Journal: Oncology reports

Article Title: PTEN enhances G2/M arrest in etoposide-treated MCF‑7 cells through activation of the ATM pathway.

doi: 10.3892/or.2016.4674

Figure Lengend Snippet: Figure 6. PTEN promotes etoposide-induced inactivation of CDC25C. (A) The control and PTEN-knockdown MCF-7 cells were treated with different doses of etoposide (ETOP) for 8 h and phospho-CDC25C (inac- tive form) was detected by western blotting. Representative images of western blotting are presented for both control and PTEN-knockdown groups. (B) The relative expression level of phosphorylated CDC25C was quantified against total CDC25C that was first normalized by β-actin. Data are presented as means ± SE. *p<0.05 in comparison to the respective phospho‑CDC25C (S216) in the control group.

Article Snippet: Anti-phospho-ATM, anti-Chk2, anti-phosph-Chk2 (T68), anti-phospho-p53 (S15) and anti-phospho-CDC25C (S216) antibodies were purchased from Cell Signaling.

Techniques: Control, Knockdown, Western Blot, Expressing, Comparison

Journal: Oncology reports

Article Title: Resveratrol synergizes with cisplatin in antineoplastic effects against AGS gastric cancer cells by inducing endoplasmic reticulum stress‑mediated apoptosis and G2/M phase arrest.

doi: 10.3892/or.2020.7708

Figure Lengend Snippet: Figure 5. RES sensitizes AGS cells to DDP by inducing G2/M cell cycle arrest. (A) PI staining and flow cytometry were performed to determine cell cycle progression after RES (20 µM) and DDP (1 µg/ml) cotreatment for 48 and 72 h. (B and C) The percentage of cells in the G2/M phase was examined. (D‑F) After RES (20 µM) and DDP (1 µg/ml) combination treatment for 48 h, the protein levels of cyclin B1, p‑CDK1 (Tyr15), CDK1, Cdc25C, p21Waf1/Cip1 and p27Kip1 were detected by western blotting. All data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001. RES, resveratrol; DDP, cisplatin; p‑CDK1, phosphorylated cyclin‑dependent kinase 1; CTRL, control; COM, combination treatment.

Article Snippet: The primary antibodies used were rabbit antibodies against CHOP (also named DDIT3; 1:1,000; product code ab179823; Abcam), poly-ADP-ribose polymerase (PARP; 1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), Bax (1:1,000, cat. no. 2774; Cell Signaling Technology, Inc.), Bcl-2 (1:1,000, cat. no. 2872, Cell Signaling Technology, Inc.), glucose-regulated protein 78 (GRP78; 1:1,000; product code ab108615; Abcam), PERK (1:1,000; cat. no. 5683; Cell Signaling Technology, Inc.), eIF2α (1:1,000; cat. no. 5324; Cell Signaling Technology, Inc.), p-eIF2α (1:1,000; cat. no. 3398; Cell Signaling Technology, Inc.), caspase-12 (1:1,000; product code ab62484; Abcam), cyclin B1 (1:1,000; cat. no. 4138; Cell Signaling Technology, Inc.), p-CDK1 (Tyr15; 1:1,000; cat. no. 4539; Cell Signaling Technology, Inc.), Cdc25C (1:1,000; cat. no. 4688; Cell Signaling Technology, Inc.), p21Waf1/Cip1 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), p27Kip1 (1:1,000; cat. no. 3686; Cell Signaling Technology, Inc.) and GAPDH (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc.), and mouse monoclonal antibody against CDK1 (1:1,000; cat. no. 9116; Cell Signaling Technology, Inc.).

