Journal: Oncology Reports

Article Title: Suppression of CDCA3 inhibits prostate cancer progression via NF-κB/cyclin D1 signaling inactivation and p21 accumulation

doi: 10.3892/or.2021.8253

Figure Lengend Snippet: Knockdown of CDCA3 induces G0/G1-phase arrest in DU145 and PC-3 cells. (A) Flow cytometry was used to analyze the cell cycle following CDCA3 knockdown in DU145 and PC-3 cells. (B) Cell cycle-associated protein levels, including cyclin D1 and p21, were analyzed via western blotting. GAPDH was used as a loading control. Data are presented as the mean ± SD of three independent experiments. **P<0.01 and ***P<0.001. CDCA3, cell division cycle-associated 3; sh-, short hairpin; NC, negative control.

Article Snippet: The following antibodies were used: Mouse monoclonal anti-GAPDH (1:2,000; cat. no. 33033M; BIOSS), rabbit polyclonal anti-CDCA3 (1:1,000; cat. no. YT0819), rabbit polyclonal anti-cleaved caspase-3 (1:1,000; cat. no. YC0006), rabbit polyclonal anti-pro-caspase-3 (1:1,000; cat. no. YT6113), and rabbit polyclonal anti-cyclin-dependent kinase inhibitor 1 (p21; 1:1,000; cat. no. YT3497; all from Immunoway Biotechnology Company), rabbit polyclonal anti-cyclin D1 (1:1,000; cat. no. 0623R; BIOSS), mouse monoclonal anti-NFκB-p65 (1:1,000; cat. no. YM311), rabbit polyclonal anti-phosphorylated (p)-NFκB-p65 (1:1,000; cat. no. YP0192), rabbit polyclonal anti-IKKα/β (1:1,000; cat. no. YT2302), and rabbit polyclonal anti-NFκB-p105/p50 (1:1,000; cat. no. YT3101; all from Immunoway Biotechnology Company), HRP-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. 40295G-HRP; BIOSS) and HRP-labeled goat anti-mouse secondary antibody (1:5,000; cat. no. 0368G-HRP; BIOSS).

Techniques: Flow Cytometry, Western Blot, Negative Control

Journal: Cancer Science

Article Title: Simvastatin inactivates β1‐integrin and extracellular signal‐related kinase signaling and inhibits cell proliferation in head and neck squamous cell carcinoma cells

doi: 10.1111/j.1349-7006.2007.00471.x

Figure Lengend Snippet: (a) Effect of simvastatin on expressions of β1‐integrin mRNA transcript and protein. Analysis of β1‐integrin mRNA expression using reverse transcription‐polymerase chain reaction (RT‐PCR). Head and neck squamous cell carcinoma cells were incubated for 48 h with 5 or 10 µM simvastatin and compared with untreated cells (incubated with vehicle). (b) Analysis of β1‐integrin, FAK, and p‐FAK (Y397) expressions using western blot. The condition of cell culture was identical with that of RT‐PCR analysis. The intensity of each band of western blot was determined using NIH Image program and the ratio of β1‐integrin and β‐actin was calculated for each treatment. The β1‐integrin/β‐actin ratio in untreated cells was set as 1. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Antibodies for immunoblotting were obtained from the following sources: rabbit polyclonal anti‐ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti‐phospho‐ERK1/2 (Santa Cruz Biotechnology); rabbit polyclonal anti‐β1‐integrin (Chemicon International Inc., Temecula, CA, USA); rabbit polyclonal anti‐FAK (Santa Cruz Biotechnology); rabbit polyclonal anti‐phosphorylated FAK (Y397; Santa Cruz Biotechnology); mouse monoclonal anti‐p21 (Santa Cruz Biotechnology); mouse monoclonal anti‐p27 (Santa Cruz Biotechnology); mouse monoclonal anti‐thymidylate synthase (Chemicon); rabbit polyclonal anti‐active‐caspase‐3 (BioVision, Mountain View, CA, USA); and mouse monoclonal anti‐β‐actin (Abcam, Cambridge, MA, USA).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot, Cell Culture

