p yap1 ser397  (Proteintech)

Journal: Journal of Translational Medicine

Article Title: TROP2 confers resistance to oxidative stress-induced cancer cell death through YAP/HMOX1 signaling

doi: 10.1186/s12967-026-07955-z

Figure Lengend Snippet: TROP2 suppression sensitized NSCLC to RSL3-induced ferroptosis in vivo. ( A ) Schematic diagram of the in vivo experimental timeline. Nude mice were subcutaneously injected with control (shCON) or TROP2-knockdown (shTROP2) PC9 cells, followed by intraperitoneal injection of RSL3 (5 mg/kg) or vehicle every other day. ( B ) Representative photographs of dissected subcutaneous tumors from each treatment group at the endpoint of the study. ( C ) Final tumor weights from each group at the endpoint. Data are presented as mean ± SD ( n = 6). ( D ) Tumor growth curves showing tumor volume over time for the four experimental groups. Data are presented as mean ± SEM ( n = 6 mice per group). ( E ) Representative IHC images of tumor sections stained for TROP2, YAP1, HMOX1, ACSL4, and SLC7A11 from the indicated groups. Scale bar, 100 μm. Data are presented as mean ± SD ( n = 6, C) or mean ± SEM ( n = 6 mice per group, D ). Statistical significance for differences among groups was determined by one-way ANOVA followed by Tukey’s post hoc test (C, and for the final time point in D). ∗ P < 0.05, ∗∗ P < 0.01

Article Snippet: After that, the membranes were blocked in 5% fat-free milk for 1 h. The membranes were incubated with the primary antibodies including TROP2 (1:1000, cat #47866, CST), GPX4 (1:1000, cat #AF7020, Beyotime), SLC7A11 (1:1000, cat #AF7992, Beyotime), ACSL4 (1:1000, cat #AG1908, Beyotime), HMOX1 (1:1000, cat #AG2181, Beyotime), YAP1 (1:1000, cat #14074, CST), P-YAP1 (1:1000, cat #13619, CST), LATS1 (1:1000, cat #3477, CST), p-LATS1 (1:1000, cat #9157, CST), Histone3 (1:1000, cat #9715, CST),α-Tubulin (1:1000, cat #2144, CST) at 4 °C overnight and the secondary antibodies at room temperature for 2 h. Protein bands were visualized using the ECL (Bio-Rad, Hercules, CA, USA).

Techniques: In Vivo, Injection, Control, Knockdown, Staining

Journal: Journal of Translational Medicine

Article Title: TROP2 confers resistance to oxidative stress-induced cancer cell death through YAP/HMOX1 signaling

doi: 10.1186/s12967-026-07955-z

Figure Lengend Snippet: TROP2 confers ferroptosis resistance by inactivating YAP to suppress HMOX1 transcription. ( A ) YAP1 mRNA levels in H292 and PC9 cells with TROP2 knockdown, as determined by qRT-PCR. ( B ) YAP1 mRNA levels in PC9 and A549 cells with TROP2 overexpression, as determined by qRT-PCR. ( C ) Representative IF images showing enhanced nuclear localization of YAP (green) in PC9 cells upon TROP2 knockdown. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( D ) Representative IF images showing suppressed nuclear localization of YAP (green) in PC9 cells upon TROP2 overexpression. Scale bar, 50 μm. ( E ) Western blot analysis of YAP protein levels in nuclear and cytoplasmic fractions of PC9 and H292 cells with or without TROP2 knockdown. ( F ) Quantitative analysis (bar graph) of the bands was shown adjacent to the representative blots (from E). ( G, H ) Cell viability assessed by CCK-8 assay in PC9 and H292 cells with TROP2 knockdown, with or without concomitant YAP inhibition, following RSL3 treatment. ( I,J ) Measurement of cellular GSH levels in PC9 and H292 cells under the indicated conditions. ( K, L ) Measurement of cellular MDA levels in PC9 and H292 cells under the indicated conditions. Data are presented as mean ± SD ( n = 3). Statistical significance was determined by two-tailed unpaired Student’s t-test ( A , B , F ); two-way ANOVA followed by Sidak’s post hoc test or three-way ANOVA followed by Sidak’s post hoc test ( G - L ). ∗ P < 0.05, ∗∗ P < 0.01

Article Snippet: After that, the membranes were blocked in 5% fat-free milk for 1 h. The membranes were incubated with the primary antibodies including TROP2 (1:1000, cat #47866, CST), GPX4 (1:1000, cat #AF7020, Beyotime), SLC7A11 (1:1000, cat #AF7992, Beyotime), ACSL4 (1:1000, cat #AG1908, Beyotime), HMOX1 (1:1000, cat #AG2181, Beyotime), YAP1 (1:1000, cat #14074, CST), P-YAP1 (1:1000, cat #13619, CST), LATS1 (1:1000, cat #3477, CST), p-LATS1 (1:1000, cat #9157, CST), Histone3 (1:1000, cat #9715, CST),α-Tubulin (1:1000, cat #2144, CST) at 4 °C overnight and the secondary antibodies at room temperature for 2 h. Protein bands were visualized using the ECL (Bio-Rad, Hercules, CA, USA).

