wtap sirna  (Santa Cruz Biotechnology)

Journal: Acta Diabetologica

Article Title: The UBR5 protein facilitates mesangial cell hypertrophy and glycolysis induced by high glucose by increasing the phosphorylation levels of AKT

doi: 10.1007/s00592-025-02464-9

Figure Lengend Snippet: WTAP promotes UBR5 m6A modification in an IGF2BP1 dependent manner. HMCs were treated with HG (25 mM glucose) for 0, 12, 24, and 48 h, and expression of WTAP was measured by A RT-qPCR and B Western blot. C–H HMCs were first transduced with lentiviral vectors for WTAP interference or overexpression and treated with HG (25 mM glucose) for 48 h. C , D RT-qPCR and E Western blotting were conducted to evaluate WTAP and UBR5 expression. F Global m6A levels were measured by ELISA. G MeRIP-PCR was used to assess UBR5 3’UTR methylation levels. H UBR5 3’UTR activity was examined by Luciferase reporter gene assays. I HMCs transduced with lentiviral vectors for WTAP interference were treated with 0.2 mM actinomycin D for 0, 2, 4, and 6 h, and RT-qPCR was performed to measure UBR5 transcription levels. HMCs were transduced with lentiviral vectors for IGF2BP1 interference, and J RT-qPCR and K Western blotting were performed to detect UBR5 expression. L HMCs transduced with lentiviral vectors for IGF2BP1 interference were treated with 0.2 mM actinomycin D for 0, 2, 4, and 6 h, and RT-qPCR was performed to measure UBR5 transcription levels. M RIP-PCR was executed to detect the binding of IGF2BP1 at the 3’UTR region of UBR5. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Sangon Biotech (Shanghai, China) customized UBR5 shRNA, WTAP shRNA, IGF2BP1 shRNA, and scrambled shRNA sequences, and subcloned them into plasmid vector pLKO.1 (Addgene, Watertown, MA, USA).

Techniques: Modification, Expressing, Quantitative RT-PCR, Western Blot, Transduction, Over Expression, Enzyme-linked Immunosorbent Assay, Methylation, Activity Assay, Luciferase, Binding Assay

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: The expression level of WTAP in GC patients by IHC.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: The relationship between the relative expression level of WTAP and clinicopathological variables in 50 patients with fresh GC tissues.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: The relationship between the relative expression level of WTAP and clinicopathological variables in 50 patients with paraffin GC tissues.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Overexpressed WTAP promoted the cell proliferation, wound healing, migration and invasion ability. The overexpression efficiency of WTAP in HGC-27 and AGS cell lines was detected by qRT-PCR (A, B) and Western blot analysis (C, D) . (E, F) The cell viability of HGC-27 and AGS cell lines were determined. (G, H) The scratch healing ability of HGC-27 and AGS cell lines was detected. Scale bar, 100 μm. (I, J) Transwell migration and invasion assays were used to evaluate HGC-27 and AGS cell migration and invasion ability. Scale bar, 100 μm.** P <0.01; *** P <0.001; **** P <0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Migration, Over Expression, Quantitative RT-PCR, Western Blot

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Knockdown of WTAP inhibited the cell proliferation, wound healing, migration and invasion ability. The knockdown efficiency of WTAP in HGC-27 and AGS cell lines was detected by qRT-PCR (A, B) and Western blot analysis (C, D) . (E, F) The cell viability of HGC-27 and AGS cell lines were determined. (G, H) The scratch healing ability of HGC-27 and AGS cell lines was examined. Scale bar, 100 μm. (I, J) Transwell migration and invasion assays were used to evaluate HGC-27 and AGS cell migration and invasion ability. Scale bar, 100 μm.* P <0.05; *** P <0.001; **** P <0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Western Blot

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: WTAP regulates the tumorigenesis and growth of GC cells in nude mice. (A) The expression level of WTAP mRNA in stable cell lines was detected by qRT-PCR. (B) The expression level of WTAP protein in stable cell lines was detected by WB. (C) The tumor size was measured every 2 days and the tumor growth curve was plotted. (D) Tumors of the two groups of nude mice were dissected and photographed on the 18th day after transplantation. (E) The tumor was weighed after dissection. (F) Representative images of HE staining of the 2 groups of xenograft tumors. Scale bar, 20 μm. (G) Representative immunohistochemical results and quantitative analysis of Ki-67 positive staining in 2 groups of xenograft tumors. Scale bar, 20 μm. Data are shown as means ± S.D. *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing, Stable Transfection, Quantitative RT-PCR, Transplantation Assay, Dissection, Staining, Immunohistochemical staining

