Journal: PloS one

Article Title: Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.

doi: 10.1371/journal.pone.0061934

Figure Lengend Snippet: Figure 1. Expression profiling of protein phosphatases and cell cycle proteins in different PCa cells. LNCaP C-33, C-81 and PC-3 PCa cells were plated at a density of 86103, 66103 and 4.86103 cells/ cm2, respectively, in duplicates for 3 days in regular medium. (Left FBS panel, lanes #1–3) Cells were replaced with fresh regular medium for 24 hr and then harvested. (Right SR panel, lanes #4–6) All three PCa cells were then steroid-starved for 48 hr in a steroid-reduced (SR) medium and then harvested. Total cell lysate proteins were analyzed for cPAcP, SHP1, SHP2, Cdc25A, Cdc25B, Cdc25C, Cyclin B1 and Cyclin D1. Cdc25C spliced forms were observed upon long exposure of films (lower panel of Cdc25C). b-actin was analyzed and used as a loading control. doi:10.1371/journal.pone.0061934.g001

Article Snippet: The Cdc25C cDNA plasmid was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Control

Journal: PloS one

Article Title: Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.

doi: 10.1371/journal.pone.0061934

Figure Lengend Snippet: Figure 2. Effects of androgens on Cdc25C and cell cycle protein levels in PCa cells. (A) LNCaP C-33 and C-81 cells were seeded at a density of 86103 and 66103 cells/cm2, respectively, in duplicates for 3 days in regular medium. Cells were steroid-starved for 48 hr in SR medium and then treated with 10 nM DHT for 24 hr and 48 hr. Total cell lysate proteins were harvested and analyzed for Cdc25C, Cdc25B, cPAcP, Cyclin B1 and Cyclin D1 proteins. b-actin was detected for serving as a loading control. (B) AS LNCaP C-33 and (C) VCaP cells were plated at a density of 86103 and 26104

Article Snippet: The Cdc25C cDNA plasmid was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Control

Journal: PloS one

Article Title: Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.

doi: 10.1371/journal.pone.0061934

Figure Lengend Snippet: Figure 4. Effect of Cdc25C protein expression on the proliferation of PCa cells. (A,B) LNCaP C-33 and (C) PC-3 cells were plated at a density of 1.26104 or 16104 cells/cm2 for 72 hr and then transfected with Cdc25C cDNA (A) or shRNA (B,C) plasmids. Control cells were transfected with the respective vector alone. (A) Cdc25C cDNA transfected C-33 cells were cultured in regular medium and the cell number was then counted after 2 days. (B) Cdc25C shRNA-transfected C-33 cells were steroid starved for 48 hr in SR medium, and then treated with or without 10 nM DHT for 2 days. (C) Cdc25C shRNA transfected PC-3 cells were cultured in regular medium for 2 days, and the cell number was counted. The ratio of cell proliferation was calculated by normalizing the experimental cell number to that of control cells transfected with vector alone, respectively. Total cell lysate proteins were analyzed for Cdc25A, Cdc25B, Cdc25C, Cyclin B1 and/or Cyclin D1 proteins. b-actin was used as a loading control. The ratios of Cdc25C, Cyclin B1 and CyclinD1 protein levels to b-actin were calculated after semi-quantification by densitometric analyses on films with different exposure time periods. *p,0.05, n = 263; Bar, standard deviation. doi:10.1371/journal.pone.0061934.g004

Article Snippet: The Cdc25C cDNA plasmid was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Transfection, shRNA, Control, Plasmid Preparation, Cell Culture, Standard Deviation

Journal: PloS one

Article Title: Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.

