Journal: PLOS One

Article Title: The Hippo-YAP/β-catenin signaling axis coordinates odontogenic differentiation in dental pulp stem cells: Implications for dentin-pulp regeneration

doi: 10.1371/journal.pone.0326978

Figure Lengend Snippet: (A) Immunofluorescence shows progressive increases in YAP and β-catenin protein levels in DPSCs over 14 days of odontogenic induction. Nuclei were stained with DAPI (blue). Scale bar: 25 µm. (B) Spatial immunohistochemical mapping in hydrogen peroxide-bleached murine incisors (mimicking inflammatory pulp conditions) demonstrates enriched YAP/β-catenin co-expression in odontoblast layers adjacent to predentin. Scale bar: 100 µm in100 × ; 25 µm in 400 × . (C) qRT-PCR quantification confirms progressive upregulation of YAP, β-catenin, and Wnt targets (Cyclin D1, c-Myc) during differentiation. Data: mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Primary antibodies against YAP (1:50; 13584-1-AP, Proteintech, USA)) and β-catenin (1:50; 51067-2-AP, Proteintech, USA) were incubated overnight at 4°C, followed by fluorescent secondary antibody (1:200; SA00013-4, Proteintech, USA).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, Quantitative RT-PCR

Journal: PLOS One

Article Title: The Hippo-YAP/β-catenin signaling axis coordinates odontogenic differentiation in dental pulp stem cells: Implications for dentin-pulp regeneration

doi: 10.1371/journal.pone.0326978

Figure Lengend Snippet: (A) Lentiviral-mediated YAP overexpression (OE-YAP) and knockdown (sh-YAP) efficiency validated by immunofluorescence. (B, D) qRT-PCR shows OE-YAP upregulates Wnt pathway components (β-catenin, c-Myc, and Cyclin D1), while sh-YAP suppresses them. (C) Immunofluorescence reveals OE-YAP increases cytoplasmic β-catenin (red), whereas sh-YAP diminishes it. Data: mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bar: 25 µm.

Article Snippet: Primary antibodies against YAP (1:50; 13584-1-AP, Proteintech, USA)) and β-catenin (1:50; 51067-2-AP, Proteintech, USA) were incubated overnight at 4°C, followed by fluorescent secondary antibody (1:200; SA00013-4, Proteintech, USA).

Techniques: Over Expression, Knockdown, Immunofluorescence, Quantitative RT-PCR

Journal: PLOS One

Article Title: The Hippo-YAP/β-catenin signaling axis coordinates odontogenic differentiation in dental pulp stem cells: Implications for dentin-pulp regeneration

doi: 10.1371/journal.pone.0326978

Figure Lengend Snippet: (A) Immunofluorescence demonstrates that β-catenin knockdown not only abolishes OE-YAP-induced β-catenin upregulation but also reduces YAP expression, indicating β-catenin-dependent stabilization of YAP. Scale bar: 25 µm. (C) qRT-PCR validates β-catenin siRNA knockdown efficacy. (B, E) ALP activity is decreased by 25.48% in β-catenin-silenced OE-YAP cells compared to controls. Scale bar: 100 µm. (D, F) Mineralization capacity is reduced by 41.52% following β-catenin ablation. Scale bar: 100 µm. (G) β - catenin siRNA downregulates YAP-induced markers and reciprocally suppresses YAP mRNA expression. Data: mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Primary antibodies against YAP (1:50; 13584-1-AP, Proteintech, USA)) and β-catenin (1:50; 51067-2-AP, Proteintech, USA) were incubated overnight at 4°C, followed by fluorescent secondary antibody (1:200; SA00013-4, Proteintech, USA).

Techniques: Immunofluorescence, Knockdown, Expressing, Quantitative RT-PCR, Activity Assay

Journal: PLOS One

Article Title: The Hippo-YAP/β-catenin signaling axis coordinates odontogenic differentiation in dental pulp stem cells: Implications for dentin-pulp regeneration

doi: 10.1371/journal.pone.0326978

Figure Lengend Snippet: (A, C) The scratch wound closure assay: β-catenin siRNA reduces OE-YAP migration by 42.87% at 48 h (representative images in A, quantification in C). (B, D) The Transwell assay: β-catenin knockdown decreases OE-YAP invasion by 56.12% (representative images in B, quantification in D). (E) CCK-8 analysis shows that β-catenin siRNA reduces OE-YAP proliferative capacity by 54.97%. Data: mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. control. Scale bar: 100 µm.

