Journal: International Journal of Molecular Sciences

Article Title: NSC 95397 Suppresses Proliferation and Induces Apoptosis in Colon Cancer Cells through MKP-1 and the ERK1/2 Pathway

doi: 10.3390/ijms19061625

Figure Lengend Snippet: Involvement of MKP-1/ERK1/2 pathway in NSC 95397-induced p21 expression and caspase-3 activation in human colon cancer. Representative Western blots showing ( A ) the expression of Cdc25A, the phosphorylation of Cdk1 at Tyr15; ( B ) the expression of MKP-1 and ERK1/2, and the phosphorylation of ERK1/2 at Thr202/Tyr 204 in SW480, SW620, and DLD-1 cells treated with 10 μM NSC 95397 for 6 h, with actin as loading control; ( C ) representative Western blots showing expression of p21 and cleaved caspase-3 in colon cancer cells that were pre-incubated with 20 μM U0126 for 2 h and then treated with 10 m μM NSC 95397 for 6 h.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-p21 (Cell Signaling Technology #2947, Danvers, MA, USA), rabbit monoclonal anti-CDK4 (Cell Signaling Technology #12790), mouse monoclonal anti-CDK6 (Cell Signaling Technology #3136), rabbit polyclonal anti-Phospho-Rb (Ser795) (Cell Signaling Technology #9301), rabbit monoclonal anti-Phospho-Rb (Ser807/811) (Cell Signaling Technology #8516), mouse monoclonal anti-Caspase-9 (Cell Signaling Technology #9608), rabbit monoclonal anti-Cleaved Caspase-9 (Cell Signaling Technology #7237), rabbit monoclonal anti-Caspase-3 (Cell Signaling Technology #9665), rabbit monoclonal anti-Cleaved Caspase-3 (Cell Signaling Technology #9664), rabbit monoclonal anti-Caspase-7 (Cell Signaling Technology #12827), rabbit monoclonal anti-Cleaved Caspase-7 (Cell Signaling Technology #8438), rabbit polyclonal anti-PARP (Cell Signaling Technology #9542), rabbit monoclonal anti-Cleaved PARP (Cell Signaling Technology #5625), rabbit polyclonal anti-cdc25A (Cell Signaling Technology #3652), rabbit polyclonal anti-Phospho-cdc2 (Tyr15) (Cell Signaling Technology #4539), rabbit polyclonal anti-MKP-1 (Bioworld Technology #BS1677, St. Louis Park, MN, USA), rabbit polyclonal anti-ERK1/2 (GeneTex #GTX59618, Irvine, CA, USA), rabbit polyclonal anti-Phospho-ERK1/2 (GeneTex #GTX61126), and mouse monoclonal anti-Actin (Merck Millipore #MAB1501, Burlington, MA, USA).

Techniques: Expressing, Activation Assay, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: NSC 95397 Suppresses Proliferation and Induces Apoptosis in Colon Cancer Cells through MKP-1 and the ERK1/2 Pathway

doi: 10.3390/ijms19061625

Figure Lengend Snippet: A schematic showing that the cytotoxic effect of NSC 95397 on colon cancer cells is activated by inhibiting MKP-1 activity followed by ERK1/2 activation. Lines ending in arrows indicate promotion and T-bars represent inhibition.

Article Snippet: The following antibodies were used: rabbit monoclonal anti-p21 (Cell Signaling Technology #2947, Danvers, MA, USA), rabbit monoclonal anti-CDK4 (Cell Signaling Technology #12790), mouse monoclonal anti-CDK6 (Cell Signaling Technology #3136), rabbit polyclonal anti-Phospho-Rb (Ser795) (Cell Signaling Technology #9301), rabbit monoclonal anti-Phospho-Rb (Ser807/811) (Cell Signaling Technology #8516), mouse monoclonal anti-Caspase-9 (Cell Signaling Technology #9608), rabbit monoclonal anti-Cleaved Caspase-9 (Cell Signaling Technology #7237), rabbit monoclonal anti-Caspase-3 (Cell Signaling Technology #9665), rabbit monoclonal anti-Cleaved Caspase-3 (Cell Signaling Technology #9664), rabbit monoclonal anti-Caspase-7 (Cell Signaling Technology #12827), rabbit monoclonal anti-Cleaved Caspase-7 (Cell Signaling Technology #8438), rabbit polyclonal anti-PARP (Cell Signaling Technology #9542), rabbit monoclonal anti-Cleaved PARP (Cell Signaling Technology #5625), rabbit polyclonal anti-cdc25A (Cell Signaling Technology #3652), rabbit polyclonal anti-Phospho-cdc2 (Tyr15) (Cell Signaling Technology #4539), rabbit polyclonal anti-MKP-1 (Bioworld Technology #BS1677, St. Louis Park, MN, USA), rabbit polyclonal anti-ERK1/2 (GeneTex #GTX59618, Irvine, CA, USA), rabbit polyclonal anti-Phospho-ERK1/2 (GeneTex #GTX61126), and mouse monoclonal anti-Actin (Merck Millipore #MAB1501, Burlington, MA, USA).