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Analysis of transcriptome sequencing and m 6 A sequencing results. (A-C) The sequencing results identified the genes significantly changed in Sh-WTAP group compared with Sh-ctrl group. (D, E) m 6 A sequencing identified the diminished m 6 A peaks in Sh-WTAP group. (F) The m 6 A consensus sequence motif was identified in HGC-27 cells. (G, H) Distribution of m 6 A IP signal in mRNA transcripts in HGC-27 cells.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Sequencing

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: MAP2K6 is a direct downstream target gene of WTAP. (A) The intersection of genes corresponding to the downregulated m 6 A peaks in m 6 A sequencing and downregulated genes in transcriptome sequencing. (B, C) The expression levels of CNTN1, LHX9, SYT1, MAP2K6 and PRUNE2 genes were detected by qRT-PCR. (D) Attenuation of WTAP diminishes m 6 A modification of MAP2K6 mRNA in HGC-27 cells. (E) MeRIP-qPCR was used to detect the change of MAP2K6 mRNA enrichment in HGC-27 cells after knocking down WTAP expression level. (F) MeRIP-qPCR was used to detect the change of MAP2K6 mRNA enrichment in AGS cells after up-regulating WTAP expression level. (G) Diagram illustrating the fusion of the wild-type MAP2K6 sequence and the MAP2K6 sequence with a mutation at the 1409th m 6 A site with the luciferase reporter gene, respectively. (H, I) Relative luciferase intensity of HGC-27/AGS cells under different treatments. (J) Schematic diagram of the catRAPID website predicting the binding site between WTAP protein and MAP2K6 mRNA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Modification, Mutagenesis, Luciferase, Binding Assay

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: WTAP positively regulates MAP2K6. (A, B) The mRNA expression levels of MAP2K6 upon WTAP knockdown or overexpression were measured by qRT-PCR. (C, D) The protein expression levels of MAP2K6 upon WTAP knockdown or overexpression were measured by WB. (E) Co-expression of WTAP and MAP2K6 in GC cells was determined by confocal immunofluorescent assay (600X). Scale bar, 50 μm. (F) The mRNA expression levels of MAP2K6 in 50 GC tissues and paired adjacent normal tissues were determined by qRT-PCR. (G) WTAP was positively correlated with MAP2K6 expression in 50 GC tissues. (H) The colocalization of WTAP and MAP2K6 in GC tissues were confirmed by immunofluorescent assay (200X). Scale bar, 150 μm.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: Expressing, Knockdown, Over Expression, Quantitative RT-PCR

Journal: Journal of Cancer

Article Title: WTAP-Mediated N6-Methyladenosine Modification Promotes Gastric Cancer Progression by Regulating MAP2K6 Expression

doi: 10.7150/jca.98559

Figure Lengend Snippet: Oncogenic function of WTAP depends on MAP2K6. (A, B) CCK-8 and colony formation assay were used to determine the effect of down-regulation of MAP2K6 expression on cell proliferation induced by overexpression of WTAP. (C, D) Wound healing assay and Transwell migration assay were used to investigate the effect of down-regulation of MAP2K6 expression on cell migration potency induced by WTAP overexpression. (E) Transwell invasion assay was used to clarify the effect of down-regulation of MAP2K6 expression on cell invasion potency induced by overexpression of WTAP. (F-J) The effects of MAP2K6 on WTAP in HGC-27 cells were detected by CCK-8 assay (F) , colony formation assay (G) , Wound healing assay (H) , Transwell migration assay (I) , Transwell invasion assay (J) .* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were digested in 25 cm 2 cell culture flasks after they reached 80-90% fusion, then the WTAP overexpression plasmid, WTAP siRNA (GenePharma Co., Ltd., Shanghai, China), WTAP shRNA (GeneCopoeia Co., Ltd., Guangzhou, China), Mitogen-activated protein kinase kinase 6 (MAP2K6) siRNA (GenePharma Co., Ltd.), MAP2K6 overexpression plasmid and their respective negative control were transfected into GC cells for 6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to its instruction.

Techniques: CCK-8 Assay, Colony Assay, Expressing, Over Expression, Wound Healing Assay, Transwell Migration Assay, Migration, Transwell Invasion Assay