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: Primary antibodies used for experiments are the following: ERK (Cell Signaling Technology, #4695, 1:1000), p-ERK (Cell Signaling Technology, #4370, 1:1000), AKT (Cell Signaling Technology, #4691, 1:1000), p-AKT (Santa Cruz Biotechnology, sc-7985-R, 1:250), MET (Proteintech, 25869-1-AP, 1:1000), Tubulin (Proteintech, 66031-1-Ig, 1:3000), YAP1 (Cell Signaling Technology, #14074, 1:1000), p-Ser397-YAP1(Cell Signaling Technology, #13619, 1:1000), MOB-1 (Cell Signaling Technology, #13730, 1:1000), p-MOB-1 (Cell Signaling Technology, #8699 S, 1:1000), LATS-1 (Cell Signaling Technology, #3477,1:1000), p-LATS-1 (Cell Signaling Technology, #8654, 1:1000).

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: Primary antibodies used for experiments are the following: ERK (Cell Signaling Technology, #4695, 1:1000), p-ERK (Cell Signaling Technology, #4370, 1:1000), AKT (Cell Signaling Technology, #4691, 1:1000), p-AKT (Santa Cruz Biotechnology, sc-7985-R, 1:250), MET (Proteintech, 25869-1-AP, 1:1000), Tubulin (Proteintech, 66031-1-Ig, 1:3000), YAP1 (Cell Signaling Technology, #14074, 1:1000), p-Ser397-YAP1(Cell Signaling Technology, #13619, 1:1000), MOB-1 (Cell Signaling Technology, #13730, 1:1000), p-MOB-1 (Cell Signaling Technology, #8699 S, 1:1000), LATS-1 (Cell Signaling Technology, #3477,1:1000), p-LATS-1 (Cell Signaling Technology, #8654, 1:1000).

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. 4 AURKA inhibition disrupts c-MET and CSC properties. a Western blot illustrating c-MET levels after treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Tubulin was used as loading control. Results are plotted as the average ± SD of all the biological replicates and were normalized to the EGF condition. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3), one-way ANOVA (n = 4). b Colony formation assay following treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Results are plotted as the average ± SD of all the biological replicates. *p < 0.05, **p < 0.01 (n = 3), one-way ANOVA. c. ALDH1 relative activity after treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Results were normalized to the control condition for each cell line and are plotted as the average± SD of all the biological replicates (n = 3) **p < 0.01, ***p < 0.001, one-way ANOVA. d SOX2 gene expression after treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Results were normalized to the control condition in each cell line and are plotted as the average ± SD of all the biological replicates (n = 3). *p < 0.05, **p < 0.01 (n = 3), one-way ANOVA. e. Spheroid size (mm3) ability after treatment with alisertib and/or cetuximab in C10 cell line. Results are plotted as the average ± SD of all the biological replicates *p < 0.05, **p < 0.01 (n = 3), one-way ANOVA. CTR Control, ALS Alisertib, CTX Cetuximab, COM Combined.

Article Snippet: Primary antibodies used for experiments are the following: ERK (Cell Signaling Technology, #4695, 1:1000), p-ERK (Cell Signaling Technology, #4370, 1:1000), AKT (Cell Signaling Technology, #4691, 1:1000), p-AKT (Santa Cruz Biotechnology, sc-7985-R, 1:250), MET (Proteintech, 25869-1-AP, 1:1000), Tubulin (Proteintech, 66031-1-Ig, 1:3000), YAP1 (Cell Signaling Technology, #14074, 1:1000), p-Ser397-YAP1(Cell Signaling Technology, #13619, 1:1000), MOB-1 (Cell Signaling Technology, #13730, 1:1000), p-MOB-1 (Cell Signaling Technology, #8699 S, 1:1000), LATS-1 (Cell Signaling Technology, #3477,1:1000), p-LATS-1 (Cell Signaling Technology, #8654, 1:1000).