Techniques: Staining, Flow Cytometry, Western Blot, Control

Journal: Oncology Letters

Article Title: Luteolin potentiates low-dose oxaliplatin-induced inhibitory effects on cell proliferation in gastric cancer by inducing G 2 /M cell cycle arrest and apoptosis

doi: 10.3892/ol.2021.13134

Figure Lengend Snippet: Changes in TRAP1, P-ERK1/2/ERK1/2 and CDC25C protein expression levels in mouse forestomach carcinoma cells induced by luteolin (20 µM) and/or oxaliplatin (5 µM). (A-C) TRAP1, P-ERK1/2/ERK1/2, CDC25C and β-actin protein expression levels were assessed by western blot analysis, and quantitative analysis of protein expression levels is shown in the histogram. *P<0.05, **P<0.01. Experiments were repeated at least in triplicate. Lut, luteolin; Oxa, oxaliplatin; TRAP1, tumor necrosis factor receptor-associated protein 1; ERK1/2, extracellular-regulated protein kinases1/2; CDC25C, cell division cycle 25 homolog C.

Article Snippet: The primary antibodies used were: β-actin mouse monoclonal antibody (cat. no. TA-09; 1:2,000; OriGene Technologies, Inc.), Bcl-2 rabbit polyclonal antibody (cat. no. ab196495, 1:1,000; Abcam), BCL-2-associated X protein (Bax) rabbit monoclonal antibody (cat. no. ab182734; 1:1,000; Abcam), cyclin A2 rabbit monoclonal antibody (cat. no. ab181591; 1:2,000; Abcam), cyclin B1 rabbit monoclonal antibody (cat. no. ab32053; 1:1,000; Abcam), cyclin-dependent kinase-1 (CDK1) rabbit monoclonal antibody (cat. no. ab133327; 1:20,000; Abcam), tumor necrosis factor receptor-associated protein 1 (TRAP1) rabbit polyclonal antibody (cat. no. 10325-1-AP; 1:2,000; ProteinTech Group, Inc.), cell division cycle 25 homolog C (CDC25C) mouse monoclonal antibody (cat. no. 66912-1-lg; 1:2,000; ProteinTech Group, Inc.), extracellular-regulated protein kinases1/2 (ERK1/2) rabbit polyclonal antibody (cat. no. 9102s; 1:1,000; Cell Signaling Technology, Inc.) and p-ERK1/2 mouse monoclonal antibody (cat. no. 9106s; 1:1,000; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: The Novel Methylation Biomarker SCARA5 Sensitizes Cancer Cells to DNA Damage Chemotherapy Drugs in NSCLC

doi: 10.3389/fonc.2021.666589

Figure Lengend Snippet: SCARA5 induced G2/M cell cycle arrest by inhibiting FOXM1. (A, B) The inhibitory effect of SCARA5 on cycle related markers was verified by q-PCRand WB. (C) After overexpression of SCARA5, the DNA damage repair marker phosphorylated histone H2AX and checkpoint protein was detected by q-PCR and WB. (D) The common transcription factors of CyclinB1, CDC25C, CHK1, and DEGs of GSE12667 was shown in Venn diagram. (E) The mRNA expression of FOXM1 was downregulated in SCARA5-expressed A549 cells. (F) Ectopic expression of FOXM1 attenuated the inhibitory effect of SCARA5 on CDC25C, Cyclin B1, and CHK1. (G) FOXM1 protein could combine with the promoters of CyclinB1 and CDC25C. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Then, membranes were incubated with primary antibodies specific for SCARA5 (#ab118894, Abcam), FOXM1 (#ab207298), CyclinB1 (Sc245), CHK1 (A5004, Bimake), CDC25C (A5133, Bimake), CDC25C (Ser216) (#4901, CST), CDK1 (SC54), Phospho-HistoneH2AX (Ser139) (#80312, CST), HSPA5 (#3177, CST).

Techniques: Over Expression, Marker, Expressing

Journal: Frontiers in Oncology

Article Title: The Novel Methylation Biomarker SCARA5 Sensitizes Cancer Cells to DNA Damage Chemotherapy Drugs in NSCLC

doi: 10.3389/fonc.2021.666589

Figure Lengend Snippet: Proposed mechanism of how SCARA5 affects HSP70/FOXM1/CCNB1 in A549. SCARA5 is expressed on the cell membrane and enters into the cytoplasm through endocytosis and locates in the endoplasmic reticulum, causing UPR, leading to the upregulation of HSP70 family protein. HSP70 inhibits the expression of FOXM1, which leads to the downregulation of CyclinB1 and CDC25C, the downstream genes of FOXM1, causing G2/M arrest and inhibits the proliferation of lung cancer cells.