Journal: International Journal of Molecular Medicine

Article Title: Aspirin promotes apoptosis and inhibits proliferation by blocking G 0 /G 1 into S phase in rheumatoid arthritis fibroblast-like synoviocytes via downregulation of JAK/STAT3 and NF-κB signaling pathway

doi: 10.3892/ijmm.2018.3883

Figure Lengend Snippet: Effects of aspirin on Bcl-2, BAX, PARP1, Cyclin D1 and P21 expression in RA-FLS. (A) After RA-FLS were exposed to various concentration of aspirin for 24 h, western blotting was used to determine Bcl-2, BAX, PARP1, Cyclin D1 and P21 protein levels. The corresponding internal control was GADPH. Changes in Bcl-2, BAX, PARP1, Cyclin D1 and P21 protein expression in RA-FLS treated with aspirin are shown: Bcl-2, PARP1, Cyclin D1 and P21 were decreased while BAX was increased, each with increasing aspirin concentration. (B) Bar graph shows the gray value analysis of Bcl-2, BAX, PARP1, Cyclin D1 and P21. Data are presented as the means ± SD (error bars) from three independent experiments. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. DMSO group. We set the value as target protein vs. GAPDH, and set the value of control group as 1. CCND1, Cyclin D1; RA-FLS, rheumatoid arthritis-fibroblast-like synoviocytes; Bcl-2, B-cell lymphoma-2; BAX, Bcl-2-associated X protein; PARP1, poly (ADP-ribose) polymerase 1.

Article Snippet: The membranes were blocked with 5% BSA in TBS-T for 1 h, and then incubated with rabbit anti-Bax (1:2,000 dilution, Proteintech Group; #S0599-2-lg), anti-Bcl-2 (1:1,000 dilution, Santa Cruz Biotechnology, Inc.; #sc-7382), anti-PARP1 (1:2,000 dilution, Proteintech Group; #13371-1-AP), anti-P21 (1:1,000 dilution, #EPR3993), anti-cyclin D1 (1:10,000 dilution, #EPR2241; both from Abcam), anti-P65 (1:2,000 dilution; #8242), anti-p-P65 (1:2,000 dilution; #3033; both from Cell Signaling Technology, Inc.), anti-P50/P105 (1:1,000 dilution, Abcam: #E381), anti-p-P50/105 (1:1,000 dilution, Affinity Company: #AF3219), anti-STAT3 (1:2,000 dilution, #EPR787Y) and anti-p-STAT3 (1:200,000 dilution, #EP2147Y; both from Abcam) antibodies for 2 h at room temperature.

Techniques: Expressing, Concentration Assay, Western Blot

Journal: Pancreas

Article Title: Histone Deacetylase Inhibition Restores Expression of Hypoxia-Inducible Protein NDRG1 in Pancreatic Cancer

doi: 10.1097/MPA.0000000000000982

Figure Lengend Snippet: Impact of TSA on cell growth and the cell cycle regulator p21 Cip1 . A, Cell proliferation was evaluated by the MTT assay. Cell viability is expressed as the ratio of the number of viable cells with TSA treatment compared to that without treatment. Cells growing in DMSO were also analyzed as vehicle control. Means (SD) of six replicates are plotted. B, Temporal expression of p21 Cip1 (21 kDa); α-tubulin was used as a loading control. The results shown are from 1 representative experiment out of 3 independently performed experiments. MTT, methylthiazolyldiphenyl-tetrazolium bromide.

Article Snippet: Proteins were transferred to nitrocellulose membranes using a semidry transfer system (Bio-Rad), blocked with 5% nonfat dry milk in 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween-20 for 1 hour, and incubated overnight at 4°C with primary antibody: sheep anti-NDRG1 1:6000, AB-160, Kinasource (Dundee, Scotland); sheep anti-phospho-NDRG1 (Thr 346, Thr 356, Thr 366) 1:3000, Kinasource PB-025; mouse anti-p21 Cip1 1:2000, Cell Signaling; rabbit anti-CAII (carbonic anhydrase 2) 1:5000, Santa Cruz Biotechnology (Heidelberg, Germany); rabbit anti-β-actin 1:1000, Sigma A5060; or mouse anti-α-tubulin 1:2000, Santa Cruz Biotechnology.

Techniques: MTT Assay, Control, Expressing