Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: Ectopic expression of GRHL3 does not influence proliferation/survival rate and cell cycle profile of T24 cells ( A , B ) Forced stable expression of GRHL3 in T24 cells resulted in significantly higher transcript and protein expression levels compared to parental T24 cells and empty vector controls (pLX304). Three biological replicates were used for RT-qPCR experiments. *** indicates p -value < 0.001. ( C ) Calculated population doublings using Thiazolyl blue tetrazolium bromide (MTT) assays were comparable between GRHL3-overexpressing T24 cells, parental T24 cells and empty vector controls after 11 days in culture (T24 vs. T24 + pLX304 vs. T24+GRHL3: 23.92 vs. 25.19 vs. 25.40 population doublings (PD) at day 11; n.s). ( D ) Cell cycle analysis was performed by Propidium iodide (PI) staining and relative amounts of cells in the respective cell cycle phases are indicated. No difference could be observed in G1, S or G2/M phases between parental cells, empty vector controls and ectopic GRHL3-expressing cells (statistically n.s.).

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Cell Cycle Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: GRHL3 overexpression reduces migration in scratch wound assay. ( A ) Representative scratch wound experiment, displaying the original scratch wound gap in each picture by red lines during gap closure over 12 h. ( B ) Quantification of the “wound” closure shows that ectopic GRHL3-expressing T24 cells migrate significantly more slowly compared to empty vector controls (T24 + pLX304) at 6, 9 and 12 h ( p = 0.0013 at 9 h). Seven replicates were performed in this experiment. ** indicates p -value < 0.01.

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Over Expression, Migration, Scratch Wound Assay Assay, Expressing, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: Ectopic overexpression of GRHL3 impairs invasion capacity of T24 cells in Boyden chamber assay. ( A ) Representative images from Boyden chamber assays showing “invaded” T24 cells stained with crystal violet, indicating lower invasive potential in GRHL3-overexpressing cells. Scale bar, 200 µm. ( B ) Analysis of the cell-covered area using software demonstrated that GRHL3-overexpressing cells (T24 + GRHL3) invaded through membranes to a significantly lower extent than empty vector controls (T24 + pLX304; p = 0.0317). SD bars are shown for a total of 70 images analyzed. * indicates p -value < 0.05.

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Over Expression, Boyden Chamber Assay, Staining, Software, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: (( Aa )–( Ae )) Representative formalin-fixed, paraffin-embedded (FFPE) hematoxylin and eosin (H&E) tissue sections of porcine bladders after 10 days in organ culture. ( Aa ) Native, untreated porcine bladder with intact morphology maintaining urothelium (black arrow), stroma and muscle tissue. ( Ab ) The de-epithelialized porcine bladder with stromal and muscle tissue, without urothelial re-growth, used as negative control. (( Ac )–( Ae )) Cell lines were seeded onto de-epithelialized bladder tissue and grown in organ culture. While non-invasive RT4 cells formed a stratified multi-layer on top of the basal membrane ( Ac , black arrow), T24 cells with empty vector controls (T24 + pLX304) showed diffuse invasion into the stromal compartment ( Ad , black dotted arrow). In contrast, T24 cells overexpressing GRHL3 (T24 + GRHL3) did not invade but instead formed a superficial multi-layer on the pig stroma, similar to non-invasive RT4 cells ( Ae ). Four biological replicates were performed for each experiment. Scale bar, 200 µm. ( B ) Anti-human leukocyte antigen (Anti-HLA) staining confirms the invasive capacity of T24 + pLX304 cells into the pig bladder stroma and muscle tissue as well as the reduced invasion ability upon overexpression of T24 + GRHL3. (H&E, hematoxylin and eosin; NC, negative control).

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Formalin-fixed Paraffin-Embedded, Organ Culture, Negative Control, Membrane, Plasmid Preparation, Staining, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells

doi: 10.3390/ijms22062959

Figure Lengend Snippet: Ectopic expression of GRHL3 drives T24 cells towards a more differentiated phenotype when cultured in organ culture conditions allowing cell–stroma interactions. ( A ) Native porcine urothelium expresses FOXA1 in organ culture. ( B ) De-epithelialized porcine bladder was used as s negative control. ( C ) RT4 cells seeded on the de-epithelialized porcine bladder form a multi-layered epithelial lining. Not all cells are FOXA1-positive (red arrow), indicating partial differentiation. ( D ) T24+pLX304 cells invade into the porcine bladder stroma and muscle and lack FOXA1 expression (black dotted arrows). ( E ) GRHL3-expressing T24 cells do not invade and express FOXA1 (red dotted arrow). ( F ) Western blot shows FOXA1 protein expression only in RT4 but not in T24 cells, irrespective of ectopic GRHL3 expression when grown in 2D culture conditions. ( G ) Similarly, FOXA1 expression in 2D cell culture is not upregulated in T24 + GRHL3 cells. *** indicates p -value < 0.001; ns = not significant.

Article Snippet: The empty vector (pLX304) was acquired from Addgene and a 1688-bp PCR product of GRHL3 cDNA was cloned by Nhel and Xbal (New England Biolabs, Ipswich, MA, USA) into the pLX304 backbone.

Techniques: Expressing, Cell Culture, Organ Culture, Negative Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Role of Nudt2 in Anchorage-Independent Growth and Cell Migration of Human Melanoma

doi: 10.3390/ijms241310513

Figure Lengend Snippet: Soft agar colony formation in cells overexpressing WT Nudt2(PLX304 − V5 − Nudt2). Stable overexpression of WT Nudt2 was generated in CHL − 1 melanoma cell and the cells were seeded at 5000 cells/250 µL in 24-well plates for 21 days. ( A ). Expression of endogenous Nudt2, overexpressed Nudt2 (V5 − Nudt2) was analyzed via Western blot. ( B ). Representative photomicrographs: colonies were stained with p-iodonitrotetrazolium violet stain. ( C ). Colony numbers were counted using the Image J software(ij153-win-java8); n = 7. The data are represented as mean ± SEM. For statistical analysis, Wilcoxon signed-rank test was used. * p < 0.01.

Article Snippet: A mixture of vsvg/viral envelope plasmid, dvpr/viral packaging vector, PLX304 empty vector (Addgene) or Nudt2 WT vector or PLX304-Luciferase-V5 vector were co-transfected into the 293T cells.

Techniques: Over Expression, Generated, Expressing, Western Blot, Staining, Software

Journal: International Journal of Molecular Sciences

Article Title: Role of Nudt2 in Anchorage-Independent Growth and Cell Migration of Human Melanoma

doi: 10.3390/ijms241310513

Figure Lengend Snippet: The lists of constructs and vectors used in this study.

Article Snippet: A mixture of vsvg/viral envelope plasmid, dvpr/viral packaging vector, PLX304 empty vector (Addgene) or Nudt2 WT vector or PLX304-Luciferase-V5 vector were co-transfected into the 293T cells.

Techniques: Construct, Plasmid Preparation, Control, Over Expression, Expressing, Luciferase, In Vivo