sirnas against jnk1  (Amaxa)

Journal: Cancers

Article Title: IL-33-Induced Transcriptional Activation of LPIN1 Accelerates Breast Tumorigenesis

doi: 10.3390/cancers13092174

Figure Lengend Snippet: COT interacts with JNK1/2 to mediate IL-33-induced LPIN1 expression. ( A ) MCF7 cells were serum starved for 24 h, pretreated with the indicated inhibitors for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, and harvested. The protein and mRNA levels were assessed using immunoblotting or RT-PCR. LY294002 (PI3K inhibitor). SP600125 (JNK inhibitor). PD98059 (ERK inhibitor). WP1066 (STAT3 inhibitor). ( B ) MCF7 cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting. ( C ) MCF7 cells were transfected with MOCK or Myc-COT. At 48 h after transfection, the cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the entire cell lysates were separated using SDS-PAGE and subjected to immunoblotting. ( D ) HEK293 cells expressing V5-JNK1, V5-JNK2 with Myc-COT were subjected to immunoprecipitation with anti-V5 antibodies, which is followed by immunoblotting with anti-Myc or anti-V5 antibodies. ( E , F ) MCF7 cells were starved for 24 h, exposed to 25 ng/mL IL-33 for the indicated time, harvested, and lysed. Immunoprecipitation was performed using JNK1 ( E ) or JNK2 ( F ) antibodies and then analyzed using immunoblotting, as indicated. ( G ) MCF7 cells were transfected with different amounts of pcDNA4/V5-JNK1 (left) or pcDNA4/V5-JNK2 (right), incubated for 48 h, harvested, and subjected to immunoblotting. ( H ) MCF7 cells were transfected with either V5-JNK1 (left) or V5-JNK2 (right). At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting. ( I ) MCF7 cells were transfected with siRNA-JNK1/2. At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting.

Article Snippet: The human IL-33 plasmid, pCMV/Myc-IL-33, and pcDNA3.1/V5-JNK1 were purchased from OriGene Technologies Inc. (Rockville, MD, USA). siRNA against JNK1 (accession number: NM_001323302.1) and JNK2 (accession number: NM_139068.2) were purchased from Mbiotech (Seoul, Korea).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, SDS Page, Transfection, Immunoprecipitation, Incubation

Journal: Cancers

Article Title: IL-33-Induced Transcriptional Activation of LPIN1 Accelerates Breast Tumorigenesis

doi: 10.3390/cancers13092174

Figure Lengend Snippet: IL-33 stimulates binding of c-Jun to the LPIN1 promoter. ( A , B ) MCF7 cells were transfected with different amounts of pcDNA4/V5-JNK1 ( A ) or pcDNA4/V5-JNK2 ( B ), incubated for 48 h, harvested, and subjected to immunoblotting. The levels of LPIN1 and β -actin mRNA were determined using RT-PCR. ( C ) MCF7 cells were transfected with siRNA-JNK1/2. At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. The levels of mRNA were determined using RT-PCR. ( D ) MCF7 cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, and then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. The levels of mRNA were determined using RT-PCR. ( E ) Schema of the putative c-Jun-binding sites within the LPIN1 promoter region. ( F ) ChIP assay with either anti-c-Jun antibody or control mouse IgG, with input chromatin as a positive control. ( G ) Cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, and then exposed to 25 ng/mL IL-33 for 24 h. Following that, the ChIP assay was performed on these samples using an anti-c-Jun antibody or control mouse IgG, with input chromatin as a positive control. The input DNA and DNA isolated from the precipitated chromatin were amplified using PCR and separated on a 1.5% agarose gel. IgG, immunoglobulin.

Article Snippet: The human IL-33 plasmid, pCMV/Myc-IL-33, and pcDNA3.1/V5-JNK1 were purchased from OriGene Technologies Inc. (Rockville, MD, USA). siRNA against JNK1 (accession number: NM_001323302.1) and JNK2 (accession number: NM_139068.2) were purchased from Mbiotech (Seoul, Korea).

