sw1990  (ATCC)

Journal: Neoplasia (New York, N.Y.)

Article Title: OTUD4 regulates pancreatic cancer progression via Hippo/YAP axis

doi: 10.1016/j.neo.2026.101285

Figure Lengend Snippet: OTUD4 is crucial for the progression of PAAD. A . Transfection of siControl or siOTUD4 #1/#2 was performed in PAAD cells. The knockdown efficiency of OTUD4 at the protein level was confirmed by Western blot analysis. B-C. The relative mRNA expression level of OTUD4 was measured by RT-qPCR following transfection with siControl or siOTUD4 #1/#2. D-E. Proliferation rate of PAAD cells transfected with siControl or siOTUD4 #1/#2 was measured using the CCK-8 assay. F-I. The migration and invasion abilities of PAAD cells following transfection with siControl or siOTUD4 #1/#2 was assessed by Transwell assay. J-M. Proliferation rate of PAAD cells transfected with siControl or siOTUD4 #1/#2 was assessed using the EdU assay. Hoechst 33342 was used for nuclear staining, and the scale bar is 200 μm. N-Q. Apoptosis of PAAD cells transfected with siControl or siOTUD4 was measured by flow cytometry following staining with FITC and PI. R-V. SW1990 cells transfected with shControl and shOTUD4 were injected subcutaneously into nude mice. The nude mice were sacrificed after 35 days of injection, and the tumor weight ( S ) and volume ( T ) were measured. IHC assays were utilized for evaluating the Ki-67 positive rate ( U-V ). n = 6. The scale bar is 400 μm for 10X and 100 μm for 40X. All the experiments were performed in triplicate. All data are presented as mean ± SDs. Statistical methods: Student’s t -test for B-C, G, I, K, M, O, Q, S and V; two-way ANOVA for D and E. ** P < 0.01; *** P < 0.001.

Article Snippet: The AsPC-1, SW1990, HEK293T cell lines were all originated from American Type Culture Collection (ATCC).

Techniques: Transfection, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, CCK-8 Assay, Migration, Transwell Assay, EdU Assay, Staining, Flow Cytometry, Injection

Journal: Neoplasia (New York, N.Y.)

Article Title: OTUD4 regulates pancreatic cancer progression via Hippo/YAP axis

doi: 10.1016/j.neo.2026.101285

Figure Lengend Snippet: The hydrolytic activity of OTUD4 is essential in regulating YAP function. A . A schematic diagram depicts the C45A mutation of OTUD4. B-C. The protein level of YAP after transfecting either siControl or siOTUD4 #1/#2 in AsPC-1 cells ( B ) and SW1990 cells ( C ) was measured through western-blot. D. RT-qPCR assays were used for evaluating the mRNA expression levels of CTGF and CYR61 in PAAD cells after transfection with siControl or siOTUD4. E-F. Dual-luciferase reporter gene assays were conducted in siControl-transfected or siOTUD4-transfected AsPC-1 (E) and SW1990 cells (F) to evaluate TEAD4 transcriptional activity. G-H. Immunoblotting assays were performed to observe the protein level of YAP after transfecting Flag or Flag-OTUD4 WT or Flag-OTUD4 C45A in AsPC-1 cells ( G ) and SW1990 cells (H) . I-J. Quantification of CTGF and CYR61 expression levels by RT-qPCR was performed in PAAD cells transfected with Flag or Flag-OTUD4 WT or Flag-OTUD4 C45A . K-L. TEAD4 transcriptional activity were measured using a dual-luciferase reporter assay after cells transfection with Flag, Flag-OTUD4 WT , or Flag-OTUD4 C45A plasmids. All the experiments were performed in triplicate. All the data are presented as the means ± SDs. Statistical methods: Student’s t -test for D, E-F, I and J-K. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The AsPC-1, SW1990, HEK293T cell lines were all originated from American Type Culture Collection (ATCC).

Techniques: Activity Assay, Mutagenesis, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Reporter Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: OTUD4 regulates pancreatic cancer progression via Hippo/YAP axis

doi: 10.1016/j.neo.2026.101285

Figure Lengend Snippet: YAP overexpression rescues OTUD4 knockdown-induced suppression of PAAD. A. SW1990 cells were transfected with siControl, siOTUD4 , or siOTUD4+Myc-YAP, respectively. The protein levels of OTUD4 and YAP were measured by immunoblotting. B. To determine the effect of OTUD4 and YAP, the mRNA levels of CTGF and CYR61 in SW1990 cells were measured using RT-qPCR after the cells were transfected with siRNAs and Myc-YAP plasmids. C. CCK-8 assay was conducted to evaluate cell proliferation of SW1990 cells after transfection with siControl, siOTUD4+Myc, or siOTUD4+Myc-YAP. D-E. To assess migration and invasion, transwell assays were performed using SW1990 cells transfected with the specified siRNA and plasmid. F-G. The EdU assay was used to further evaluate the proliferative capacity of SW1990 cells following transfection with indicated siRNA and plasmid. The nuclei were stained with Hoechst 33342.The scale bar is 200 μm. H-I. Flow cytometry was performed to analyze apoptosis in SW1990 cells transfected with specific siRNAs and plasmids. J-N. SW1990 cells transfected with shControl, shOTUD4, or shOTUD4 + Myc-YAP were subcutaneously injected into nude mice. After 35 days, the mice were euthanized ( J ), and tumor weight ( K ) and volume ( L ) were measured. In addition, IHC staining was performed to assess the expression of Ki-67 ( M ), and the percentage of Ki-67-positive cells was quantified ( N ). n = 6. All the experiments were performed in triplicate. All the data are presented as the means ± SDs. Statistical methods: Student’s t -test for B, E, J, I, K and N . Two-way ANOVA for C and L. ** P < 0.01 *** P < 0.001.