doi: 10.1371/journal.pone.0061934

Figure Lengend Snippet: Figure 5. Effects of inhibition of protein biosynthesis and degradation pathways on Cdc25C protein levels. (A) Effects of de novo biosynthesis inhibitors on Cdc25C protein levels by androgens. LNCaP C-33 cells were plated at a density of 1.66104 cells/cm2 for 3 days in regular medium. Cells were steroid starved for 48 hr in a SR medium, and then treated with 5 mg/mL Actinomycin D (Act D) or 10 mg/mL cycloheximide (CHX) for 24 hr alone or 10 nM DHT was added 30 min post treatment with Act D and CHX. Total cell lysate proteins were analyzed for Cdc25C, AR and PSA protein level. b-actin was analyzed as a loading control. (B & C) Effects of inhibitors of proteasomal degradation pathway on Cdc25C protein level. LNCaP C-33 cells were seeded in regular medium for 72 hr, steroid starved for 48 hr and the cells were treated with or without 10 nM DHT or different concentrations of proteasomal inhibitors (B) MG132 (0.01, 0.05 and 0.1 mM) and (C) Epoximicin (0.1 and 1.0 M). Cells were harvested and analyzed for Cdc25C protein levels. b-actin was analyzed serving as a loading control. (D) Effect of androgens on the ubiquitination of Cdc25C protein. Steroid-starved LNCaP C-33 cells were treated with 10 nM DHT or solvent alone for 48 hr. An aliquot of total cellular lysate proteins was immunoprecipitated (IP) by reacting with anti-Cdc25C Ab or normal IgG and followed by Protein A-Sepharose beads. The immune complexes were analyzed by immunoblotting (IB) with anti-Cdc25C Ab (left panel) or anti-ubiquitin Ab (right panel). The positions of ubiquitinated Cdc25C protein (Ubi-Cdc25C) are indicated by arrows; while an additional band was detected by anti-ubiquitin Ab (Complex Ib). (E & F) Effects of inhibitors of lysosomal degradation pathway on Cdc25C protein levels. LNCaP C-33 cells were seeded as described in B & C, after steroid starvation, cells were treated with or without 10 nM DHT or different concentrations of lysosomal protease inhibitors. (E) Leupeptin (10 to 200 mM) and (F) E64D (0.01 to 1.0 mM) for 48 hr. Cells were harvested and analyzed for Cdc25C protein levels. b-actin was analyzed as a loading control. doi:10.1371/journal.pone.0061934.g005

Article Snippet: The Cdc25C cDNA plasmid was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Inhibition, Control, Ubiquitin Proteomics, Solvent, Immunoprecipitation, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Translationally controlled tumor protein induces mitotic defects and chromosome missegregation in hepatocellular carcinoma development.

doi: 10.1002/hep.24709

Figure Lengend Snippet: Fig. 5. TCTP decreases Cdk1 activity through ubiquitin-proteasome degradation of Cdc25. (A) Western blotting analysis was used to compare Cdk1-Tyr15 expression between Vec-7703 and TCTP-7703 cells arrested at prometaphase (0 hours) and released for the indicated time points. Expression of protein was quantified by ImageJ software, and the ratio of Cdk1-Tyr15/b-actin was calculated and is shown in the line chart. (B and C) Expressions of Cdc25C at protein (upper panel) or messenger RNA level (lower panel) were detected in Vec-7703 and TCTP-7703 cells by western blotting analysis (B) or by reverse-transcriptase PCR (C). (D) Expression of TCTP and Cdc25C proteins in QGY-7703 cells transfected with the plasmid encoding HA-Ub, with or without TCTP expression construct, and treated with nocodazole for 6 hours in the presence or absence of MG132 (20 lM). HA-Ub and b-actin were used as a loading control. (E) Cell lysates described in (D) were immunoprecipitated with an anti- Cdc25C antibody and were then analyzed for HA-Ub and Cdc25C expression by western blotting analysis. (F) Under the treatment of CHX (50 lg/mL) for the indicated time points, Cdc25C stability during M progression was analyzed in cells released from prometaphase arrest. Expres- sion level of Cdc25C was quantified by ImageJ software. The value is expressed as the average of two independent experiments.

Article Snippet: Cells were transfected with plasmids expressing Cdc25C (OriGene, Rockville, MD), TCTP, and hemagglutinin (HA)tagged ubiquitin (HA-Ub) (Sigma-Aldrich, St. Louis, MO), either alone or in combination.

