pcmv6 plasmid with full-length socs2 sequence  (OriGene)

Journal: Oncology Letters

Article Title: Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation

doi: 10.3892/ol.2020.11794

Figure Lengend Snippet: Manipulation of SOCS2 expression alters the proliferation of AGS cells. Knockdown of SOCS2 in AGS-WT cells promoted cell proliferation in AGS cells. Cells were treated with SOCS2-siRNA to knock down SOCS2. Cells treated with a non-specific control siRNA were used as controls. AGS cell treatment with SOCS2-siRNA significantly knocked down SOCS2 expression in AGS-WT cells at (A) RNA and (B) protein levels. *P<0.05 vs. Ctrl siRNA. (C) A significantly higher cell proliferation rate was detected by using an MTS assay in cells treated with siRNA-SOCS2 when compared with cells treated with Ctrl-siRNA. *P<0.05 vs. Ctrl siRNA. Overexpression of SOCS2 inhibited cell AGS cell proliferation. Cells were transfected with pCMV6-SOCS2 (full-length-SOCS2) to overexpress SOCS2. Cells treated with the pCMV6 empty vector were used as controls. SOCS2 overexpression in AGS-WT cells following transfection of pCMV6-SOCS2 was confirmed via reverse transcription-quantitative PCR and western blotting, respectively, at (D) RNA and (E) protein levels. *P<0.05 vs. pCMV6-Ctrl. A significantly lower cell proliferation rate was detected using an MTS assay in cells transfected with pCMV6-SOCS2 compared with pCMV6 empty vector-treated cells (F) Representative blotting images are presented in B and E. Densitometric levels of positive band for SOCS2 were quantified and are expressed as the ratio to GAPDH. *P<0.05 vs. pCMV6-Ctrl. SOCS, suppressor of cytokine signaling; WT, wild type; siRNA, small interfering RNA; Ctrl, control; OD, optical density.

Article Snippet: The pCMV6 plasmid with full-length SOCS2 sequence (pCMV6-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knockdown, Control, MTS Assay, Over Expression, Transfection, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Modulation of SOCS2 expression alters the proliferation ability of SW480 cells. (A-C) siRNA (si-SOCS2) was used to successfully knock down SOCS2 expression in SW480 cells. Knockdown of SOCS2 had minor impact on METTL3 expression but significantly increased LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D) Knockdown of SOCS2 enhanced SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the si-Ctrl group). (E-G) Transfection of the pCMV6-SOCS2 plasmid upregulated SOCS2 expression and decreased LGR5 expression in SW480 cells (*P<0.05, compared with the pCMV6-Entry group). SOCS2 overexpression had minor impact on METTL3 expression (P>0.05, compared with the pCMV6-Entry group). (H) SOCS2 overexpression decreased SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the pCMV6-Entry group). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knockdown, MTS Assay, Transfection, Plasmid Preparation, Over Expression

Journal: Oncology Letters

Article Title: Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation

doi: 10.3892/ol.2020.11794

Figure Lengend Snippet: The sequences of real-time PCR primers.

Article Snippet: The pCMV6 plasmid with full-length SOCS2 sequence (pCMV6-SOCS2) was purchased from Origene (RC203163).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation

doi: 10.3892/ol.2020.11794

Figure Lengend Snippet: Expression of effector molecules associated with AGS cell proliferation in METTL3-KO AGS cells. (A) Expression of selected genes associated with AGS cell proliferation were measured using reverse transcription-quantitative PCR. Several of these genes, including SOCS1, SOCS2, SOCS3 and LGR5, exhibited decreased expressions in METTL3-KO cells when compared with METTL3-WT cells. (B) Inhibition of RAD17, LGR5 and SOCS2 RNA stability in METTL3-KO AGS cells. AGS-WT and AGS METTL3-KO cells were cultured in the presence of actinomycin for 2 h. Levels of RAD17, LGR5 and SOCS2 were measured in cells following actinomycin treatment and compared with cells tested prior to actinomycin administration. Reduced RNA levels represents degradation rate. A lower degradation rate was observed for RAD17, LGR5 and SOCS2 in METTL3-KO AGS cells compared with AGS-WT. *P<0.01 vs. AGS-WT. METTL3-KO, methyltransferase like 3 knocked down; WT, wild type; SOCS, suppressor of cytokine signaling; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; RAD17, RAD17 checkpoint clamp loader component.

Article Snippet: The pCMV6 plasmid with full-length SOCS2 sequence (pCMV6-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Inhibition, Cell Culture

Journal: Oncology Letters

Article Title: Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation

doi: 10.3892/ol.2020.11794

Figure Lengend Snippet: Expression of SOCS1, SOCS2 and SOCS3 at the RNA and protein levels in METTL3-WT and METTL3-KO AGS cells. Elevated RNA expressions of (A) SOCS1, (B) SOCS2 and (C) SOCS3 were detected in METTL3-KO AGS cells, as determined via reverse transcription-quantitative PCR and compared with METTL3-WT AGS cells. METTL3-KO resulted in increased levels of (E) SOCS2, but not (D) SOCS1 and (F) SOCS3 (F) protein in AGS cells, as determined via western blotting and compared with METTL3-WT AGS cells. β-actin was also immunoblotted to ensure equal loading of proteins to each lane. Representative blotting images are presented. Densitometric levels of positive bands for SOCS1, SOCS2 and SOCS3 were quantified and are expressed as the ratio to β-actin. *P<0.01 vs. AGS-WT. SOCS, suppressor of cytokine signaling; METTL3, methyltransferase like 3; WT, wild type; KO, knocked down.

Article Snippet: The pCMV6 plasmid with full-length SOCS2 sequence (pCMV6-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Letters

Article Title: Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation

doi: 10.3892/ol.2020.11794

Figure Lengend Snippet: Manipulation of SOCS2 expression alters the proliferation of AGS cells. Knockdown of SOCS2 in AGS-WT cells promoted cell proliferation in AGS cells. Cells were treated with SOCS2-siRNA to knock down SOCS2. Cells treated with a non-specific control siRNA were used as controls. AGS cell treatment with SOCS2-siRNA significantly knocked down SOCS2 expression in AGS-WT cells at (A) RNA and (B) protein levels. *P<0.05 vs. Ctrl siRNA. (C) A significantly higher cell proliferation rate was detected by using an MTS assay in cells treated with siRNA-SOCS2 when compared with cells treated with Ctrl-siRNA. *P<0.05 vs. Ctrl siRNA. Overexpression of SOCS2 inhibited cell AGS cell proliferation. Cells were transfected with pCMV6-SOCS2 (full-length-SOCS2) to overexpress SOCS2. Cells treated with the pCMV6 empty vector were used as controls. SOCS2 overexpression in AGS-WT cells following transfection of pCMV6-SOCS2 was confirmed via reverse transcription-quantitative PCR and western blotting, respectively, at (D) RNA and (E) protein levels. *P<0.05 vs. pCMV6-Ctrl. A significantly lower cell proliferation rate was detected using an MTS assay in cells transfected with pCMV6-SOCS2 compared with pCMV6 empty vector-treated cells (F) Representative blotting images are presented in B and E. Densitometric levels of positive band for SOCS2 were quantified and are expressed as the ratio to GAPDH. *P<0.05 vs. pCMV6-Ctrl. SOCS, suppressor of cytokine signaling; WT, wild type; siRNA, small interfering RNA; Ctrl, control; OD, optical density.

Article Snippet: The pCMV6 plasmid with full-length SOCS2 sequence (pCMV6-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knockdown, Control, MTS Assay, Over Expression, Transfection, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA