snai2  (Cell Signaling Technology Inc)

Journal: Cancers

Article Title: Characterisation of Bespoke Patient-Derived In Vitro Models of Ewing Sarcoma

doi: 10.3390/cancers18030512

Figure Lengend Snippet: Expression of EWSR1-gene fusion downstream targets, cell proliferation, cell migration and structure of the cytoskeleton in PDES, paired tumours and cell lines. ( A ) Expression of Ki67 in the PDES CCRG1-L-008 and TC-32 cell lines was detected by IF and HCI. Nuclei were stained with DAPI, and three regions were imaged per well across three independent experiments. IgG control cells were negative for fluorescence. White arrows = co-localisation of DAPI and Ki67, white scale bar = 200 µm. ( B ) Protein expression of downstream targets of the EWSR1 fusion (CD73, ITGB1, LOX, NKx2.2, SLUG/SNAI2, and SOX2) and the non-fusion target, ZEB2, in RD-ES cells and CCRG1-L-008 by IF and HCI. White scale bar = 200 µm, white arrows = positive staining in PDES. ( C ) Tumours paired with PDES (CCRG1-L-070, CCRG1-L-087, and CCRG1-L-088) analysed by IHC. Red scale bar = 200 µm. ( D ) There was a positive correlation between the percentage of cells positive for CD73, ITGB1, LOX, NKx2.2, SLUG/SNAI2, SOX2, ZEB2, and Ki67 in PDES (detected by HCI) and paired tumours (detected by IHC); Pearson’s correlation coefficient (R 2 = 0.70, p < 0.0001). Filled triangle = CD73, filled diamond = ITGB1, open triangle = Ki67, open circle = LOX, filled square = NKx2.2, open diamond = SLUG/SNAI2, filled square = SOX2, open square = ZEB2. ( E ) Detection of single base substitutions (SBS) and ( F ) small insertion and deletion (ID) mutational signatures from the COSMIC database in PDES and cell lines. After removal of common variants from WGS data using dbSNP and gnomAD, SBS5, ID1, ID2 and ID12 were the most common signatures in PDES and ES cell lines. ES cell lines contained additional SBS signatures (SBS7c, SBS8, SBS10c, SBS18, SBS30, and SBS40). ( G ) The migration of PDES and cell lines from a 3D spheroid, visualised using light microscopy (Olympus). The migration index (MI) was calculated using image J, expressing the total migrated area relative to the size of the spheroid core at 0 h. Migration was observed in 100% of PDES (25/25) and ES cell lines (6/6). PDES were more migratory than ES cell lines (* = p < 0.02). Light microscopy representative images of migrating SK-N-MC (MI = 11.6 ± 1) and PDES CCRG1-L-011 (MI = 149 ± 5) are shown. Black arrow = migration area. ( H ) The actin cytoskeleton of the RD-ES cell line and PDES (CCRG1-L-008, CCRG1-L-014, CCRG1-L-017, and CCRG1-L-066) was determined by IF, staining cells with phalloidin (red) and DAPI (blue). PDES had a more extensive actin cytoskeleton than ES cell lines. White arrow = actin cytoskeleton. The white scale bar = 100 µm.

Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies for Ki67 (0.46 μg/mL, M7240 clone MIB-1, Dako, Agilent Technologies), CD73 (100 μg/mL, A13821, Antibodies.com), ITGB1 (10.83 μg/mL, ab52971, Abcam Plc.), NKx2.2 (10 μg/mL, ab210463 488-conjugated, Abcam Plc.), LOX (10 μg/mL, ab197061, 647-conjugated, Abcam Plc.), SLUG/SNAI2 (1.75 μg/mL, #9585, C19G7, Cell Signalling Technology, London, UK), SOX2 (0.48 μg/mL, #5049, 488-conjugated, Cell Signalling Technology), ZEB2 (5 μg/mL, PA5-20980, Thermo Fisher Scientific) or corresponding isotype control (Normal Rabbit Serum Ig mix, 086199 (Thermo Fisher Scientific), Negative Control Mouse IgG1, X0931 (Dako), Alexa Fluor 647 and 488 Rabbit IgG isotype controls (ab199093 and ab199091, Abcam Plc.)).

Techniques: Expressing, Migration, Staining, Control, Fluorescence, Light Microscopy

Journal: Cancers

Article Title: Characterisation of Bespoke Patient-Derived In Vitro Models of Ewing Sarcoma