Article Snippet: Then, membranes were incubated with primary antibodies specific for SCARA5 (#ab118894, Abcam), FOXM1 (#ab207298), CyclinB1 (Sc245), CHK1 (A5004, Bimake), CDC25C (A5133, Bimake), CDC25C (Ser216) (#4901, CST), CDK1 (SC54), Phospho-HistoneH2AX (Ser139) (#80312, CST), HSPA5 (#3177, CST).

Techniques: Membrane, Expressing

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: LNCaP C-33, C-81 and PC-3 PCa cells were plated at a density of 8×10 3 , 6×10 3 and 4.8×10 3 cells/cm 2 , 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). β-actin was analyzed and used as a loading control.

Article Snippet: The Cdc25C shRNA plasmid and control plasmid, and the horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, 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: (A) LNCaP C-33 and C-81 cells were seeded at a density of 8×10 3 and 6×10 3 cells/cm 2 , 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. β-actin was detected for serving as a loading control. (B) AS LNCaP C-33 and (C) VCaP cells were plated at a density of 8×10 3 and 2×10 4 cells/cm 2 , respectively, for 3 days, and then steroid starved for 48 hr in SR medium. Cells were treated with 10 nM DHT with or without 10 µM Casodex for 48 hr. (B) For C-33 cells, total cell lysate proteins were analyzed for AR, Cdc25A, Cdc25B, Cdc25C, cPAcP, PSA, Cyclin D1 and Cyclin B1 protein levels. (C) For VCaP cells, total cell lysate proteins were analyzed for AR, Cdc25C, Cyclin D1, Cyclin B1 and PSA protein levels. β-actin was analyzed in each experiment and used as a loading control. (D) Effect of DHT vs . EGF on Cdc25C protein level in C-33 cells. Steroid-starved C-33 cells were treated with 10 nM DHT or 10 ng/ml EGF for 24 hr, and control cells were treated with solvent alone. All cells were harvested for analyzing Cdc25C protein level. As controls, cPAcP, ErbB-2 and its tyrosine phosphorylation at Y877, AR and its phosphorylation at S81, and Cyclin B1 were also analyzed. Tubulin was detected for serving as a loading control.

Article Snippet: The Cdc25C shRNA plasmid and control plasmid, and the horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Control, Solvent, Phospho-proteomics

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: (A,B) LNCaP C-33 and (C) PC-3 cells were plated at a density of 1.2×10 4 or 1×10 4 cells/cm 2 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. β-actin was used as a loading control. The ratios of Cdc25C, Cyclin B1 and CyclinD1 protein levels to β-actin were calculated after semi-quantification by densitometric analyses on films with different exposure time periods. * p <0.05, n = 2×3; Bar, standard deviation.

Article Snippet: The Cdc25C shRNA plasmid and control plasmid, and the horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: 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: (A) Effects of de novo biosynthesis inhibitors on Cdc25C protein levels by androgens. LNCaP C-33 cells were plated at a density of 1.6×10 4 cells/cm 2 for 3 days in regular medium. Cells were steroid starved for 48 hr in a SR medium, and then treated with 5 µg/mL Actinomycin D (Act D) or 10 µg/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. β-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 µM) and (C) Epoximicin (0.1 and 1.0 M). Cells were harvested and analyzed for Cdc25C protein levels. β-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 µM) and (F) E64D (0.01 to 1.0 µM) for 48 hr. Cells were harvested and analyzed for Cdc25C protein levels. β-actin was analyzed as a loading control.

Article Snippet: The Cdc25C shRNA plasmid and control plasmid, and the horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

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