Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long noncoding RNA NEAT1 promotes laryngeal squamous cell cancer through regulating miR-107/CDK6 pathway

doi: 10.1186/s13046-016-0297-z

Figure Lengend Snippet: NEAT1 regulates CDK6 through modulating miR-107. a TargetScan database predicated CDK6 as a target of miR-107. b miR-107 decreased luciferase activity of 3′UTR but not that of 3′UTR-NC and 3′UTR-MU. miR-NC indicated scramble miRNA used as the negative control for miR-107. ** P < 0.01. c Starbase database predicted the target site between CDK6 and miR-107. d Real-time PCR showed that miR-107 level significantly increased after NEAT1 knockdown in Hep-2 cells. ** P < 0.01. e Western blot analysis showed that CDK6 protein level decreased after NEAT1 knockdown in Hep-2 cells. Lane 1 and 2: control Hep-2 cells; Lane 3 and 4: Hep-2 cells transduced with NEAT1 siRNA. GAPDH was loading control. f Western blot analysis showed that CDK6 protein level decreased after NEAT1 knockdown in Hep-2 cells, but was restored after the knockdown of miR-107. GAPDH was loading control

Article Snippet: Hep-2 cells were collected and CDK6 expression was detected as described previously using CDK6 antibody (1:400 dilution, Boster, Wuhan, China) [ ].

Techniques: Luciferase, Activity Assay, Negative Control, Real-time Polymerase Chain Reaction, Knockdown, Western Blot, Control, Transduction

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