Journal: Oncology reports

Article Title: miR-3619-5p inhibits prostate cancer cell growth by activating CDKN1A expression.

doi: 10.3892/or.2016.5250

Figure Lengend Snippet: Figure 6. miR-3619-5p inhibits cell cycle related gene expression primarily by regulating CDKN1A expression. DU145 and PC3 cells were transfected with 50 nM of the indicated RNAs for 72 h. (A) Expression of p21 mRNA was assessed by real-time PCR. GAPDH served as a loading control. (B) Induction of p21 protein expression was detected by western blot analysis. GAPDH were also detected and served as a loading control. (C) Expression of cyclin D1, CDK4 and CDK6 mRNA was detected by real-time PCR. GAPDH served as a loading control. (D) Expression of cyclin D1, CDK4 and CDK6 mRNA was determined by western blot analysis. α-Tublin served as a loading control. *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: After blocking the membranes were incubated overnight at 4 ̊C with appropriate dilutions of specific primary antibodies as follows: p21 (1/2000) (Cell Signaling Technology), cyclin D1 (1/2000) (Affinity, USA), CDK4 (1/1000) (Affinity), CDK6 (1/2000) (Affinity), GAPDH (1/500) (Boster, Wuhan, China) and α-tubulin (1/500) (Boster).

Techniques: Gene Expression, Expressing, Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of Cancer

Article Title: Histone Deacetylase Inhibitor-Induced CDKN2B and CDKN2D Contribute to G2/M Cell Cycle Arrest Incurred by Oxidative Stress in Hepatocellular Carcinoma Cells via Forkhead Box M1 Suppression

doi: 10.7150/jca.60027

Figure Lengend Snippet: Synergistic effect of oxidative stress and HDACi on the expression of CDK and CDKN. To validate MACE results, oxidative stress-induced HCC cells (HepG2 and Hep3B) were used to perform qPCR and western blot. A. mRNA expressions of Cdk4 and 6 were analyzed by qPCR in HepG2 cells. Expression of Cdk4/6 was significantly decreased by oxidative stress induced by tBHP. B. HepG2 cells were treated with vehicle, tBHP (25 µmol/L), SAHA (1 µmol/L), MS-275 (1 µmol/L), or combination with tBHP and HDACi. Expressions of Cdk4 and 6 were slightly decreased by single treatment with HDACi and synergistically decreased by tBHP and HDACi. C. tBHP-induced expressions of Cdkn2b and Cdkn2d were increased in a dose-dependent manner. D. Expressions of Cdkn2b and Cdkn2d were increased by single treatment with HDACi and synergistically increased by cotreatment with tBHP and HDACi. E. Expressions of Cdk4 and 6 were decreased by tBHP in a dose-dependent manner in Hep3B cells. F. Expressions of Cdk4 and 6 were decreased synergistically by co-treatment with tBHP and HDACi. G. Expressions of Cdkn2b and Cdkn2d were increased by tBHP dose-dependently. H. Expressions of Cdkn2b and Cdk2d were synergistically increased by co-treatment with tBHP and HDACi. Graphs are representative of the mean±SD from three independent experiments (* p <0.05, ** p <0.01, vs. Vehicle). I. A representative blot of CDK1, CDK2, CDK4, CDK6, CDKN2B, and CDKN2D in HepG2 cells. Protein levels of CDK4 and 6 were decreased by tBHP whereas CDKN2B and CDKN2D were increased by tBHP in a dose-dependent manner. J. Protein levels of CDK1 and CDK2 were rarely changed by tBHP, HDACi, or combined treatment. Protein levels of CDK4 and 6 were dramatically decreased by combinatorial treatment with tBHP and HDACi. In contrast to CDK4 and 6, co-treatment with tBHP and HDACi increased the protein levels of CDKN2B and CDKN2D.

Article Snippet: Antibodies to detect human CDK4, CDK6, CDKN2B, CDKN2D, AURKA, PLK1, CCNB1, and phospho-CCNB1 (pSer147) were purchased from Cusabio (Hubei, China).

Techniques: Expressing, Western Blot

Journal: Journal of Cancer

Article Title: Histone Deacetylase Inhibitor-Induced CDKN2B and CDKN2D Contribute to G2/M Cell Cycle Arrest Incurred by Oxidative Stress in Hepatocellular Carcinoma Cells via Forkhead Box M1 Suppression

doi: 10.7150/jca.60027

Figure Lengend Snippet: Double knockdown of CDKN2B and CDKN2D reduces the effect of tBHP and SAHA. Small interfering RNAs were transfected into the HepG2 cells for 24 h, which were subsequently incubated with tBHP and SAHA for CDKN2B and CDKN2D inductions. A. Western blot showed specific knockdown of CDKN2B and CDKN2D by siRNA. Co-treatment with tBHP and SAHA reduced the expressions of Cdk4 (B) and Cdk6 (C) , which were significantly restored by CDKN2B and CDKN2D double knockdown. Graphs are representative of the mean±SD from three independent experiments (* p <0.05, vs. Vehicle, # p <0.05, vs. tBHP+SAHA). Representative blot images (D) and bands density analysis showed that double knockdown of CDKN2B and CDKN2D reversed the tBHP and SAHA-induced suppression of phospho-FOXM1 (E) , AURKA and PLK1 (F) , as well as phospho-CCNB1 (G) . Graphs are representative of the mean±SD from three independent experiments (* p <0.05, ** p <0.01, vs. Vehicle, # p <0.05, ## p< 0.01 vs. tBHP+SAHA).

Article Snippet: Antibodies to detect human CDK4, CDK6, CDKN2B, CDKN2D, AURKA, PLK1, CCNB1, and phospho-CCNB1 (pSer147) were purchased from Cusabio (Hubei, China).

Techniques: Knockdown, Transfection, Incubation, Western Blot