Journal: International journal of molecular sciences

Article Title: MicroRNA PC-3p-2869 Regulates Antler Growth and Inhibits Proliferation and Migration of Human Osteosarcoma and Chondrosarcoma Cells by Targeting CDK8, EEF1A1, and NTN1.

doi: 10.3390/ijms241310840

Figure Lengend Snippet: Figure 5. miR-PC-2869 regulates the expression of CDK8, EEF1A1, and NTN1. (A) The target sites of miR-PC-2869 in CDK8, EEF1A1, and NTN1 are conserved between humans and red deer (Cervus elaphus). Blue bases represent the complementary seed-matching region. (B) Western blot analysis of CDK8, EEF1A1, and NTN1 protein expression after transfection with miR-PC-2869 mimics or NC mimics in MG63, SW1353, and antler cartilage cells. The Western blot bands are representative images of three independent biological replicates, and fold-changes in densitometric values in three replicate experiments are presented as mean ± standard deviation. Ce, Cervus elaphus. Hs, Homo sapiens. NC, negative control. CA, antler cartilage cells.

Article Snippet: Following blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4 ◦C with specific primary antibodies against CDK8 (cat. no. A5548; 1:1000; ABclonal, Wuhan, China), EEF1A1 (cat. no. ab157455; 1:1000; Abcam), NTN1 (cat. no. A1500; 1:1000; ABclonal), GAPDH (cat. no. sc-47724; 1:3000; Santa Cruz Biotechnology, Dallas, TX, USA; or cat. no. AC033; 1:20,000; ABclonal), c-myc (cat. no. A5011; 1:1000; Bimake), or cyclin D1 (cat. no. A5035; 1:1000; Bimake).

Techniques: Expressing, Western Blot, Transfection, Standard Deviation, Negative Control

Journal: International journal of molecular sciences

Article Title: MicroRNA PC-3p-2869 Regulates Antler Growth and Inhibits Proliferation and Migration of Human Osteosarcoma and Chondrosarcoma Cells by Targeting CDK8, EEF1A1, and NTN1.

doi: 10.3390/ijms241310840

Figure Lengend Snippet: Figure 6. CDK8, EEF1A1, or NTN1 knockdown repressed the proliferation and migration of antler cartilage cells. (A–C) Western blot analysis of the interference efficiency of CDK8, EEF1A1, or NTN1 siRNA in antler cartilage cells. The Western blot bands are representative images of three independent biological replicates, and fold-changes in densitometric values in three replicate experiments are presented as mean ± standard deviation. (D–F) The proliferation of antler cartilage cells transfected with si-CDK8 (D), si-EEF1A1 (E), si-NTN1 (F), or si-NC was detected by CCK-8 assay. (G–I) The effect of si-CDK8 (G), si-EEF1A1 (H), or si-NTN1 (I) on antler cartilage cell migration was measured by transwell migration assays. Scale bar represents 200 µm. si-NC, negative control siRNA. ** p < 0.01, *** p < 0.001.

Article Snippet: Following blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4 ◦C with specific primary antibodies against CDK8 (cat. no. A5548; 1:1000; ABclonal, Wuhan, China), EEF1A1 (cat. no. ab157455; 1:1000; Abcam), NTN1 (cat. no. A1500; 1:1000; ABclonal), GAPDH (cat. no. sc-47724; 1:3000; Santa Cruz Biotechnology, Dallas, TX, USA; or cat. no. AC033; 1:20,000; ABclonal), c-myc (cat. no. A5011; 1:1000; Bimake), or cyclin D1 (cat. no. A5035; 1:1000; Bimake).

