed by t test (*p < 0.05, **p < 0.01, ***p < 0.001) and the results were presented as mean ± SEM (n = 3)

Article Snippet: Cell line and culturing conditions Human neuroblastoma cell line SH-SY5Y and human embryonic kidney cell line 293T were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Negative Control, Transfection

Journal: Biology direct

Article Title: c-Jun promotes neuroblastoma cell differentiation by inhibiting APC formation via CDC16 and reduces neuroblastoma malignancy.

doi: 10.1186/s13062-025-00630-1

Figure Lengend Snippet: Fig. 6 CDC16 overexpression abolished cell differentiation mediated by c-Jun overexpression. (A). Immunoblots showing the expression of differentia tion markers in control cells (NC), c-Jun overexpressed cells (c-Jun OE) and cells co-overexpressed c-Jun and CDC16 (c-Jun OE + CDC16 OE) after RA- induced differentiation. (B-D). Relative protein level of TUBB3 and GAP43 (ratio to GAPDH) analyzed by ordinary one-way ANOVA combined with Sidak’s multiple comparisons test. (E). Neural differentiation of NC, c-Jun OE and c-Jun OE + CDC16 OE SH-SY5Y cells. Cells were stained with TUBB3 and DAPI and images were acquired using a fluorescence microscope (Nikon). Number of cells with neurite outgrowth (> 20 μm) was counted using Image J software with cell counter plugin. Data were analyzed by ordinary one-way ANOVA combined with Sidak’s multiple comparisons test (mean ± SEM, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Scale bar: 50 μm

Article Snippet: Cell line and culturing conditions Human neuroblastoma cell line SH-SY5Y and human embryonic kidney cell line 293T were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Cell Differentiation, Western Blot, Expressing, Control, Staining, Fluorescence, Microscopy, Software

Journal: Biology direct

Article Title: c-Jun promotes neuroblastoma cell differentiation by inhibiting APC formation via CDC16 and reduces neuroblastoma malignancy.

doi: 10.1186/s13062-025-00630-1

Figure Lengend Snippet: Fig. 7 CDC16 overexpression rescued cell cycle arrest in G1 stage induced by c-Jun overexpression. (A-B). Cell cycle analysis by flow cytometry for negative control (NC), c-Jun OE and c-Jun OE + CDC16 OE cells. Percentage of cells at different cell cycle stage was analyzed with ordinary one-way ANOVA combined with Sidak’s multiple comparisons (mean ± SEM, n = 3; *p < 0.05, **p < 0.01). (C). Schematic representation of cell cycle progression and expression of cyclins at different stage. c-Jun overexpression inhibited cell cycle progression from G1 to S stage, increased G1 to G0 stage and promoted cell differentiation, whereas c-Jun and CDC16 co-overexpression promoted cell cycle progression. (D). Western blot showing the expression of cell cycle related markers in NC, c-Jun OE and C-Jun OE + CDC16 OE cells. Relative protein level was analyzed as ratio to GAPDH and heatmap was represented as mean ± SEM (n = 3). (E). qPCR result showing the mRNA expression of cell cycle related markers (cyclin D1, cyclin E1, cyclin A2, cyclin B and CDKs) in NC, c-Jun OE and c-Jun OE + CDC16 OE cells. Data were analyzed with ordinary one-way ANOVA combined with Sidak’s multiple comparisons (mean ± SD, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). (F). CDC16 shRNA was constructed to pLVX-shRNA2-GFP plasmid and then transfect into HEK293T cells. Cells expressing pLVX-shRNA2-GFP-CDC16 shRNA showed green fluorescence and expression of CDC16 was detected by Western blot. (G). Represent images of SH-SY5Y cells transfected with CDC16 shRNA lentivirus (left: 100µL and right: 200µL). (H-I). Western blot showing the expression of cell cycle related markers in NC, c-Jun OE and c-Jun OE + CDC16 knockdown cells. Relative protein level was analyzed as ratio to GAPDH (mean ± SEM, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Cell line and culturing conditions Human neuroblastoma cell line SH-SY5Y and human embryonic kidney cell line 293T were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Cell Cycle Assay, Flow Cytometry, Negative Control, Expressing, Cell Differentiation, Western Blot, shRNA, Construct, Plasmid Preparation, Fluorescence, Transfection, Knockdown

Journal: Biology direct

Article Title: c-Jun promotes neuroblastoma cell differentiation by inhibiting APC formation via CDC16 and reduces neuroblastoma malignancy.

doi: 10.1186/s13062-025-00630-1

Figure Lengend Snippet: 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 repre sented as mean ± SEM of three independent experiments. (C-E). Transwell assay to detect the effect of CDC16 on the inhibition of cell migration and inva sion 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

Article Snippet: Cell line and culturing conditions Human neuroblastoma cell line SH-SY5Y and human embryonic kidney cell line 293T were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Migration, Proliferation Assay, Knockdown, Transwell Assay, Inhibition, Western Blot, Expressing, Phospho-proteomics