Article Snippet: Primary antibodies against YAP (1:50; 13584-1-AP, Proteintech, USA)) and β-catenin (1:50; 51067-2-AP, Proteintech, USA) were incubated overnight at 4°C, followed by fluorescent secondary antibody (1:200; SA00013-4, Proteintech, USA).

Techniques: Scratch-induced Wound-closure Assay, Migration, Transwell Assay, Knockdown, CCK-8 Assay, Control

Journal: PLOS One

Article Title: The Hippo-YAP/β-catenin signaling axis coordinates odontogenic differentiation in dental pulp stem cells: Implications for dentin-pulp regeneration

doi: 10.1371/journal.pone.0326978

Figure Lengend Snippet: (A) Immunofluorescence demonstrates diminished expression of both YAP and β-catenin in WIF-1-treated OE-YAP cells. Scale bar: 25 µm (B) qRT-PCR confirms WIF-1-mediated downregulation of β-catenin and its downstream targets c-Myc and Cyclin D1 in OE-YAP cells. (CD) WIF-1 treatment abolishes YAP-mediated enhancement of ALP activity and mineralization capacity. Scale bar: 100 µm. Data: mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Primary antibodies against YAP (1:50; 13584-1-AP, Proteintech, USA)) and β-catenin (1:50; 51067-2-AP, Proteintech, USA) were incubated overnight at 4°C, followed by fluorescent secondary antibody (1:200; SA00013-4, Proteintech, USA).

Techniques: Immunofluorescence, Expressing, Quantitative RT-PCR, Activity Assay

Journal: PLOS One

Article Title: The Hippo-YAP/β-catenin signaling axis coordinates odontogenic differentiation in dental pulp stem cells: Implications for dentin-pulp regeneration

doi: 10.1371/journal.pone.0326978

Figure Lengend Snippet: (A) Immunohistochemistry of implanted DPSCs grafts shows OE-YAP upregulates dentinogenic markers (DSPP, DMP1, RUNX2, OCN, and ALP) and β-catenin compared to controls. Scale bar: 100 µm (100×); 25 µm (400×). (B) Quantification confirms 1.36- to 1.62-fold induction of odontogenic markers and 1.27-fold β-catenin increase in OE-YAP grafts. (C) Schematic illustration of the tooth slice/scaffold design for ectopic transplantation. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Primary antibodies against YAP (1:50; 13584-1-AP, Proteintech, USA)) and β-catenin (1:50; 51067-2-AP, Proteintech, USA) were incubated overnight at 4°C, followed by fluorescent secondary antibody (1:200; SA00013-4, Proteintech, USA).

Techniques: Immunohistochemistry, Transplantation Assay

Journal: Cancer Cell International

Article Title: USP24 promotes hepatocellular carcinoma progression by deubiquitinating and stabilizing YAP1

doi: 10.1186/s12935-025-03796-w

Figure Lengend Snippet: Identification of USP24 as a regulator for YAP1 in HCC. A Analysis and generation of heat maps of CNV values of 56 USPs in the STAD dataset using cBioPortal. B After silencing USPs (CNV≥2%) in Bel-7402 cells, YAP1/TEAD4-driven transcriptional activity was accessed, and it is represented by the ratio of 8×GTIIC-luciferase activity with 8 TBSs to GTIIC luciferase activity with 8 mutant TBSs. C Bel-7402 and Huh-7 cells were treated with increasing concentrations of WP1130 for 12 h, and the indicated proteins were measured by western blotting. D USP24 was depleted by shRNAs (shUSP24-1 and shUSP24-2) in HCC cells and the indicated proteins were examined by western blotting. Control shRNA (shNC) was used as negative control. E qRT-PCR analysis of the indicated genes in Bel-7402 and Huh-7 cells with USP24 knockdown. F Increasing amounts of Flag-USP24 plasmids were co-transfected with Myc-YAP1 plasmid into HEK293T cells and the indicated proteins were examined by western blotting. G HEK293T cells were transfected with Flag-USP24 or vector plasmids. A CHX chase experiment was performed and Myc-tagged YAP1 protein was determined by western blotting (upper panel). The lower panel shows the relative protein amounts of different groups. H Bel-7402 cells stably expressing control shRNA or USP24 shRNAs were treated with or without CHX (20 mg/mL) and harvested at the indicated times, and indicated proteins was determined by western blotting (upper panel). The lower panel shows the relative protein amounts of different groups. Data are presented as mean ± SD, and P values were calculated using Student t test. * P < 0.05 and ** P < 0.01