Techniques: Activity Assay, Activation Assay, Inhibition

Journal: Veterinary Research

Article Title: Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway

doi: 10.1186/s13567-023-01178-6

Figure Lengend Snippet: Sequences of DNA primers .

Article Snippet: Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃.

Techniques: Sequencing, Clone Assay

Journal: Veterinary Research

Article Title: Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway

doi: 10.1186/s13567-023-01178-6

Figure Lengend Snippet: Sequences of RNA oligonucleotides .

Article Snippet: Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃.

Techniques:

Journal: Veterinary Research

Article Title: Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway

doi: 10.1186/s13567-023-01178-6

Figure Lengend Snippet: Let-7d is involved in the regulation of MAPK by targeting MKP1. A KEGG pathway analysis of let-7d target genes. B GO term analysis of let-7d target genes. C Conservation of the let-7d target sequence in the MKP1 3’-UTR among different species and conservation of the let-7d sequence among different species. D The relative level of MKP1 in chicken embryonic lungs. E The mRNA and protein levels of MKP1 in CP-II cells. F The dual-luciferase reporter assay was performed in CP-II cells. The ratio of Renilla activity: Firefly activity represents luciferase activity. The data are presented as the means ± SDs ( n = 4). Different lowercase letters represent p < 0.05. * p < 0.05, ** p < 0.01.

Article Snippet: Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃.

Techniques: Sequencing, Luciferase, Reporter Assay, Activity Assay

Journal: Veterinary Research

Article Title: Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway

doi: 10.1186/s13567-023-01178-6

Figure Lengend Snippet: Let-7d negatively regulates MKP1. A, B . The levels of let-7d in CP-II cells. (C-F). The expression of MKP1 in different treatment groups was detected by qPCR C, D and Western blotting E, F . The data are the mean ± S.D. of at least 3 independent experiments. * p < 0.05, ** p < 0.01. ns > 0.05.

Article Snippet: Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃.

Techniques: Expressing, Western Blot

Journal: Veterinary Research

Article Title: Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway

doi: 10.1186/s13567-023-01178-6

Figure Lengend Snippet: The regulatory effect of MKP1 on MAPK pathway-related genes. WB was used to detect protein levels after MKP1 overexpression or knockdown. GAPDH served as the loading control. A, B The expression of MKP1. The expression of p38, ERK or JNK in the MKP1 overexpression groups C and knockdown groups ( D ). The data are the mean ± S.D. of at least 3 independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃.

Techniques: Over Expression, Expressing

Journal: Veterinary Research

Article Title: Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway

doi: 10.1186/s13567-023-01178-6

Figure Lengend Snippet: MKP1 regulates inflammatory cytokine production in CP-II. CP-II cells were transfected with pcDNA3.1-MKP1 (over MKP1), empty vector (pcDNA3.1), Si-MKP1 or Si-NC for 24 h and then infected with 200 µL of MG-HS. The relative protein expression of inflammatory cytokines in different treatment groups was detected by ELISA kits. The data are presented as the means ± SDs ( n = 4). * p < 0.05, ** p < 0.01. ns > 0.05.

Article Snippet: Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway

doi: 10.1186/s13567-023-01178-6

Figure Lengend Snippet: The effect of MKP1 on CP-II cell proliferation and apoptosis. CP-II cells were transfected with pcDNA3.1-MKP1 (over MKP1), empty vector (pcDNA3.1), Si-MKP1 or Si-NC for 24 h and then infected with 200 µL of MG-HS. Overexpression of MKP1 was denoted as Over-MKP1; the negative control for MKP1 overexpression was denoted as pcDNA3.1; the MKP1 inhibitor was denoted as Si-MKP1; and the negative control for MKP1 inhibitor was denoted as Si-NC. A CCK-8 kit was used to detect cell proliferation at different times ( A, B ). After 48 h of treatment, the apoptotic cell ratio was analysed by flow cytometry. ( C ). A CCK-8 kit was used to detect cell proliferation at different times. All data from the experiments carried out independently three times are shown as the mean value ± SD, and * p < 0.05, ** p < 0.01 indicated significant differences.

Article Snippet: Equal amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1 h. Then, primary antibodies for rabbit anti-MKP1 (ABclonal, A2919), p-ERK (ABclonal, AP0472), p-JNK (ABclonal, A4867), P38 (ABclonal, A0227), GAPDH (Abmart, M20024) or β-actin (Abmart, T40104) (at 1:5000 dilution) protein were incubated overnight at 4 ℃.

Techniques: Transfection, Plasmid Preparation, Infection, Over Expression, Negative Control, CCK-8 Assay, Flow Cytometry