Techniques: Knockdown, Migration, Western Blot, Standard Deviation, Transfection, CCK-8 Assay, Negative Control

Journal: International journal of molecular sciences

Article Title: MicroRNA PC-3p-2869 Regulates Antler Growth and Inhibits Proliferation and Migration of Human Osteosarcoma and Chondrosarcoma Cells by Targeting CDK8, EEF1A1, and NTN1.

doi: 10.3390/ijms241310840

Figure Lengend Snippet: Figure 7. Downregulation of CDK8, EEF1A1, or NTN1 inhibits proliferation, colony formation, and migration of osteosarcoma cells. (A–C) Western blot analysis of MG63 cells transfected with CDK8 (A), EEF1A1 (B), or NTN1 (C) siRNA. The Western blot bands are representative images of three independent biological replicates, and fold-changes in densitometric values in three replicate experiments are presented as mean ± standard deviation. (D–L) Effects of CDK8, EEF1A1, or NTN1 knockdown on the proliferation (D–F), colony formation capacities (G–I), and migration (J–L) of MG63 cells were evaluated using CCK-8 assay, colony formation assay, and transwell migration assay, respectively. Scale bar represents 200 µm. si-NC, negative control siRNA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Following blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4 ◦C with specific primary antibodies against CDK8 (cat. no. A5548; 1:1000; ABclonal, Wuhan, China), EEF1A1 (cat. no. ab157455; 1:1000; Abcam), NTN1 (cat. no. A1500; 1:1000; ABclonal), GAPDH (cat. no. sc-47724; 1:3000; Santa Cruz Biotechnology, Dallas, TX, USA; or cat. no. AC033; 1:20,000; ABclonal), c-myc (cat. no. A5011; 1:1000; Bimake), or cyclin D1 (cat. no. A5035; 1:1000; Bimake).

Techniques: Migration, Western Blot, Transfection, Standard Deviation, Knockdown, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Negative Control

Journal: International journal of molecular sciences

Article Title: MicroRNA PC-3p-2869 Regulates Antler Growth and Inhibits Proliferation and Migration of Human Osteosarcoma and Chondrosarcoma Cells by Targeting CDK8, EEF1A1, and NTN1.

doi: 10.3390/ijms241310840

Figure Lengend Snippet: Figure 8. Silencing of CDK8, EEF1A1, or NTN1 suppressed the proliferation, colony formation, and migration of chondrosarcoma cells. (A–C) Western blot analysis of SW1353 cells transfected with si-CDK8, si-EEF1A1, si-NTN1, or si-NC. The Western blot bands are representative images of three independent biological replicates, and fold-changes in densitometric values in three replicate experi- ments are presented as mean ± standard deviation. (D–L) The effects of CDK8, EEF1A1, or NTN1 knockdown on the proliferation (D–F), colony formation capacities (G–I), and migration (J–L) of SW1353 cells were assessed by CCK-8 assay, colony formation assay, and transwell migration assay, respectively. Scale bar represents 200 µm. si-NC, negative control siRNA. ** p < 0.01; *** p < 0.001.

Article Snippet: Following blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4 ◦C with specific primary antibodies against CDK8 (cat. no. A5548; 1:1000; ABclonal, Wuhan, China), EEF1A1 (cat. no. ab157455; 1:1000; Abcam), NTN1 (cat. no. A1500; 1:1000; ABclonal), GAPDH (cat. no. sc-47724; 1:3000; Santa Cruz Biotechnology, Dallas, TX, USA; or cat. no. AC033; 1:20,000; ABclonal), c-myc (cat. no. A5011; 1:1000; Bimake), or cyclin D1 (cat. no. A5035; 1:1000; Bimake).

Techniques: Migration, Western Blot, Transfection, Standard Deviation, Knockdown, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Negative Control

Journal: International journal of molecular sciences

Article Title: MicroRNA PC-3p-2869 Regulates Antler Growth and Inhibits Proliferation and Migration of Human Osteosarcoma and Chondrosarcoma Cells by Targeting CDK8, EEF1A1, and NTN1.

doi: 10.3390/ijms241310840

Figure Lengend Snippet: Figure 9. MiR-PC-2869 blocks the expression of c-myc and cyclin D1 by targeting CDK8, EEF1A1, and NTN1. (A–D) Western blot analysis of MG63, SW1353, and antler cartilage cells transfected with miR-PC-2869 mimics (A), si-CDK8 (B), si-EEF1A1 (C), or si-NTN1 (D). The Western blot bands are representative images of three independent biological replicates, and fold-changes in densitometric values in three replicate experiments are presented as mean ± standard deviation. NC, negative control mimics. si-NC, negative control siRNA. CA, antler cartilage cells. (E) The schematic diagram illustrates the molecular mechanism through which miR-PC-2869 inhibits the proliferation and migration of osteosarcoma, chondrosarcoma, and antler cartilage cells.