Article Snippet: The membrane was then blotted with specific primary antibodies against YAP1 (cat. no. 14074; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), USP24 (cat. no. ab129064; 1:2000 dilution; Abcam, PLC., Cambridge, UK), Flag (cat. no. AE005; 1:1000 dilution; ABclonal Biotech Co., Ltd., Wuhan, China), Myc (cat. no. M047-3; 1:1000 dilution; MBL, Nagoya, Japan), HA (cat. no. 3724; 1:1000 dilution; Cell Signaling Technology) and β-Actin (cat. no.66009-1-lg; 1:10,000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA).

Techniques: Activity Assay, Luciferase, Mutagenesis, Western Blot, Control, shRNA, Negative Control, Quantitative RT-PCR, Knockdown, Transfection, Plasmid Preparation, Stable Transfection, Expressing

Journal: Cancer Cell International

Article Title: USP24 promotes hepatocellular carcinoma progression by deubiquitinating and stabilizing YAP1

doi: 10.1186/s12935-025-03796-w

Figure Lengend Snippet: USP24 interacts with YAP1. A HEK293T cells were transfected with plasmids encoding Flag-USP24 and/or Myc-YAP1. After 48 h of transfection, cell extracts were prepared and immunoprecipitated with anti-Flag or anti-Myc antibodies. The protein interactions were analyzed by western blotting. B HEK293T cells were transfected with plasmids encoding Flag-USP24 or Myc-YAP1. After 48 h of transfection, cell extracts were prepared and immunoprecipitated with anti-Flag or anti-Myc antibodies, and the indicated proteins were examined by western blotting. C Whole-cell lysates from Bel-7402 or Huh-7 cells were subjected to immunoprecipitation assays with a control IgG or an anti-YAP1 antibody. The immunoprecipitates were detected by western blotting. D Confocal microscopic analysis of USP24 and YAP1 subcellular localization. Bel-7402 cells were fixed and immunostained with antibodies against the indicated proteins. Representative images from biological triplicate experiments are shown. Scale bar, 20 µM

Article Snippet: The membrane was then blotted with specific primary antibodies against YAP1 (cat. no. 14074; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), USP24 (cat. no. ab129064; 1:2000 dilution; Abcam, PLC., Cambridge, UK), Flag (cat. no. AE005; 1:1000 dilution; ABclonal Biotech Co., Ltd., Wuhan, China), Myc (cat. no. M047-3; 1:1000 dilution; MBL, Nagoya, Japan), HA (cat. no. 3724; 1:1000 dilution; Cell Signaling Technology) and β-Actin (cat. no.66009-1-lg; 1:10,000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control

Journal: Cancer Cell International

Article Title: USP24 promotes hepatocellular carcinoma progression by deubiquitinating and stabilizing YAP1

doi: 10.1186/s12935-025-03796-w

Figure Lengend Snippet: USP24 deubiquitinates YAP1. A HEK293T cells were co-transfected with the specified plasmids. After 48 h of transfection, cell extracts were prepared for immunoprecipitation assays with anti-Myc followed by western blotting with anti-HA antibody. B HEK293T cells were co-transfected with the indicated plasmids, treated with or without WP1130 (10 µM) for 8 h before cells were harvested. Further, cellular extracts were prepared for immunoprecipitation assays with anti-Myc followed by western blotting with anti-HA antibody. C HEK293T cells stably expressed shRNAs specifically against USP24 and co-transfected with the indicated plasmids. Cellular extracts were immunoprecipitated with anti-Myc followed by western blotting with anti-HA antibody

Article Snippet: The membrane was then blotted with specific primary antibodies against YAP1 (cat. no. 14074; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), USP24 (cat. no. ab129064; 1:2000 dilution; Abcam, PLC., Cambridge, UK), Flag (cat. no. AE005; 1:1000 dilution; ABclonal Biotech Co., Ltd., Wuhan, China), Myc (cat. no. M047-3; 1:1000 dilution; MBL, Nagoya, Japan), HA (cat. no. 3724; 1:1000 dilution; Cell Signaling Technology) and β-Actin (cat. no.66009-1-lg; 1:10,000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Stable Transfection

