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

Journal: Cancers

Article Title: USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer

doi: 10.3390/cancers12113090

Figure Lengend Snippet: USP1 is a novel DUB of TAZ. ( A ) Schematic representation of siRNA screen using Dharmacon siRNA DUB library prepared using BioRender. Three siRNAs targeting a single DUB were transfected per well. ( B ) Dot plot quantification of relative TAZ expression upon transfection with siRNA library from immunoblots. ( C ) Spearman correlation plot of mRNA expression of WWTR1 vs. USP1 from TCGA data. ( D ) Immunoblot analysis of USP1 and TAZ expression in a panel of breast cancer cell lines (MCF7 and T47D – luminal, SKBR3 – HER2 overexpression, MCF10A – non-transformed epithelial, MDA-MB-231, MDA-MB-468 and HCC1143, D3H2 lung metastasis of MDA-MB-231 cells– TNBC). GAPDH was used as a loading control. Immunoblots were quantified and normalized to GAPDH.

Article Snippet: USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector.

Techniques: Transfection, Expressing, Western Blot, Over Expression, Transformation Assay, Control

Journal: Cancers

Article Title: USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer

doi: 10.3390/cancers12113090

Figure Lengend Snippet: USP1 acts as a post-translational modifier of TAZ. ( A ) Immunoblot analysis for USP1 and TAZ expression in USP1 knockdown MCF10A and MDA-MB-231 cells. GAPDH was used as a loading control. ( B ) Relative mRNA expression of USP1 and TAZ in USP1 knockdown MCF10A and MDA-MB-231 cells. ( C ) Immunoblot analysis for TAZ expression in MCF10A and MDA-MB-231 upon treatment with 15µM ML323. GAPDH was used as a loading control. ( D ) Immunoblot analysis for TAZ expression in USP1 knockdown MCF10A and MDA-MB-231 cells with MG132treatment. GAPDH was used as a loading control. ( n = 2) ( E ) Immunoblot analysis for USP1 and TAZ in USP1 knockdown MDA-MD-231 cells with CHX treatment at various time points. GAPDH was used as a loading control. ( n = 3) ( F ) Immunoblot analysis for USP1 and TAZ expression in USP1 overexpressing MCF7 cells with CHX treatment at various time points. GAPDH was used as a loading control. ( n = 2) Data are shown as the mean ± SD. Unpaired two-tailed student t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. NS: no significance. Immunoblots were quantified and normalized to GAPDH.

Article Snippet: USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector.

Techniques: Western Blot, Expressing, Knockdown, Control, Two Tailed Test

Journal: Cancers

Article Title: USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer

doi: 10.3390/cancers12113090

Figure Lengend Snippet: USP1 and TAZ form a complex and alters TAZ ubiquitination. ( A ) Interaction between exogenous USP1 and TAZ in 293T cells. Cellular extracts were immunoprecipitated with Flag or HA beads and probed for antibodies against indicated proteins. GAPDH was used as a loading control. ( B ) Interaction between exogenous TAZ and endogenous USP1 or exogenous USP1 and endogenous TAZ. GAPDH was used as a loading control. ( C ) Immunofluorescence staining of Flag-USP1 and TAZ in MDA-MB-231 cells (Scale bar = 50 µm). ( D ) Increased ubiquitination of TAZ upon knockdown of USP1 and immunoprecipitation with His-beads. GAPDH was used as a loading control for input. ( E ) Immunoblot analysis of TAZ-WT or lysine mutants in the presence or absence of USP1. GAPDH was used as a loading control. ( F ) Decreased ubiquitination of K45/46R mutation after co-immunoprecipitation with HA-beads. GAPDH was used as a loading control for input. Immunoblots were quantified and normalized to GAPDH.

Article Snippet: USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Control, Immunofluorescence, Staining, Knockdown, Western Blot, Mutagenesis

Journal: Cancers

Article Title: USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer

doi: 10.3390/cancers12113090

Figure Lengend Snippet: Loss of function of USP1 alters cell proliferation and migration. ( A ) Cell proliferation assay of MCF10A and MDA-MB-231 cells upon knockdown of USP1. ( B ) Quantification of clonogenicity assays after 11 days upon knockdown of USP1 in MCF10A and MDA-MB-231 cells. ( C ) Quantification of cell migration after knockdown of USP1 in MCF10A and MDA-MB-231 cells. ( D ) Images and quantification of tumor size of shCtrl, shUSP1-1 or shUSP1-2 MDA-MB-231 cells injected into the mammary fat pad of SCID mice, n = 6. ( E ) Immunoblot analysis of TAZ expression in shUSP1 MCF10A cells. GAPDH was used as a loading control. ( F ) Cell proliferation of control or TAZ-4SA transduced shUSP1 MCF10A cells. ( G ) Quantification of clonogenicity assay after 11 days of control or TAZ-4SA transduced shUSP1 MCF10A cells. Data are shown as the mean ± SD. Unpaired two-tailed student t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. Western blots were quantified and normalized to GAPDH.

Article Snippet: USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector.

