Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: 20-hydroxyecdysone inhibits the proliferation and induces apoptosis in bladder cancer cells. (A) Several bladder cancer cell lines (J82, 5637, T24, UMUC3 and SW780) and normal human urothelial cell line (SV-HUC-1) were incubated with different concentrations (from 0 to 100 μM) of 20-HE for 24 h. Then, all cells were collected for cell viability calculation using CCK-8 analysis. (B) IC 50 for each cell line was quantified and shown. (C-K) Bladder cancer cell lines T24 and UMUC3 were incubated with 20-HE (2.5, 5 or 10 μM). After treatment for 24 h, T24 and UMUC3 cells were harvested for studies as below. (C) Cell viability quantification by CCK-8 assay. (D&E) Colony formation and (F&G) EdU staining analysis were performed to examine the proliferation of bladder cancer cells (Scale bar = 50 μm). Quantification for each was displayed. (H&I) Apoptosis of bladder cancer cells was measured using flow cytometry analysis. (J) RT-qPCR analysis for Bcl-2 mRNA expression levels. (K) Western blot results for cleaved Caspase-3 protein expression levels. Data are displayed as mean ± SD ( n = 3 or 4 in each group). * p < 0.05, ⁎⁎ p < 0.01 ⁎⁎⁎ p < 0.001 vs the Ctrl group.

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Incubation, CCK-8 Assay, Staining, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot

Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: 20-hydroxyecdysone suppresses the migration and invasion in bladder cancer cells. (A-E) T24 and UMUC3 cells were exposed to 24 h of 20-HE (2.5, 5 or 10 μM), and were then harvested for studies as follows. (A&B) Transwell assay was conducted to examine the migratory and invasive properties of bladder cancer cells (Scale bar = 50 μm). The number of cells in migration and invasion was quantified and listed. (C) Wound closure healing analysis was used to examine the migration of bladder cancer cells (Scale bar = 100 μm). (D) The percentage of wound closure for each condition was quantified. (E) RT-qPCR analysis was conducted to examine the mRNA expression changes of EMT markers, including E-Cadherin, ZO-1, N—Cadherin, ZEB-1, Vimentin and MMP13. Data are displayed as mean ± SD ( n = 3 or 4 in each group). * p < 0.05, ⁎⁎ p < 0.01 ⁎⁎⁎ p < 0.001 vs the Ctrl group.

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Migration, Transwell Assay, Quantitative RT-PCR, Expressing

Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: 20-hydroxyecdysone reduces tumor growth and metastasis in the xenograft mouse model of bladder cancer. (A) Tumor samples from each group were shown. (B) Tumor volume was measured. (C) Tumor weights were recorded. (D) H&E staining (Scale bar = 100 μm), TUNEL staining (Scale bar = 50 μm) and IHC staining for KI-67 (Scale bar = 50 μm) expression calculation in tumor sections. Quantification for (E) TUNEL and (F) KI-67 positive cells was displayed. (G) Lung metastases that developed in the T24 cell lines using an IVIS imaging system. (H) Representative luciferase signals were captured and analyzed. (I) Lung tissues (up) and H&E staining of pulmonary tissues (down; Scale bar = 200 μm) were displayed. Tumor area was marked by the yellow dotted line. (J) The number of nodules on lung tissues was examined. (K) Tumor-to-lung ratios were examined. (L) IHC staining for N-cadherin in the tumor tissues from xenograft mouse model and lung metastasis model was conducted (Scale bar = 50 μm). N-cadherin positive expression levels were quantified. Data are displayed as mean ± SD ( n = 4 in each group). * p < 0.05, ⁎⁎ p < 0.01 ⁎⁎⁎ p < 0.001 vs the Ctrl group (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Staining, TUNEL Assay, Immunohistochemistry, Expressing, Imaging, Luciferase

Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: The enrichment analysis of DEGs mediated by 20-hydroxyecdysone in bladder cancer cells, and USP21 expression changes in bladder cancer patients. (A) Volcano plots for DEGs of 20-HE-treated (10 μM) vs Ctrl group in T24 cells. (B) Heat map showing the expression of DEGs in the 20-HE and Ctrl group in T24 cells. (C) Biological Process (BP) terms, Cellular Component (CC) terms, Molecular Function (MF) terms and KEGG analysis of RNA sequencing data for the bladder cancer cell line T24 (20-HE vs Ctrl). (D) Chord dendrogram and (E) bubble diagram indicating a top GO cluster plot of the expression spectrum of significantly expressed genes. (F&G) Protein-protein interaction network and MCODE components for enriched terms of DEGs in T24 cells (20-HE vs Ctrl) using Metascape online assay system. (H) USP21 expression levels in tumor samples and normal tissues from patients with bladder cancer by the use of TCGA database. (I&J) USP21 expression levels in bladder cancer patients at different pathologic stages or histologic grades based on TCGA dataset. (K&L) Correlation between the gene expression of USP21 and RELA (p65) and NFKBIB in bladder cancer patients by the TCGA database. Data are displayed as mean ± SD ( n = 4 or 5 in each group).

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Expressing, RNA Sequencing, Gene Expression

Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: 20-hydroxyecdysone down-regulates USP21 expression levels in bladder cancer. (A-D) T24 and UMUC3 cells were incubated with 20-HE (2.5, 5 or 10 μM) for 24 h, and were then harvested for following experiments. (A) RT-qPCR and (B) western blot assays were used to examine USP21 gene and protein expression levels. (C&D) IF staining was conducted to assess USP21 expression in T24 and UMUC3 cells (Scale bar = 15 μm). (E) USP21 mRNA and (F) protein expression levels in xenograft tumor samples were measured by RT-qPCR and western blot analysis, respectively. (G) IHC staining was included to examine USP21 positive expression levels in tumor tissues (Scale bar = 50 μm). Data are displayed as mean ± SD ( n = 3 or 4 in each group). ⁎⁎ p < 0.01 ⁎⁎⁎ p < 0.001 vs the Ctrl group.

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry

Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: 20-hydroxyecdysone reduces NF-κB/p65 stability in bladder cancer. (A-D) T24 and UMUC3 cells were treated with 20-HE (2.5, 5 or 10 μM). After 24 h, all cells were harvested for following assays. (A) RT-qPCR for NF-κB/p65 transcriptional expression calculation in bladder cancer cells. (B) Western blot results for p-NF-κB/p65 and NF-κB/p65 in bladder cancer cells. (C) IF staining for p-NF-κB/p65 and (D) NF-κB/p65 expression levels in T24 and UMUC3 cells. Quantification for p-NF-κB/p65 and NF-κB/p65 fluorescence intensity was exhibited (Scale bar = 15 μm). (E) IHC staining for p-NF-κB/p65 and NF-κB/p65 expression levels in xenograft tumor tissues from each group of mice (Scale bar = 50 μm). Data are displayed as mean ± SD ( n = 3 or 4 in each group). ⁎⁎ p < 0.01 ⁎⁎⁎ p < 0.001 vs the Ctrl group.

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Fluorescence, Immunohistochemistry

Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: USP21 directly interacts with p65 and maintains its stability in bladder cancer cells, which can be abrogated by 20-hydroxyecdysone. (A) RT-qPCR analysis of USP21 expression in T24 and UMUC3 cell lines transfected with USP21 shRNAs and shCtrl. (B) Western blotting results for USP21 and NF-κB/p65 in bladder cancer cells with shUSP21 and shCtrl transfection. (C) RT-qPCR analysis of p65 in T24 and UMUC3 cells with USP21 knockdown. (D) Coomassie blue staining on T24 cell line protein samples immunoprecipitated with IgG and anti-USP21 antibody. (E) Identification of USP12 binding partners via a combination of Co-IP and LC-MS/MS analysis. The Venn diagram shows the number of binding partners of USP21 in the two groups. Top ten overlapping proteins with a ratio of >2 were provided. (F) Co-IP assay was performed to explore the interaction between USP21 and the 10 candidates in T24 cells. (G) Immunoblotting to show the interaction between USP21 and p65 in T24 and UMUC3 cells by IP assay. IgG was used as the control. (H) GST pull-down assay was performed to examine the physical interaction between USP21 and p65 in T24 and UMUC3 cells. (I) Double IF staining for USP21 and p65 in T24 and UMUC3 cells. The nuclei were counterstained with DAPI (Scale bar = 15 μm). (J) Western blot analysis of p65 expression in T24 and UMUC3 cells transfected with varying concentrations of Flag-USP21. (K) Western blot analysis of p65 derived from T24 and UMUC3 cells transfected with Flag-USP21 and Flag-empty vector. Cells were incubated with CHX (100 μg/ml) for the shown time. (L) HA-ubiquitin (Ub) was co-expressed with Flag-USP21 in T24 and UMUC3 cells. Western blot analysis was then used to examine the ubiquitination of p65. (M) Western blot analysis for p65 ubiquitination linkage in T24 and UMUC3 cells transfected with Flag-USP21, Myc-p65, and the indicated ubiquitin plasmids. (N) Western blot results for K48-linked p65 ubiquitination in T24 and UMUC3 cells transfected with Flag-USP21, Myc-p65, and the shown ubiquitin plasmids with or without 24 h of 20-HE (10 μM) treatment. Data are displayed as mean ± SD ( n = 3 in each group). ⁎⁎ p < 0.01 ⁎⁎⁎ p < 0.001 vs the shCtrl group.

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Knockdown, Staining, Immunoprecipitation, Binding Assay, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Control, Pull Down Assay, Derivative Assay, Plasmid Preparation, Incubation, Ubiquitin Proteomics

Journal: Translational Oncology

Article Title: 20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

doi: 10.1016/j.tranon.2024.101958

Figure Lengend Snippet: 20-hydroxyecdysone-inhibited cell proliferation and EMT process are diminished by USP21. (A-I) T24 and UMUC3 cells were transfected with USP21 overexpression plasmids, and were then treated with or without 20-HE (10 μM) for an additional 24 h. Next, all cells were harvested for studies as below. (A) Cell viability was measured using CCK-8 analysis. (B&C) EdU staining was conducted to examine cell proliferation (Scale bar = 50 μm). (D) Caspase-3 activity was assessed. (E) Cleaved Caspase-3 expression levels were measured by western blot analysis. (F) Transwell analysis was performed to examine the migration and invasion of cells (Scale bar = 50 μm). (G) The number of migrated and (H) invasive cells was quantified. (I) Western blot analysis for p-NF-κB/p65 and NF-κB/p65 protein expression levels in bladder cancer cell lines. (J) Working model demonstrating the effects of 20-hydroxyecdysone on bladder cancer progression. In brief, USP21 expression is up-regulated in bladder cancer cells, and can deubiquitinate K48-linked polyubiquitination of p65, thereby maintaining p65 stability. These events contribute to the proliferation and metastasis of bladder cancer. However, 20-HE can strongly restrain USP21 expression levels, and herein destabilize p65; meanwhile, 20-HE can directly inhibit p65 via the transcriptional level, which restrains bladder cancer progression ultimately. Data are displayed as mean ± SD ( n = 3 or 4 in each group). * p < 0.05, ⁎⁎ p < 0.01 ⁎⁎⁎ p < 0.001.

Article Snippet: 20-hydroxyecdysone (also known as Crustecdysone; purity 99.63 %; #HY-N6979) and Chlorhexidine (CHX; #HY-B1248) were purchased from MedChemExpress (Shanghai, China).

Techniques: Transfection, Over Expression, CCK-8 Assay, Staining, Activity Assay, Expressing, Western Blot, Migration