doi: 10.3390/cancers18030512

Figure Lengend Snippet: Comparison of downstream targets of the EWSR1 fusion and the genome of PDES and cell lines. ( A ) RNA levels of downstream targets of the EWSR1-FLI1 (as described in ), detected using RNA sequencing and normalised using DESeq2. Levels of 10/17 activated targets were significantly increased, and 9/11 repressed targets were significantly decreased in PDES. Blue circles = ES cell lines, black circle = PDES. Grey line = RNAs shared by PDES and cell lines, black line = RNAs differentially expressed in PDES and cell lines. ( B ) SLUG/SNAI2- and SOX2-positive cells in 12 PDES and A673, TC-32 and RD-ES cell lines, detected by IF and HCI. Results are shown as mean ± SEM, comparing PDES and cell lines. ( C ) The percentage of cells positive for both SOX2 and SLUG/SNAI2 expression (mean ± SEM) in 8 PDES detected using IF and HCI. The proteins were co-expressed in <7% of cells in PDES. Representative images show co-localisation of SOX2 (green) and SLUG/SNAI2 (red) in CCRG1-L-070 cells (white arrow) and in CCRG1-L-070 and CCRG1-L-087 (red arrow) expression of SLUG/SNAI2 only. Scale bar = 200 µm. ( D ) Dendrogram of SNPs from 7 PDES and 6 cell lines detected by WGS. Consistent with a common genetic driver (EWSR1 fusion), PDES and cell lines clustered together. ( E ) Significantly more coding mutations were detected by WGS in cell lines compared to PDES. ( F ) The total size of exon copy number variants in megabases (Mb) was greater in cell lines compared to PDES. * = < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies for Ki67 (0.46 μg/mL, M7240 clone MIB-1, Dako, Agilent Technologies), CD73 (100 μg/mL, A13821, Antibodies.com), ITGB1 (10.83 μg/mL, ab52971, Abcam Plc.), NKx2.2 (10 μg/mL, ab210463 488-conjugated, Abcam Plc.), LOX (10 μg/mL, ab197061, 647-conjugated, Abcam Plc.), SLUG/SNAI2 (1.75 μg/mL, #9585, C19G7, Cell Signalling Technology, London, UK), SOX2 (0.48 μg/mL, #5049, 488-conjugated, Cell Signalling Technology), ZEB2 (5 μg/mL, PA5-20980, Thermo Fisher Scientific) or corresponding isotype control (Normal Rabbit Serum Ig mix, 086199 (Thermo Fisher Scientific), Negative Control Mouse IgG1, X0931 (Dako), Alexa Fluor 647 and 488 Rabbit IgG isotype controls (ab199093 and ab199091, Abcam Plc.)).

Techniques: Comparison, RNA Sequencing, Expressing

Journal: Frontiers in Oncology

Article Title: TRIM31 acts as an intermediate molecule in the process by which Snai2 impairs the proliferation of cervical cancer cells

doi: 10.3389/fonc.2025.1537991

Figure Lengend Snippet: Relationship between TRIM31 and Snai2. (A) Differential gene expression by RNA sequencing of SiHa-Snai2 and SiHa-Vec cells is presented as the number of genes (A) and volcano plot (B) . (C) Relative expression of TRIM31 determined by RNA sequencing in SiHa-Snai2 cells compared with SiHa-Vec cells. (D) The correlation between Snai2 and TRIM31 in cervical cancer in GEPIA online database. (E) The RNA expression level of TRIM31 determined by real-time PCR. (F) The protein expression level of TRIM31 in SiHa-Snai2-overexpressing cells and control SiHa cells. (G) The correlation between the Snai2 and TRIM31mRNA level in cervical cancer patient tissues was analyzed via TIMER2.0 online database. ** p < 0.01.

Article Snippet: After sonication, the chromatin-protein complexes were immunoprecipitated with 5 μg of anti-Snai2 antibodies (#9585, Cell Signaling Technology) or 1 μg of mouse IgG.

Techniques: Gene Expression, RNA Sequencing, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in Oncology

Article Title: TRIM31 acts as an intermediate molecule in the process by which Snai2 impairs the proliferation of cervical cancer cells

doi: 10.3389/fonc.2025.1537991

Figure Lengend Snippet: TRIM31 may communicate with Snai2 and the Wnt/β-catenin pathway. (A) Western blot of β-catenin, cyclin D1 and c-myc (downstream of Wnt/β-catenin) in C-33 A-TRIM31 and C-33 A-GFP cells with the analyzed relative density. (B) Western blot analysis of the relative density of β-catenin, cyclin D1 and c-myc (downstream of Wnt/β-catenin) in SiHa-shTRIM31 and SiHa-GFP cells. (C) Western blot of β-catenin, cyclin D1 and c-myc in C-33 A-TRIM31 cells treated with the Wnt/β-catenin pathway inhibitor XAV. (D) Western blot of β-catenin, cyclin D1 and c-myc in SiHa-Snai2 cells, which rescued TRIM31 expression. (E) Schematic representation of the reporter construct containing the firefly luciferase coding sequence fused to the TRIM31 promoter region, which was separated into 6 parts. (F) Luciferase reporter assays showing six TRIM31 promoter region fragments recognized by Snai2 in SiHa-Snai2 cells. (G) Chromatin immunoprecipitation showed that Snai2 bound to the E-box of the P1 and P2 fragments of the TRIM31 promoter region. * p < 0.05, ** p < 0.01.

Article Snippet: After sonication, the chromatin-protein complexes were immunoprecipitated with 5 μg of anti-Snai2 antibodies (#9585, Cell Signaling Technology) or 1 μg of mouse IgG.

Techniques: Western Blot, Expressing, Construct, Luciferase, Sequencing, Chromatin Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: TRIM31 acts as an intermediate molecule in the process by which Snai2 impairs the proliferation of cervical cancer cells

doi: 10.3389/fonc.2025.1537991

Figure Lengend Snippet: Summary of the mechanism by which Snai2 may impair Wnt/β-catenin pathway activity by transcriptionally inhibiting TRIM31.

Article Snippet: After sonication, the chromatin-protein complexes were immunoprecipitated with 5 μg of anti-Snai2 antibodies (#9585, Cell Signaling Technology) or 1 μg of mouse IgG.

Techniques: Activity Assay