Article Snippet: The AsPC-1, SW1990, HEK293T cell lines were all originated from American Type Culture Collection (ATCC).

Techniques: Over Expression, Knockdown, Transfection, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Migration, Plasmid Preparation, EdU Assay, Staining, Flow Cytometry, Injection, Immunohistochemistry, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: OTUD4 regulates pancreatic cancer progression via Hippo/YAP axis

doi: 10.1016/j.neo.2026.101285

Figure Lengend Snippet: OTUD4 interacts with YAP to promote its stability. A. Immunofluorescence (IF) analysis was performed to determine the subcellular localization of OTUD4 and YAP. OTUD4 is shown in green, and YAP is displayed in red. Nuclei were stained with DAPI. The scale bar represents 50 μm. B. The endogenous interaction between OTUD4 and YAP was investigated by co-IP. Immunoprecipitation was performed using anti-OTUD4 or anti-YAP on lysates of SW1990 cells, and the associated proteins were detected by Western blotting. C. A schematic diagram of the molecular docking structure, which reveals the interaction between the OTU domain of OTUD4 and the WW domain of YAP, was visualized using PyMOL. D-F. Schematic representations of the individual domains of OTUD4 and YAP are provided ( D ). Plasmids encoding full-length OTUD4, YAP, and their respective truncated domains (OTUD4 residues: 1-155, Δ34-155 and 156-1114; YAP residues: 1-171, 1-292, 171-504 and 292-504) were transfected into HEK-293T cells. Co-IP assays show the WW domain of YAP ( E ) and the OTU domain of OTUD4 ( F ) are responsible for their interaction. G, J. MG132 treatment abolished the decrease in YAP expression induced by siOTUD4 in both AsPC-1 ( G ) and SW1990 ( J ) cells. After transfection with siControl or siOTUD4, the cells were treated with 10 μM MG132 for 12 hours. H-I, K-N. The effect of OTUD4 knockdown or overexpression on YAP stability was assessed using cycloheximide (CHX) assays. OTUD4 knockdown reduced YAP stability ( H-I, K-L ), whereas OTUD4 overexpression enhanced it. In contrast, the C45A mutant of OTUD4 failed to promote YAP stabilization ( M-N ). A concentration of 100 μM CHX was used in all experiments. All the experiments were performed in triplicate. All the data are presented as the means ± SDs. Statistical methods: two-way ANOVA for I, L and N . * P < 0.05; *** P < 0.001.

Article Snippet: The AsPC-1, SW1990, HEK293T cell lines were all originated from American Type Culture Collection (ATCC).

Techniques: Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Transfection, Expressing, Knockdown, Over Expression, Mutagenesis, Concentration Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: OTUD4 regulates pancreatic cancer progression via Hippo/YAP axis

doi: 10.1016/j.neo.2026.101285

Figure Lengend Snippet: OTUD4 stabilizes YAP via decreasing its K48-linked polyubiquitination. A-B. Knockdown of OTUD4 specifically enhanced K48-linked polyubiquitination of YAP without altering K63-linked polyubiquitination. To assess ubiquitination patterns, SW1990 cells were transfected with plasmids encoding HA-tagged wild-type ubiquitin or K48 or K63 ubiquitin ( A ) or K48R mutant ubiquitin or K63R mutant ubiquitin ( B ). Following transfection, cells were treated with 20 μM MG132 for 8 hours before harvesting. YAP was immunoprecipitated using an anti-YAP antibody, and its ubiquitination levels were detected by immunoblotting assay with an anti-HA antibody. C-D. Overexpression of OTUD4 reduced K48-linked polyubiquitination of YAP without affecting K63-linked polyubiquitination. SW1990 cells were co-transfected with plasmids encoding Flag-OTUD4 WT and Myc-YAP, along with HA-tagged wild-type ubiquitin or K48 or K63 ubiquitin ( C ) or K48R mutant ubiquitin or K63R mutant ubiquitin ( D ). After transfection, the cells were treated with 20 μM MG132 for 8 hours, harvested, and subjected to immunoprecipitation using an anti-YAP antibody. Ubiquitination levels of YAP were then examined by Western blot with an anti-HA antibody. E-F. Overexpression of OTUD4 decreased K48-linked polyubiquitination of YAP without altering K63-linked polyubiquitination; however, overexpression of the C45A mutant of OTUD4 did not affect either K48-linked or K63-linked polyubiquitination. SW1990 cells were co-transfected with plasmids encoding either Flag-OTUD4 WT or Flag-OTUD4 C45A and Myc-YAP, together with HA-tagged ubiquitin or K48 ubiquitin or K63 ubiquitin ( E ) or K48R mutant ubiquitin or K63R mutant ubiquitin ( F ). After transfection, cells were treated with 20 μM MG132 for 8 hours, harvested, and subjected to immunoprecipitation using an anti-YAP antibody. YAP ubiquitination levels were then analyzed by Western blot with an anti-HA antibody.

Article Snippet: The AsPC-1, SW1990, HEK293T cell lines were all originated from American Type Culture Collection (ATCC).

Techniques: Knockdown, Ubiquitin Proteomics, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Over Expression