Techniques: Inhibition, Western Blot, Control, Colony Assay, Activity Assay, 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: a Western blot illustrating c-MET levels in the HCA46, SW48 and C10 cell lines. Tubulin was used as a loading control. Results are plotted as the average ± SD of all the biological replicates and were normalized to the HCA46 cell line ( n = 3). b Colony formation assay of CRC cell lines HCA46, SW48 and C10.Results are plotted as the average ± SD of all the biological replicates. ** p < 0.01 ( n = 3), one-way ANOVA. c ALDH1 relative activity in CRC cell lines HCA46, SW48 and C10. Results were normalized to the cetuximab-sensitive HCA46 cell line. Results are plotted as the average ± SD of all the biological replicates. ** p < 0.01, *** p < 0.001, one-way ANOVA. d Western blot illustrating c-MET expression in SW48 and C10 cell line transduced with YAP1 and YAP1 S397A plasmids, along with the empty vector. Results were normalized to the YAP1-trasduced cell lines. e Colony formation assay of SW48 and C10 cell line transduced with YAP1 and YAP1 S397A plasmids, along with the empty vector. Results are plotted as the average ± SD of all the biological replicates, ** p < 0.01 ( n = 3), one-way ANOVA. f ALDH1 relative activity in SW48 and C10 cell line transduced with YAP1 and YAP1 S397A plasmids, along with the empty vector. Results were normalized to the empty vector condition in each cell line. Results are plotted as the average ± SD of all the biological replicates, * p < 0.05, ** p < 0.01 ( n = 3), one-way ANOVA. g SOX2 gene expression in SW48 and C10 cell line transduced with YAP1 and YAP1 S397A plasmids, along with the empty vector. Results were normalized to the empty vector condition in each cell line. Results are plotted as the average ± SD of all the biological replicates * p < 0.05, ** p < 0.01 ( n = 3), one-way ANOVA.

Article Snippet: Primary antibodies used for experiments are the following: ERK (Cell Signaling Technology, #4695, 1:1000), p-ERK (Cell Signaling Technology, #4370, 1:1000), AKT (Cell Signaling Technology, #4691, 1:1000), p-AKT (Santa Cruz Biotechnology, sc-7985-R, 1:250), MET (Proteintech, 25869-1-AP, 1:1000), Tubulin (Proteintech, 66031-1-Ig, 1:3000), YAP1 (Cell Signaling Technology, #14074, 1:1000), p-Ser397-YAP1(Cell Signaling Technology, #13619, 1:1000), MOB-1 (Cell Signaling Technology, #13730, 1:1000), p-MOB-1 (Cell Signaling Technology, #8699 S, 1:1000), LATS-1 (Cell Signaling Technology, #3477,1:1000), p-LATS-1 (Cell Signaling Technology, #8654, 1:1000).

Techniques: Western Blot, Control, Colony Assay, Activity Assay, Expressing, Transduction, Plasmid Preparation, 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: a Western blot illustrating c-MET levels after treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Tubulin was used as loading control. Results are plotted as the average ± SD of all the biological replicates and were normalized to the EGF condition. * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 3), one-way ANOVA ( n = 4). b Colony formation assay following treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Results are plotted as the average ± SD of all the biological replicates. * p < 0.05, ** p < 0.01 ( n = 3), one-way ANOVA. c . ALDH1 relative activity after treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Results were normalized to the control condition for each cell line and are plotted as the average± SD of all the biological replicates ( n = 3) ** p < 0.01, *** p < 0.001, one-way ANOVA. d SOX2 gene expression after treatment with alisertib and/or cetuximab in SW48 and C10 cell lines. Results were normalized to the control condition in each cell line and are plotted as the average ± SD of all the biological replicates ( n = 3). * p < 0.05, ** p < 0.01 ( n = 3), one-way ANOVA. e . Spheroid size (mm 3 ) ability after treatment with alisertib and/or cetuximab in C10 cell line. Results are plotted as the average ± SD of all the biological replicates * p < 0.05, ** p < 0.01 ( n = 3), one-way ANOVA. CTR Control, ALS Alisertib, CTX Cetuximab, COM Combined.

Article Snippet: Primary antibodies used for experiments are the following: ERK (Cell Signaling Technology, #4695, 1:1000), p-ERK (Cell Signaling Technology, #4370, 1:1000), AKT (Cell Signaling Technology, #4691, 1:1000), p-AKT (Santa Cruz Biotechnology, sc-7985-R, 1:250), MET (Proteintech, 25869-1-AP, 1:1000), Tubulin (Proteintech, 66031-1-Ig, 1:3000), YAP1 (Cell Signaling Technology, #14074, 1:1000), p-Ser397-YAP1(Cell Signaling Technology, #13619, 1:1000), MOB-1 (Cell Signaling Technology, #13730, 1:1000), p-MOB-1 (Cell Signaling Technology, #8699 S, 1:1000), LATS-1 (Cell Signaling Technology, #3477,1:1000), p-LATS-1 (Cell Signaling Technology, #8654, 1:1000).

Techniques: Western Blot, Control, Colony Assay, Activity Assay, Gene Expression