Techniques: Knockdown, Quantitative RT-PCR, Over Expression, Staining, Western Blot, CCK-8 Assay, Inhibition, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: Identification of YM fusion breakpoint and detection of endogenous YM fusion transcript and protein in ES-2 ovarian cancer cell line (A) Amplification of YAP1-MAML2 (YM) fragments using primer sets spanning YAP1 exons 3–5 and MAML2 exon 2. RT-PCR was performed using RNA from ES-2 cells, and amplified products were visualized by agarose gel electrophoresis followed by ethidium bromide staining. (B) Sequence of the fusion junction is shown. (C) Schematic representation of the fusion event, which results in the creation of a novel YM chimeric gene and a fusion protein comprising 328 aa from exons 1–5 of YAP1 (1–328 aa of YAP1) and 982 aa from exons 2–5 of MAML2 (172–1153 aa of MAML2). (D) Detection of endogenous YM protein in ES-2 cells using an anti-MAML2 C-terminal TAD antibody. H292 cells, which express a different fusion protein, CRTC1-MAML2, were used as a control. Note that cells also express native MAML2 proteins.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Sequencing, Control

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: The YM fusion exhibits nuclear speckles, interacts with the TEAD transcription factors, and activates YAP1/TEAD-responsive reporter (A) Expression of the GFP-YM construct was confirmed by transfection of COS7 cells with an empty vector or a vector expressing GFP-YM followed by western blotting using anti-MAML2 TAD and anti-GFP antibodies. (B) GFP-tagged YM exhibited a nuclear speckle pattern (top). The same cells were stained with DAPI to label the nuclei (bottom). Scale bar: 10 μm. (C) Immunofluorescence analysis of ES-2 MAML2 knockout (KO) cells, using anti-MAML2 TAD antibodies, followed by DAPI staining revealed that the endogenous YM fusion protein displays a pattern of nuclear dots (top). The specific MAML2 KO was confirmed via western blot (WB) analysis (bottom). (D) YM-TEAD protein interaction in ES-2 cells was revealed via coIP/WB analysis. Whole-cell protein lysates (750 μg) from parental ES-2 cells were subjected to immunoprecipitation (IP) with anti-TEAD or rabbit IgG (negative control) using Protein A/G beads. IP products were analyzed for TEAD and YM by western blotting. A total of 75 μg whole-cell protein lysate served as an input. (E) The YM fusion was able to activate a YAP-responsive promoter. 293FT cells were plated at 1 × 10 5 cells/well in 24-well plates overnight and transfected with 10 ng Renilla luciferase control vector, 200 ng YAP/TEAD-responsive firefly reporter vector (8xGTIIC-Luc), and 200 ng of pCMV2 empty vector, pCMV2-MAML2, pCMV2-YAP2, or pCMV2-YM fusion. After 48 h, luciferase assays were performed, and YAP1 promoter firefly luciferase activity was shown as fold activation relative to the basal level with an empty pCMV2 vector (n = 2, mean ± SD).

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Expressing, Construct, Transfection, Plasmid Preparation, Western Blot, Staining, Immunofluorescence, Knock-Out, Immunoprecipitation, Negative Control, Luciferase, Control, Activity Assay, Activation Assay

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: The YM fusion has transforming activity (A) HEK293T cells were transfected with the expression constructs (pCMV2 vector, pCMV2-MAML2, pCMV2-YAP1, and pCMV2-YM), and protein expression was assessed by western blotting using the specified antibodies. (B) RK3E cells were transfected as described above and subsequently evaluated for colony formation at day 15 post-transfection using crystal violet staining.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Activity Assay, Transfection, Expressing, Construct, Plasmid Preparation, Western Blot, Staining

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: RNA-seq analysis of YM target genes and pathways (A) Differentially expressed genes in ES-2 cells after the depletion of YM/MAML2 are shown in Volcano plot. A few representative YAP1 target genes were indicated. (B and C) The real-time qPCR assays of YM-regulated genes identified from RNA-seq study were conducted using YM/MAML2 knockdown cells (shM2-1 and shM2-3) and the MAML2-KO and their respective control cells (ES-2 shCtl and ES-2 sgCtl) (n = 3, mean ± SD). (D) IPA analysis identified several transcription regulators of the transcriptomic response to YM/MAML2 knockdown. The changes in the activation or inhibition status of transcription regulators were plotted based on their Z scores. A positive Z score indicates activation, while a negative Z score denotes inhibition. (E) GSEA showed that the YAP conserved signature, MYC target, and E2F target gene sets were negatively enriched, whereas the inflammatory response genes were positively enriched in fusion-depleted cells compared to controls.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: RNA Sequencing, Knockdown, Control, Activation Assay, Inhibition

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: The YM-positive ES-2 cells were sensitive to YAP1 inhibitors (A and B) Cells were cultured in 24-well plates (4 × 10 4 cells per well for ES-2 and 7.5 × 10 4 cells per well for Heya-8 and H3118) overnight and then treated with YAP1 inhibitors (CA3 or VPF) at the indicated concentrations for 48 h. Cells were stained using crystal violet (A), and viable cell numbers were determined by Trypan blue assay (B) ( n = 2, mean ± SD). (C) ES-2 cells were seeded at 500 cells per well in 12-well plates and treated with CA3 or VPF at the indicated concentrations ( n = 3, mean ± SD). After 14 days of culture, colonies were fixed and stained with crystal violet. The colony areas were quantified using ImageJ and presented as a percentage of the colony area relative to the corresponding DMSO-treated control groups. ∗<0.05, ∗∗<0.005, ∗∗∗,0.0005.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Cell Culture, Staining, 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. 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. 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: 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, Staining