Article Snippet: Following blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4 ◦C with specific primary antibodies against CDK8 (cat. no. A5548; 1:1000; ABclonal, Wuhan, China), EEF1A1 (cat. no. ab157455; 1:1000; Abcam), NTN1 (cat. no. A1500; 1:1000; ABclonal), GAPDH (cat. no. sc-47724; 1:3000; Santa Cruz Biotechnology, Dallas, TX, USA; or cat. no. AC033; 1:20,000; ABclonal), c-myc (cat. no. A5011; 1:1000; Bimake), or cyclin D1 (cat. no. A5035; 1:1000; Bimake).

Techniques: Expressing, Western Blot, Transfection, Standard Deviation, Negative Control, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Transfection efficiency determined by flow cytometry . The transfection efficiency was 97.2% 6 h after transfecting with CDK8-siRNA of HCT116. The ratio of Lipofectin 2000 to siRNA was 4 μL: 4 μL, and the concentration of CDK8-siRNA is 80 pmol/L.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Transfection, Flow Cytometry, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin mRNA expression of CDK-siRNA transfected HCT116 cells detected by RT-PCR . 48 h later of CDK8-siRNA transfection, RT-PCR was performed to detect CDK8 and β-catenin mRNA expression. A: CDK8-siRNA group; B: scrambled siRNA group; C: non-siRNA group; D, E and F represented corresponding internal reference, and M: marker. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Representative Western blots of CDK8 and β-catenin expression level in CDK-siRNA transfected HCT116 cells . 72 h later of CDK8-siRNA transfection of HCT116 cells, protein expression of CDK8 (A) and β-catenin (B) was determined by western blot assay. a: non-siRNA group; b: scrambled siRNA group; c: CDK-siRNA group. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Western Blot, Expressing, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Cell proliferation activity after transfection of CDK8-siRNA assessed by MTT assay . Curves of cell growth after transfection for 24, 48 and 72 h by MTT assay. Results are given as means ± SD from three independent experiments. P < 0.05.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Activity Assay, Transfection, MTT Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Effect of CDK8-siRNA transfection on the apoptosis and cell cycle of HCT116 cells . 48 h after transfection, cell apoptosis (A) and cell cycle (B) were determined by flow cytometry. Quadrants D2-D4 represent necrotic/late apoptotic cells, viable cells, and early apoptotic cells, respectively. Results are given as means ± SD from three independent experiments.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Transfection, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin mRNA expression in colon tumor and adjacent normal tissues detected by real-time PCR . Fresh tumor and corresponding adjacent tissues from 12 patients were resected under sterile conditions and then snapfrozen in liquid nitrogen immediately. 200 mg tissue was taken out from liquid nitrogen and plused 1 ml Trizol when RNA was extaracted. Real-time PCR is performed for the expression levels of CDK8 (A) and β-catenin (B). Results are given as means ± SD from three independent experiments. P < 0.05.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin protein expression in colon tumor and adjacent normal tissues detected by IHC . The expression of CDK8 (left) and β-catenin (right) was stained brown and present in tumor tissue and adjacent normal tissues. Representative sites with negative (a, 400 X ), moderate positive (c, 400 × ), strongly positive (e, 400 ×) expression of CDK8 and corresponding weakly positive (b, 400 ×), moderate positive (d, 400 ×), strongly positive (f, 400 ×) expression of β-catenin.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Expressing, Staining