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: British journal of cancer

Article Title: Inhibition of the AURKA/YAP1 axis is a promising therapeutic option for overcoming cetuximab resistance in colorectal cancer stem cells.

doi: 10.1038/s41416-024-02649-z

Figure Lengend Snippet: Fig. 1 YAP1 activity correlates with Ser397 phosphorylation during primary resistance to cetuximab in CRC cell lines. a Plot representing the distribution of YAP1 expression in CRC cell lines sensitive to cetuximab compared to the resistant ones ns=non-significant *p < 0.05, two- tailed t-Student’s test. b Plot depicting the distribution of YAP1 activity score in cetuximab-sensitive CRC cell lines compared to the resistant ones. c Correlation analysis between AURKA expression and YAP1 activity score. d Correlation analysis between AURKA and YAP1 expression levels. e Western blot illustrating the basal levels of total amount and phosphorylated YAP1 (Ser397) in CRC cell lines HCA46, SW48 and C10. Tubulin was used as a loading control. Results are plotted as the average ± SD of all the biological replicates and were normalised to the HCA46 cell line (n = 3). ns=non-significant, **p < 0.01, ***p < 0.001, one-way ANOVA. f Gene expression levels of CTGF and CYR61 in CRC cell lines HCA46, SW48 and C10 (n = 3). ns=non-significant, **p < 0.01, ***p < 0.001, one-way ANOVA.

Article Snippet: To generate the vector for YAP1 overexpression, YAP1-V5 in pLX304 (Addgene #42555; http://n2t.net/addgene:42555) gifted by William Hahn was used. pLX304 plasmid was used as empty vector.

Techniques: Activity Assay, Phospho-proteomics, Expressing, Two Tailed Test, Western Blot, Control, Gene Expression

Journal: British journal of cancer

Article Title: Inhibition of the AURKA/YAP1 axis is a promising therapeutic option for overcoming cetuximab resistance in colorectal cancer stem cells.

doi: 10.1038/s41416-024-02649-z

Figure Lengend Snippet: Fig. 2 AURKA inhibitor alisertib disrupts YAP1 Ser397 phosphorylation and overcomes cetuximab resistance. a Western blots showing the total and phosphorylated forms of YAP1, AKT and ERK after cetuximab and/or alisertib treatment. Cells were stimulated with 40 ng/mL EGF after both treatments to induce ERK and AKT phosphorylation. Tubulin was used as loading control. Results are plotted as the average ± SD of all the biological replicates and were normalised to the EGF condition (n = 3, n = 4 for p-ERK in SW48 cell line).ns=non-significant, **p < 0.01, ***p < 0.001, one-way ANOVA. b Proliferation levels of SW48 and C10 cell lines treated with cetuximab and alisertib, either alone or in combination, relative to control (n = 3). ns=non-significant, **p < 0.01, ***p < 0.001, one-way ANOVA. CTR Control, ALS Alisertib, CTX Cetuximab, COM Combined.

Article Snippet: To generate the vector for YAP1 overexpression, YAP1-V5 in pLX304 (Addgene #42555; http://n2t.net/addgene:42555) gifted by William Hahn was used. pLX304 plasmid was used as empty vector.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: British journal of cancer

Article Title: Inhibition of the AURKA/YAP1 axis is a promising therapeutic option for overcoming cetuximab resistance in colorectal cancer stem cells.

doi: 10.1038/s41416-024-02649-z

Figure Lengend Snippet: Fig. 5 Alisertib effectively overcomes cetuximab resistance in PDX models with high levles of YAP1 phosphorylation. a Schematic representation outlining the criteria for selecting the tumor for the PDX model based on Western blot screening of YAP1 Ser397 phosphorylation. The tumor with the highest YAP1 phosphorylation levels was chosen for implantation. Mice were treated with 10 mg/kg/day alisertib five days a week and/or 0.4 mg/mice/day cetuximab twice a week for 21 days. Tumor volume and body weight was measured three times a week. Created with BioRender.com b Tumor volume measurement during 21-day treatment with alisertib and/or cetuximab. Representative images of tumors extrated from each group of mice. Results are plotted as the average ± SD of all the tumor volumes for each condition, ***p < 0.001 (n = 6), two-way ANOVA. c T/C (treated/control) ratio of tumors after the 21-day treatment with alisertib, cetuximab and combination. d Ki67 staining of tumors treated with alisertib, cetuximab, the combination of both or neither. *p < 0.05 (n = 3), Welch and Brown-Forsythe ANOVA. CTR Control, ALS Alisertib, CTX Cetuximab, COM Combined.

Article Snippet: To generate the vector for YAP1 overexpression, YAP1-V5 in pLX304 (Addgene #42555; http://n2t.net/addgene:42555) gifted by William Hahn was used. pLX304 plasmid was used as empty vector.

Techniques: Phospho-proteomics, Western Blot, Control, Staining

Journal: British journal of cancer

Article Title: Inhibition of the AURKA/YAP1 axis is a promising therapeutic option for overcoming cetuximab resistance in colorectal cancer stem cells.

doi: 10.1038/s41416-024-02649-z

Figure Lengend Snippet: Fig. 6 Alisertib reduces c-MET expression levels and impairs CSC features in vivo. a Western blot analysis of total (n = 3) and phosphorylated (n = 4) levels of LATS-1, YAP1, ERK and MOB-1 in tumors treated with alisertib and/or cetuximab, normalized to the control. Results are plotted as the average ± SD of all the biological replicates, **p < 0.01, one-way ANOVA. Differences in p-ERK expression were compared by using Welch and Brown-Forsythe ANOVA since residuals did not meet the homoscedasticity criteria of one-way ANOVA. b Analysis of SOX2 expression levels in tumors after treatment with alisertib and/or cetuximab, normalized to non-treated tumors. Results are plotted as the average ± SD of all the biological replicates, *p < 0.05 (n = 4), one-way ANOVA. c Assessment of ALDH1 activity after treatment with alisertib and/or cetuximab, normalized to non-treated tumors. Results are plotted as the average ± SD of all the biological replicates, *p < 0.05 (n = 4), one-way ANOVA. d. Evaluation of the gene expression levels of SOX2 in tumors after being treated with alisertib and/or cetuximab. Results are plotted as the average ± SD of all the biological replicates, *p < 0.05 (n = 4), one-way ANOVA. CTR Control, ALS Alisertib, CTX Cetuximab, COM Combined.

Article Snippet: To generate the vector for YAP1 overexpression, YAP1-V5 in pLX304 (Addgene #42555; http://n2t.net/addgene:42555) gifted by William Hahn was used. pLX304 plasmid was used as empty vector.

Techniques: Expressing, In Vivo, Western Blot, Control, Activity Assay, Gene Expression