Techniques: Migration, Proliferation Assay, Knockdown, Injection, Western Blot, Expressing, Control, Two Tailed Test

Journal: Cancers

Article Title: USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer

doi: 10.3390/cancers12113090

Figure Lengend Snippet: USP1 overexpression increases cell proliferation in a partially TAZ-dependent manner. ( A ) Immunoblot analysis for USP1 and TAZ in USP1 transduced MDA-MB-231, 293T and MCF7 cells. GAPDH was used as a loading control. ( B ) Cell proliferation assay of USP1 overexpressing MDA-MB-231 cells. ( C ) Quantification of clonogenicity assay after 11 days in USP1 overexpressing MDA-MB-231 cells. ( D ) Representative images and quantification of spheroid size in control or USP1 overexpressing MDA-MB-231 cells (Scale bar = 50 µm). ( E ) Immunoblot analysis of TAZ knockdown in USP1 overexpressing MDA-MB-231 cells. GAPDH was used as a loading control. ( F ) Cell proliferation assay for shCtrl or shTAZ in USP1 overexpressing MDA-MB-231. ( G ) Quantification of clonogenicity assay after 11 days for shCtrl or shTAZ in USP1 overexpressing MDA-MB-231. Data are shown as the mean ± SD. Unpaired two-tailed student t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. Immunoblots were quantified and normalized to GAPDH.

Article Snippet: USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector.

Techniques: Over Expression, Western Blot, Control, Proliferation Assay, Knockdown, Two Tailed Test

Journal: Cancers

Article Title: USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer

doi: 10.3390/cancers12113090

Figure Lengend Snippet: USP1 and TAZ expression are correlated in TNBC. ( A ) Stratification of GSE2034 into USP1 low- and USP1 high- subsets. ( B ) Cordenosi YAP/TAZ gene signature is highly correlated with USP1 high-subset patients. ( C ) Kaplan-Meier relapse free survival analysis curves for USP1 and TAZ in breast cancer patients. ( D ) The dot plot represents the correlation of USP1 and TAZ in all the TMAs stained indicating a positive correlation between staining intensity of ALL samples. Pearson correlation between USP1 and TAZ protein levels were quantified by Aperio Image Scope of TNBC patients treated at RPCCC. Data are shown as the mean ± SD. Unpaired two-tailed student t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. ( E ) Representative images of H&E and IHC staining of tumor microarrays (TMAs) from TNBC patients treated at Roswell Park Comprehensive Cancer Center.

Article Snippet: USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector.

Techniques: Expressing, Staining, Two Tailed Test, Immunohistochemistry

Journal: Cancers

Article Title: USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer

doi: 10.3390/cancers12113090

Figure Lengend Snippet: Key resources table.

Article Snippet: USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector.

Techniques: Recombinant, Magnetic Beads, Western Blot, Lysis, Extraction, In Vitro, Transfection, Sequencing

Journal: International Journal of Molecular Medicine

Article Title: lncRNA KCNQ1OT1 promotes proliferation and invasion of glioma cells by targeting the miR-375/YAP pathway

doi: 10.3892/ijmm.2020.4760

Figure Lengend Snippet: KCN-mediated YAP upregulation enhances epithelial-mesenchymal transition in glioma cells. (A) Analysis of the expression level of YAP using western blotting following transfections with siNC or siKCN. (B) Following transfection with YAP1 overexpression plasmid the mRNA and protein expression levels of YAP in U251 and U87-MG cells were examined by reverse transcription-quantitative PCR and western blotting. (C) The proliferation rates of U251 and U87-MG glioma cells were measured following transfections with siNC or siKCN and pcDNA or YAP overexpression plasmid. ** P<0.01, *** P<0.001 vs. the siNC+YAP, siKCN and siKCN+YAP groups. (D) The effects of KCN-siRNA and YAP overexpression on colony formation with the magnification of 40×. Effects of the KCN/YAP pathway on the (E) migration and (F) invasion capacities of glioma cells. Magnification, ×200. (G) Effects of the KCN/YAP axis on the expression of YAP-downstream proteins according to western blotting with U251 cells, using GAPDH as a loading control. * P<0.05, ** P<0.01, *** P<0.001. KCN, KCNQ1OT1; si, small interfering; NC, negative control; YAP, Yes-associated protein; p-, phosphorylated; TAZ, transcriptional activator with PDZ-binding motif; MMP9, matrix metalloproteinase 9.

Article Snippet: The overexpression vector of YAP1 (pcDNA-YAP1) was kindly provided by Professor Yosef Shaul (Department of Molecular Genetics, Weizmann Institute of Science; Addgene plasmid #18881; http://n2t.net/addgene:18881 ; RRID: Addgene_18881).

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Migration, Control, Negative Control, Binding Assay

Journal: Cell

Article Title: HIF Regulates Multiple Translated Endogenous Retroviruses: Implications for Cancer Immunotherapy

doi: 10.1016/j.cell.2025.01.046

Figure Lengend Snippet: Key resource table

Article Snippet: The pLX304-EV-IRES-GFP (Addgene 187640) and pLX304-VHL-IRES-Tdtomato (Addgene 187643) vectors were used to generate isogenic 786-O, A-498 and RCC4 cell lines. pLenti-EF1α-HIF2α dPA (P405A, P531A) vector (Addgene 232954) was used to generate 786-O cells with overexpressed HIF2α mutant.

Techniques: Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Purification, Sample Prep, Sequencing, Amplification, Software, Binding Assay, Transferring, Reverse Transcription, Low Protein Binding, Antibody Purification, Adjuvant, Enzyme-linked Immunospot