Journal: Cancer Cell International

Article Title: USP24 promotes hepatocellular carcinoma progression by deubiquitinating and stabilizing YAP1

doi: 10.1186/s12935-025-03796-w

Figure Lengend Snippet: USP24 is upregulated in tumor samples and correlates with the protein levels of YAP1. A Scatter plots showing the correlations between the USP24 expression level and YAP1 expression level in HCC patients by Spearman correlation analyses using GEPIA platform from TCGA database. B The USP24 and YAP1 protein level in various HCC cell lines were determined by western blotting. C Overall staining of USP24 in the tissue microarray. The microarray contained 90 pairs of HCC tissues and 90 matched normal tissues, and the detached spots (red circles) were excluded, which including 1 HCC tissue and 2 normal tissues, leaving 177 samples retained for analysis. D The USP24 staining score of HCC tissues ( n = 89) and matched normal tissues ( n = 88). E Representative IHC images of USP24 expression in HCC tissues and matched normal tissues. Scale bar, 50 µM. * P < 0.05

Article Snippet: The membrane was then blotted with specific primary antibodies against YAP1 (cat. no. 14074; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), USP24 (cat. no. ab129064; 1:2000 dilution; Abcam, PLC., Cambridge, UK), Flag (cat. no. AE005; 1:1000 dilution; ABclonal Biotech Co., Ltd., Wuhan, China), Myc (cat. no. M047-3; 1:1000 dilution; MBL, Nagoya, Japan), HA (cat. no. 3724; 1:1000 dilution; Cell Signaling Technology) and β-Actin (cat. no.66009-1-lg; 1:10,000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA).

Techniques: Expressing, Western Blot, Staining, Microarray

Journal: Cancer Cell International

Article Title: USP24 promotes hepatocellular carcinoma progression by deubiquitinating and stabilizing YAP1

doi: 10.1186/s12935-025-03796-w

Figure Lengend Snippet: USP24 regulates HCC cell proliferation in vitro and in vivo. A Bel-7402, Huh-7, or PLC/PRF/5 cells were infected with control shRNA or USP24 shRNAs, and cell proliferation was monitored by trypan blue exclusion assay. B Cell morphology (96 h) was captured under the microscope. Original magnification, ×200. C Bel-7402 or Huh-7 cells were transfected with the indicated plasmids and seeded into 6-well plates at a density of 2000 cells/well. After 14 days, colony formation was detected by crystal violet staining. D PLC/PRF/5 cells were transfected with the indicated plasmids and seeded into 6-well plates at a density of 4000 cells/well. After 14 days, colony formation was detected by crystal violet staining. E HCC cells were infected with either shCtrl + vector, shUSP24-2 + vector, or shUSP24-2 + YAP1, and cell proliferation was monitored by CCK-8 assay. F , G Huh-7 ( F ) and PLC/PRF/5 ( G ) cells were infected with either shCtrl + vector, shUSP24-2 + vector, or shUSP24-2 + YAP1, and cell proliferation was monitored by colony formation assay. H , I Bel-7402 cells stably transfected with either shNC or shUSP24-2 were subcutaneously injected into BALB/c nude mice ( n = 6 for each group) to establish an HCC xenograft mouse model. After 20 days, tumors from 12 of the mice were extracted and photographed. Tumor images ( H , upper panel), tumor volume ( H , lower panel) and weight ( I ) were assessed. J Expression patterns of Ki-67, TUNEL and YAP1 were examined by IHC analysis in the xenograft tumors of each group, Scale bar, 20 µM. Data are presented as mean ± SD, and P values were calculated using Student t test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The membrane was then blotted with specific primary antibodies against YAP1 (cat. no. 14074; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), USP24 (cat. no. ab129064; 1:2000 dilution; Abcam, PLC., Cambridge, UK), Flag (cat. no. AE005; 1:1000 dilution; ABclonal Biotech Co., Ltd., Wuhan, China), Myc (cat. no. M047-3; 1:1000 dilution; MBL, Nagoya, Japan), HA (cat. no. 3724; 1:1000 dilution; Cell Signaling Technology) and β-Actin (cat. no.66009-1-lg; 1:10,000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA).

Techniques: In Vitro, In Vivo, Infection, Control, shRNA, Trypan Blue Exclusion Assay, Microscopy, Transfection, Staining, Plasmid Preparation, CCK-8 Assay, Colony Assay, Stable Transfection, Injection, Expressing, TUNEL Assay