Journal: Molecular Medicine Reports

Article Title: MicroRNA-328 directly targets p21-activated protein kinase 6 inhibiting prostate cancer proliferation and enhancing docetaxel sensitivity

doi: 10.3892/mmr.2015.4390

Figure Lengend Snippet: PAK6 expression in BPH and Pca tissue samples. (A) Pca samples were stained with anti-PAK6 (magnification, ×400). (B) BPH and (C–F) Pca tissue samples were stained with anti-PAK6 (magnification, ×200). Tissue samples with Gleason score of (C) <7; (D) 7 and (E) >7. (F) CRPC tissues. (G) PAK6 overexpression in CRPC samples was dectected by western blotting. PAK6, p21-activated protein kinase 6; BPH, benign prostate hyperplasia; Pca, prostate cancer; CRPC, castration-resistant Pca; ADPC, androgen-dependent Pca.

Article Snippet: A PAK6 3′-UTR-luciferase reporter was created by ligating the PAK6 3′-UTR PCR product into the Xho I and Not I restriction sites of the psiCHECK-2TM Vector (Promega Corp., Madison, WI, USA).

Techniques: Expressing, Staining, Over Expression, Western Blot

Journal: Molecular Medicine Reports

Article Title: MicroRNA-328 directly targets p21-activated protein kinase 6 inhibiting prostate cancer proliferation and enhancing docetaxel sensitivity

doi: 10.3892/mmr.2015.4390

Figure Lengend Snippet: PAK6 was directly targeted by miR-328. (A) miR-328 may bind to target sequences located in the nucleotides of the 3′-UTR of PAK6 mRNA (miRanda). (B) Quantitative reverse transcription-polymerase chain reaction identified decreased miR-328 expression levels in CRPC tissue samples compared with in ADPC tissues ( * P<0.05). (C) PAK6 protein was inhibited by treatment with miR-328 mimics, as evaluated by western blotting. (D) PAK6 3′-UTR was cloned into a luciferase reporter cloning site in the psiCHECK-2 dual luciferase vector. Luciferase activity markedly decreased with increasing miR-328 expression level ( * P<0.05). PAK6, p21-activated protein kinase 6; miR, microRNA; CRPC, castration-resistant prostate cancer; ADPC, androgen-dependent prostate cancer; NC, negative control; WT, wild-type; MUT, mutant.

Article Snippet: A PAK6 3′-UTR-luciferase reporter was created by ligating the PAK6 3′-UTR PCR product into the Xho I and Not I restriction sites of the psiCHECK-2TM Vector (Promega Corp., Madison, WI, USA).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Expressing, Western Blot, Clone Assay, Luciferase, Cloning, Plasmid Preparation, Activity Assay, Negative Control, Mutagenesis

Journal: Nutrients

Article Title: Polygonum cuspidatum Extract (Pc-Ex) Containing Emodin Suppresses Lung Cancer-Induced Cachexia by Suppressing TCF4/TWIST1 Complex-Induced PTHrP Expression

doi: 10.3390/nu14071508

Figure Lengend Snippet: Mammalian two-hybrid system-based high-throughput screening (HTS) for identification of natural compounds inhibiting TCF4–TWIST1. (Administrator) (A) Confirmation of interaction of TCF4 and TWIST1 by using a mammalian two-hybrid system. Principle of the mammalian two-hybrid system (upper panel). HEK293T cells were transiently transfected with GAL4-TWIST1, VP16-TCF4-full length (FL), VP16-TCF4-N-terminal domain (N), -middle domain (M) and C-terminal domain (C) expressing plasmids with pGL4.31-luciferase vector, and then cells were further incubated for 48 h. Luciferase activities were analyzed by using luciferase assay. ( B ) High-throughput screening for identification of natural compounds inhibiting the interaction of TCF4 and TWIST1. HEK293T cells were transfected with GAL4-TWIST1, VP16-TCF4-full length (FL) and pGL4.31-luciferase vector, and then transfected cells were incubated for 48 h prior to treatment of natural compounds library. Post-transfection, cells were incubated with 5 µg/mL of the natural compound for 24 h. Luciferase activities were analyzed by using luciferase assay. ( C ) Measurement of cell viability in hit compounds-treated cells. HEK293T cells were incubated for 24 h with each hit compound (5 µg/mL) as indicated. Cell viability was measured by crystal violet staining and assay. ( D ) Emodin is a potential small molecule targeting the interaction of TCF4 and TWIST1. HEK293T cells transfected with GAL4-TWIST1, VP16-TCF4-full length (FL) and pGL4.31-luciferase plasmids were incubated for 24 h with emodin as indicated, then luciferase activities were analyzed. The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis was performed by using a one-way ANOVA Tukey post hoc test.

Article Snippet: CheckMate TM /Flexi ® Vector Mammalian Two-Hybrid System Kit (Promega, Madison, WI, USA) was used to generate a mammalian two-hybrid luciferase assay system.

Techniques: High Throughput Screening Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Incubation, Staining