Techniques: Activity Assay, Ubiquitin Proteomics, Western Blot, Expressing, Software, Reverse Transcription, Transfection, Plasmid Preparation, Construct, Control, Immunoprecipitation

Journal: Hepatology (Baltimore, Md.)

Article Title: Translationally controlled tumor protein induces mitotic defects and chromosome missegregation in hepatocellular carcinoma development.

doi: 10.1002/hep.24709

Figure Lengend Snippet: Fig. 6. Silencing TCTP expression inhibits its tumorigenicity and improper mitosis progression. (A) Representatives of foci formation induced by 8024-control and 8024-shTCTP cells. The number of foci was calculated and is summarized in the bar chart. Bars represent the mean 6 SD of three independent experiments (**P < 0.01; independent Student’s t test). (B) Images of tumors formed in nude mice induced by 8024- shTCTP cells and 8024-control. Average tumor volume at 5 weeks is expressed as mean 6 SD for each group (n ¼ 6) (*P < 0.05; independ- ent Student’s t test). (C) Flow cytometry was used to detect cell-cycle distribution of 8024-control and 8024-shTCTP cells treated with thymi- dine-nocodazole block (0 hours), then released for 2 or 6 hours. (D) The hypertetraploid population (indicated by circle) was examined in cells treated with or without nocodazole for 2 days. (E) Cdk1-Tyr15 and Cdc25C were detected by western blotting analysis in cells released from prometaphase arrest. (F) Expression levels of Cdc25C and b-actin were quantified, and the ratio of Cdc25C/b-actin was calculated for the indi- cated time points.

Article Snippet: Cells were transfected with plasmids expressing Cdc25C (OriGene, Rockville, MD), TCTP, and hemagglutinin (HA)tagged ubiquitin (HA-Ub) (Sigma-Aldrich, St. Louis, MO), either alone or in combination.

Techniques: Expressing, Control, Flow Cytometry, Blocking Assay, Western Blot

Journal: Scientific Reports

Article Title: CDC25C downregulation suppresses HCC growth via mitochondrial stress-induced autophagy and apoptosis

doi: 10.1038/s41598-026-36351-2

Figure Lengend Snippet: The suppressed levels of CDC25C in cell line and xenograft model. ( A ) The CDC25C gene was detected via agarose gel electrophoresis, with β-actin serving as an internal control. The marker used was a 1000 bp Gene Ruler, and the expected product sizes were 970 bp for CDC25C and 709 bp for β-actin. The negative control prepared from a sample containing just the Master-Mix, in which there was no DNA contamination. ( B ) The relative expression of CDC25C mRNA was evaluated using qRT-PCR. ( C ) The expression level of CDC25C protein was assessed through Western blotting and quantified using ImageJ software, with GAPDH serving as the internal control. Data are presented as means ± SDs from three independent experiments. c p <0.001.

Article Snippet: CDC25C , Mouse , #66912-1-Ig , Proteintech , , 1:1000.

Techniques: Agarose Gel Electrophoresis, Control, Marker, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Software

Journal: Scientific Reports

Article Title: CDC25C downregulation suppresses HCC growth via mitochondrial stress-induced autophagy and apoptosis

doi: 10.1038/s41598-026-36351-2

Figure Lengend Snippet: Downregulation of CDC25C inhibited the malignant biological behaviors of Hepa1-6 cells. ( A ) The colony formation ability was assessed using a plate cloning assay at 100× magnification, with the colony formation rate calculated by counting the number of colonies containing more than 50 cells. ( B ) The lateral migration ability of cells was measured using a wound healing assay at 100× magnification, and the migration rate (scratch healing rate) was calculated by measuring the width of the scratch. ( C , D ) The longitudinal migration and invasion abilities of the cells were evaluated using Transwell assays, quantifying the number of cells that passed through the filter. The scale bar represents 20 μm. Data are presented as means ± SDs from three independent experiments. a p <0.05, b p <0.01, c p <0.001.

Article Snippet: CDC25C , Mouse , #66912-1-Ig , Proteintech , , 1:1000.