Techniques: Binding Assay, Transfection, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control, Isolation, Amplification, Agarose Gel Electrophoresis

Journal: Cancers

Article Title: IL-33-Induced Transcriptional Activation of LPIN1 Accelerates Breast Tumorigenesis

doi: 10.3390/cancers13092174

Figure Lengend Snippet: COT interacts with JNK1/2 to mediate IL-33-induced LPIN1 expression. ( A ) MCF7 cells were serum starved for 24 h, pretreated with the indicated inhibitors for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, and harvested. The protein and mRNA levels were assessed using immunoblotting or RT-PCR. LY294002 (PI3K inhibitor). SP600125 (JNK inhibitor). PD98059 (ERK inhibitor). WP1066 (STAT3 inhibitor). ( B ) MCF7 cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting. ( C ) MCF7 cells were transfected with MOCK or Myc-COT. At 48 h after transfection, the cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the entire cell lysates were separated using SDS-PAGE and subjected to immunoblotting. ( D ) HEK293 cells expressing V5-JNK1, V5-JNK2 with Myc-COT were subjected to immunoprecipitation with anti-V5 antibodies, which is followed by immunoblotting with anti-Myc or anti-V5 antibodies. ( E , F ) MCF7 cells were starved for 24 h, exposed to 25 ng/mL IL-33 for the indicated time, harvested, and lysed. Immunoprecipitation was performed using JNK1 ( E ) or JNK2 ( F ) antibodies and then analyzed using immunoblotting, as indicated. ( G ) MCF7 cells were transfected with different amounts of pcDNA4/V5-JNK1 (left) or pcDNA4/V5-JNK2 (right), incubated for 48 h, harvested, and subjected to immunoblotting. ( H ) MCF7 cells were transfected with either V5-JNK1 (left) or V5-JNK2 (right). At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting. ( I ) MCF7 cells were transfected with siRNA-JNK1/2. At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting.

Article Snippet: The human IL-33 plasmid, pCMV/Myc-IL-33, and pcDNA3.1/V5-JNK1 were purchased from OriGene Technologies Inc. (Rockville, MD, USA). siRNA against JNK1 (accession number: NM_001323302.1) and JNK2 (accession number: NM_139068.2) were purchased from Mbiotech (Seoul, Korea).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, SDS Page, Transfection, Immunoprecipitation, Incubation

Journal: Cancers

Article Title: IL-33-Induced Transcriptional Activation of LPIN1 Accelerates Breast Tumorigenesis

doi: 10.3390/cancers13092174

Figure Lengend Snippet: IL-33 stimulates binding of c-Jun to the LPIN1 promoter. ( A , B ) MCF7 cells were transfected with different amounts of pcDNA4/V5-JNK1 ( A ) or pcDNA4/V5-JNK2 ( B ), incubated for 48 h, harvested, and subjected to immunoblotting. The levels of LPIN1 and β -actin mRNA were determined using RT-PCR. ( C ) MCF7 cells were transfected with siRNA-JNK1/2. At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. The levels of mRNA were determined using RT-PCR. ( D ) MCF7 cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, and then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. The levels of mRNA were determined using RT-PCR. ( E ) Schema of the putative c-Jun-binding sites within the LPIN1 promoter region. ( F ) ChIP assay with either anti-c-Jun antibody or control mouse IgG, with input chromatin as a positive control. ( G ) Cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, and then exposed to 25 ng/mL IL-33 for 24 h. Following that, the ChIP assay was performed on these samples using an anti-c-Jun antibody or control mouse IgG, with input chromatin as a positive control. The input DNA and DNA isolated from the precipitated chromatin were amplified using PCR and separated on a 1.5% agarose gel. IgG, immunoglobulin.

Article Snippet: The human IL-33 plasmid, pCMV/Myc-IL-33, and pcDNA3.1/V5-JNK1 were purchased from OriGene Technologies Inc. (Rockville, MD, USA). siRNA against JNK1 (accession number: NM_001323302.1) and JNK2 (accession number: NM_139068.2) were purchased from Mbiotech (Seoul, Korea).

Techniques: Binding Assay, Transfection, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control, Isolation, Amplification, Agarose Gel Electrophoresis