Journal: Cancers

Article Title: Cancer Cell Biomechanical Properties Accompany Tspan8-Dependent Cutaneous Melanoma Invasion

doi: 10.3390/cancers16040694

Figure Lengend Snippet: EMT-TFs regulate the expression of Tspan8, crucial for cutaneous melanoma invasion. ( a ) qPCR analysis of SNAI2, ZEB2, or TSPAN8 transcript expression levels 72 h after SNAI2 ( left panel ), ZEB2 ( right panel ), or control ( both panels ) siRNA transfection in non-invasive (IC8) or invasive (T1C3 and C-09.10) melanoma cells. ( b ) SNAI2, ZEB2, and Tspan8 expression measured by Western blot analysis 72 h after SNAI2 ( left panel ), ZEB2 ( right panel ), or control ( both panels ) siRNA transfection in T1C3 or C-09.10 melanoma cells. ( c ) ( Top panel ), qPCR analysis of TSPAN8 transcript expression level in C-09.10-ZEB1 compared to C-09.10-CT melanoma cells. ( Bottom panel ), ZEB1 and Tspan8 expression measured by Western blot analysis in C-09.10-CT and C-09.10-ZEB1 melanoma cells. ( d ) ZEB1 chromatin immunoprecipitation (ChIP) assays performed in C-09.10 melanoma cells, using IgG antibody as a negative control. Enrichment of TSPAN8 promoter region was analyzed by qPCR in comparison with a negative control promoter region located –1 kb upstream of the beginning of pTSPAN8 or a positive control using MITF promoter region. Results are representative of three independent experiments. ( e ) ( Upper left panel ), qPCR analysis of SNAI2, ZEB1, or TSPAN8 transcript expression levels in healthy skin, melanoma, or local metastasis conditions of mitf::Xmrk/+ medaka. ( Upper right panel ), ZEB1 and Tspan8 expression measured by Western blot analysis in healthy skin versus melanoma conditions of mitf::Xmrk/+ medaka. ( Lower panel ), Tspan8 staining in areas of invasive melanoma by immunohistochemistry on melanoma sections from a mitf::Xmrk/+ medaka fish, using a control antibody (serum before immunization, ( left image ) or a custom antibody directed against Tspan8 (serum after immunization, ( right image ). ( f ) ( Left panel ), quantification of Tspan8 expression level, based on the immunoscore previously established (47), in ZEB1 low ( n = 6) and ZEB1 high ( n = 7) melanoma samples. ( Right panel ), ZEB1 ( top ) and Tspan8 ( bottom ) expression analyzed by immunohistochemistry in ZEB1 int melanoma samples ( n = 5). GAPDH was used as a housekeeping gene in qPCR analysis, where data are shown as the mean ± SEM of three independent experiments, and β-actin was used as a loading control in Western blot analysis, where results are representative of two independent experiments. Statistical significance of qPCR data was assessed by two-tailed Student’s t -test for paired samples, where mean differences were considered significant when p < 0.05 (* p < 0.05; ** p < 0.01). The original western blot figures can be found in .

Article Snippet: The used antibodies were as follows: mouse monoclonal TS29 clone (1/500) for human Tspan8 detection; sc-166476 mouse antibody (1/500, Santa Cruz, CA, USA) for human SNAI2 detection; rabbit monoclonal antibody HPA027524 (RRID:AB_1844977, Sigma, Burbank, CA, USA) for human ZEB2 detection; rabbit monoclonal antibody HPA003456 (1/500, RRID: AB_10603840, Sigma, USA) for human ZEB1 detection; custom antibody elaborated from rabbit serum before and after immunization (Covalab, Bron, France) for Medaka Tspan8 detection; mouse monoclonal anti-actin clone C4 antibody (1/5000; MAB1501, Millipore, Darmstadt, Germany) for both human and medaka β-actin detection.

Techniques: Expressing, Control, Transfection, Western Blot, Chromatin Immunoprecipitation, Negative Control, Comparison, Positive Control, Staining, Immunohistochemistry, Two Tailed Test

Journal: Cancers

Article Title: Cancer Cell Biomechanical Properties Accompany Tspan8-Dependent Cutaneous Melanoma Invasion

doi: 10.3390/cancers16040694

Figure Lengend Snippet: Tspan8 modulation regulates stiffness and morphological properties of cutaneous melanoma cells. ( a ) Global stiffness measurements of different melanoma cell constructs according to their Tspan8 expression levels measured by Western blot analysis ( bottom panels ). ( Top left panel ), non-invasive melanoma cells with an empty vector versus melanoma cells with a vector allowing ectopic expression of Tspan8. ( Top middle panel ), melanoma cells expressing very low level of Tspan8 versus the enrichment of the same cells expressing Tspan8. ( Top right panel ), melanoma cells strongly expressing Tspan8 with a control shRNA compared to the same cells transfected with a shRNA directed against TSPAN8. ( b ) Morphological analysis (aspect ratio and circularity) of melanoma cells according to their Tspan8 expression profile in correlation with ( a ). ( c ) Global stiffness measurement of melanoma cells expressing or not expressing ZEB1 and Tspan8 according to the ectopic expression of ZEB1 and transfection of control or TSPAN8 siRNA. ( d ) Global stiffness measurement of melanoma cells expressing or not expressing Tspan8 in human skin reconstructs (HSR). ( Left panel ), measurement at the surface of whole HSR with cells not expressing Tspan8 and cells ectopically expressing Tspan8 ( left ), along with cells expressing Tspan8 against the same cells expressing a shRNA against Tspan8 ( right ). ( Right panel ), measurement of melanoma cells on cryosections of HSR with cells not expressing Tspan8 and cells ectopically expressing Tspan8 ( left ) or cells expressing Tspan8 against the same cells expressing a shRNA against Tspan8 ( right ). β-actin was used as a loading control in Western blot analysis, where results are representative of two independent experiments. Statistical significance was assessed by two-tailed Student’s t -test or Wilcoxon test, depending on the normality of the paired samples. Mean differences were considered significant when p < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001; ns non-significant). The original western blot figures can be found in .

Article Snippet: The used antibodies were as follows: mouse monoclonal TS29 clone (1/500) for human Tspan8 detection; sc-166476 mouse antibody (1/500, Santa Cruz, CA, USA) for human SNAI2 detection; rabbit monoclonal antibody HPA027524 (RRID:AB_1844977, Sigma, Burbank, CA, USA) for human ZEB2 detection; rabbit monoclonal antibody HPA003456 (1/500, RRID: AB_10603840, Sigma, USA) for human ZEB1 detection; custom antibody elaborated from rabbit serum before and after immunization (Covalab, Bron, France) for Medaka Tspan8 detection; mouse monoclonal anti-actin clone C4 antibody (1/5000; MAB1501, Millipore, Darmstadt, Germany) for both human and medaka β-actin detection.

Techniques: Construct, Expressing, Western Blot, Plasmid Preparation, Control, shRNA, Transfection, Two Tailed Test