Techniques: Cloning, Migration, Wound Healing Assay

Journal: Scientific Reports

Article Title: CDC25C downregulation suppresses HCC growth via mitochondrial stress-induced autophagy and apoptosis

doi: 10.1038/s41598-026-36351-2

Figure Lengend Snippet: Downregulation of CDC25C altered the morphology of the subcellular structure of Hepa1-6 cells. The images of Hepa1-6 cells captured under transmission electron microscopy. The scale bar represents a measurement of 2 μm at lower magnification and 200 nm at higher magnification. White arrows indicate healthy mitochondria, orange arrows denote autophagosomes, blue arrow highlight cellular vacuolation, and red arrows signify swollen and dysfunctional mitochondria.

Article Snippet: CDC25C , Mouse , #66912-1-Ig , Proteintech , , 1:1000.

Techniques: Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: CDC25C downregulation suppresses HCC growth via mitochondrial stress-induced autophagy and apoptosis

doi: 10.1038/s41598-026-36351-2

Figure Lengend Snippet: Downregulation of CDC25C induced a mitochondrial stress response. ( A , B ) Mitochondrial calcium concentrations were measured in Hepa1-6 and AML12 cells using the Rhod-2/AM fluorescent probe, with ImageJ software employed to quantify the red fluorescence intensity. ( C , D ) ROS levels were assessed in Hepa1-6 cells and AML12 using the MitoSOX fluorescent probe, and the red fluorescence intensity was quantified using ImageJ software. ( E ) The relative mRNA expression levels of CHOP, HSP60, ClpP, and LONP1 were evaluated via qRT-PCR. ( F , G ) The relative protein expression levels of CHOP, HSP60, ClpP, and LONP1 were determined through Western blotting and quantified using ImageJ software, with GAPDH serving as the internal control. The scale bar represents 10 μm. Data are presented as means ± SDs from three independent experiments. a p <0.05, b p <0.01, c p <0.001. ‘ns’ indicates no statistical significance.

Article Snippet: CDC25C , Mouse , #66912-1-Ig , Proteintech , , 1:1000.

Techniques: Software, Fluorescence, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: CDC25C downregulation suppresses HCC growth via mitochondrial stress-induced autophagy and apoptosis

doi: 10.1038/s41598-026-36351-2

Figure Lengend Snippet: Downregulation of CDC25C induced an autophagic response. ( A ) The relative mRNA expression levels of LC3, p62, and Beclin1 were evaluated using qRT-PCR. ( B , C ) The relative protein expression levels of LC3, p62, and Beclin1 were determined through Western blotting and quantified using ImageJ software, with GAPDH serving as the internal control. Data are presented as means ± SDs from three independent experiments. a p < 0.05, b p < 0.01, c p <0.001.

Article Snippet: CDC25C , Mouse , #66912-1-Ig , Proteintech , , 1:1000.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software, Control

Journal: Scientific Reports

Article Title: CDC25C downregulation suppresses HCC growth via mitochondrial stress-induced autophagy and apoptosis

doi: 10.1038/s41598-026-36351-2

Figure Lengend Snippet: Downregulation of CDC25C induced a mitochondria-mediated apoptosis. ( A ) Hoechst 33258 staining was employed to morphologically identify apoptotic cells. ( B ) Annexin V/TMRE costaining was utilized for apoptosis detection via flow cytometry to investigate the apoptosis rate and quantified using ImageJ software. ( C ) The relative mRNA expression levels of Cyt c, Caspase-3 and Caspase-9 were evaluated using qRT-PCR. ( D , E ) The relative protein expression levels of Cyt c, Caspase-3 and Caspase-9 were determined through Western blotting and quantified using ImageJ software, with GAPDH serving as the internal control. The scale bar represents 50 μm. Data are presented as means ± SDs from three independent experiments. a p < 0.05, b p < 0.01, c p <0.001. ‘ns’ indicates no statistical significance.

Article Snippet: CDC25C , Mouse , #66912-1-Ig , Proteintech , , 1:1000.

Techniques: Staining, Flow Cytometry, Software, Expressing, Quantitative RT-PCR, Western Blot, Control