Journal: Oncotarget

Article Title: Long non-coding RNA AK058003, as a precursor of miR-15a, interacts with HuR to inhibit the expression of Υ-synuclein in hepatocellular carcinoma cells

doi: 10.18632/oncotarget.14276

Figure Lengend Snippet: ( A ) HepG2 and SK-HeP1 stably over-expressing lncRNA-AK058003 cell lines were showed to have significantly increased expression levels of lncRNA-AK058003 compared to vector controls. ( B ) siRNA mediated knock-down of lncRNA-AK058003 (siAK058003) in HepG2 and SK-HeP1 cell lines were decreased significantly compared to scramble controls. ( C ) LncRNA-AK058003 up-regulated and ( D ). lncRNA-AK058003 down-regulated HepG2 and SK-HeP1 cell lines were seeded into 96 well plates and cell proliferation was assessed by EdU immunofluorescence staining. The proliferation ability value is in red. Original magnification, ×400. The barplot on the right shows the percentage of EdU-positive nuclei, indicating lncRNA-AK058003 notably restrained cell multiplication. ( E ) CCK8 assays showed that cell proliferation was suppressed by lncRNA-AK058003 overexpression in SK-HeP1 cells. The right curves show no significant difference in lncRNA-AK058003 down-regulated HepG2 cells. ( F , G ) FACS analysis showing a significant increase in the G1 phase in HepG2 and SK-HeP1 cells overexpressing lncRNA-AK058003. ( H ) The total protein expression of CDK4, CDK6, and cyclinD1 were detected by western blot analysis. Representative images of ( I ) transwell migration and ( J ) invasion assays in up-regulated and siRNA mediated knock-down of lncRNA-AK058003. Original magnification, × 400. ( K ) The metastasis related proteins (MMP9, E-cadherin) were performed in lncRNA-AK058003-overexpressing and lncRNA-AK058003- down-regulating HepG2 and SK-HeP1 cells by Western blot analysis. Data are shown as the mean ± S.E.M. based on three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membrane were probed with the following antibodies: SNCG antibodies (Abcam, ab6169), CDK4 (Bioworld, BS6462), CDK6 (Bioss, bs-0568R), CyclinD1 (CST, 2978P), MMP9 (Bioworld, bs6893) and E-cadherin (ProteinTech, 20874-1-AP).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Over Expression, Western Blot, Migration

Journal: The Journal of biological chemistry

Article Title: Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 with cyclin A-Cdk2 and cyclin D2-Cdk4.

doi: 10.1074/jbc.272.41.25863

Figure Lengend Snippet: FIG. 1. Analysis of Cdk2- and p27-associated kinase activity in p27 inducible Mv1Lu cells. Tet-p27 cells were grown in media con- taining the indicated concentration of tetracycline for 18 h before they were assayed as follows. a, [125I]iododeoxyuridine incorporation into DNA at the end of the incubation (data are averages of triplicate determinations and are plotted as percent relative to the incorporation in the presence of 1 mg/ml tetracycline). b, immunoblotting of total cell extract (100 mg of protein) with p27 antibodies. c–e, p27 immunopre- cipitation followed by immunoblotting with anti-Cdk4 (c), anti-Cdk6 (d), or anti-Cdk2 (e). For reasons unknown, the recovery of p27-bound Cdk was low in extracts from cells incubated without tetracycline (data not shown). f and g, Cdk2 immunoprecipitation followed by histone H1 (f) or Rb (g) kinase assays. h, immunoblotting of total cell extract (200 mg of protein) with Rb antiserum. Rbphos indicates the hyperphospho- rylated form of Rb. i and j, p27 immunoprecipitation followed by histone H1 (i) or Rb (j) kinase assays. NRS, normal rabbit serum. Cdk2, Cdk2 immunoprecipitation, followed by kinase assay. k, left, in a separate experiment, Mv1Lu cell extracts were subjected to four cycles of immu- nodepletion with Cdk4 and Cdk6 antisera. These extracts were then immunoprecipitated with p27 antiserum (lane 5), followed by Cdk6 and Cdk4 immunoblotting. Lanes 1–4 are the immunocomplexes from each depletion round, analyzed by Cdk4 and Cdk6 immunoblotting. k, right, p27 was immunoprecipitated from lysates that had been subjected to four cycles of immunodepletion with Cdk4/Cdk6 or normal rabbit se- rum. The Rb kinase activity associated with the p27 immunocomplexes was then determined.

Article Snippet: Immunocomplexes were analyzed by Western immunoblot analysis with p27, Cdk4 (Pharmigen), or Cdk6 (Santa Cruz) antibodies by standard techniques.

Techniques: Activity Assay, Concentration Assay, Incubation, Western Blot, Immunoprecipitation, Kinase Assay, Immunodepletion

Journal: Cancer cell international

Article Title: MicroRNA-550a-3-5p controls the brain metastasis of lung cancer by directly targeting YAP1.

doi: 10.1186/s12935-021-02197-z

Figure Lengend Snippet: Fig. 7 The effects of miR-550a-3-5p on cleaved-PARP, pRB, CDK6, YAP1, CTGF, and CYR61. A Western blotting images. B Cleaved-PARP protein expression. C pRB protein expression. D CDK6 protein expression. E YAP1 protein expression. F CTGF protein expression. (G) CYR61 protein expression. *P < 0.05, compared with the control group; #P < 0.05, compared with the miR-550a-3-5p mimics group

Article Snippet: After blocking with 5% skim milk, the membranes were incubated with anti-cleaved-Poly(ADP-ribose) polymerase (PARP) antibody (1:1000, Abcam), anti-RB transcriptional corepressor 1 (pRB) antibody (1:1000, Abcam), anti-cyclin dependent kinase 6 (CDK6) antibody (1:1000, Abcam), anti-Yes1 associated transcriptional regulator (YAP1) antibody (1:1000, Abcam), anti- connective tissue growth factor (CTGF) antibody (1:1000, Proteintech Group, Inc.), anti-cysteine rich angiogenic inducer 61 (CYR61) antibody (1:1000, Abcam), and anti-GAPDH antibody (1:10,000, Proteintech Group, Inc.) overnight at 4 °C.

Techniques: Western Blot, Expressing, Control

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: Oncology reports

Article Title: Enhanced anticancer effects of a mixture of low-dose mushrooms and Panax ginseng root extracts in human colorectal cancer cells.

doi: 10.3892/or.2017.5796

Figure Lengend Snippet: Figure 4. Amex7 prevents cell cycle progression by regulating cell cycle regulatory proteins. HT-29 cells were treated with 4% Amex7 for 6, 12, and 24 h. (A) Cell cycle distribution was analyzed by flow cytometry and (B) was quantified using a FACScan flow cytometer. (C) Expression of G1/S checkpoint regula- tors (cyclin D1, cyclin E, CDK2, CDK4, and CDK6) and G2/M checkpoint regulators (cyclin A2, cyclin B1, and p-cdc2) were measured by immunoblotting and (D) were quantified using Multi Gauge V3.0 software in HT-29 cells. Densitometric quantification was normalized to GAPDH. (-) indicates the control group and (+) indicates Amex7-treated group. Values are mean ± SD of four determination. *p<0.05, †p<0.01, §p<0.001 vs. each control group.

Article Snippet: Anti-cyclin-dependent kinase 4 (CDK4) and anti-cyclin-dependent kinase 6 (CDK6) were obtained from Bethyl (Montgomery, TX, uSA).

Techniques: Flow Cytometry, Expressing, Western Blot, Software, Control